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Anna Huttenlocher

Picture of Anna HuttenlocherProfessor of Medical Microbiology & Immunology and Pediatrics
4205 Microbial Sciences Building
1550 Linden Drive
Office: (608) 265-4642
Laboratory: 265-4669
Email: huttenlocher@wisc.edu
Overview · Personnel · Publications · Lab Website
  • Ingram PN, Hind LE, Jiminez-Torres JA, Huttenlocher A, Beebe DJ (2018) An Accessible Organotypic Microvessel Model Using iPSC-Derived Endothelium. Adv Healthc Mater 7(2): View Abstract · Pubmed Record

    While organotypic approaches promise increased relevance through the inclusion of increased complexity (e.g., 3D extracellular microenvironment, structure/function relationships, presence of multiple cell types), cell source is often overlooked. Induced pluripotent stem cell (iPSC)-derived cells are potentially more physiologically relevant than cell lines, while also being less variable than primary cells, and recent advances have made them commercially available at costs similar to cell lines. Here, the use of induced pluripotent stem cell-derived endothelium for the generation of a functional microvessel model is demonstrated. High precision structural and microenvironmental control afforded by the design approach synergizes with the advantages of iPSC to produce microvessels for modeling endothelial biology in vitro. iPSC microvessels show endothelial characteristics, exhibit barrier function, secrete angiogenic and inflammatory mediators, and respond to changes in the extracellular microenvironment by altering vessel phenotype. Importantly, when deployed in the investigation of neutrophils during innate immune recruitment, the presence of the iPSC endothelial vessel facilitates neutrophil extravasation and migration toward a chemotactic source. Relevant cell sources, such as iPSC, combine with organotypic models to open the way for improved and increasingly accessible in vitro tissue, disease, and patient-specific models.

  • Rosowski EE, Huttenlocher A (2018) Motile Collectors: Platelets Promote Innate Immunity. Immunity 48(1):16-18 View Abstract · Pubmed Record

    Platelets migrate in vitro but the significance of platelet migration in vivo remains unclear. In a recent issue of Cell, Gaertner et al. (2017) demonstrate that active platelet migration in vivo promotes mechano-scavenging of bacterial pathogens and neutrophil activation.

  • LeBert D, Squirrell JM, Freisinger C, Rindy J, Golenberg N, Frecentese G, Gibson A, Eliceiri KW, Huttenlocher A (2018) Damage-induced reactive oxygen species regulateand dynamic collagen-based projections to mediate wound repair. Elife 7: (PMC5790375) View Abstract · Pubmed Record

    Tissue injury leads to early wound-associated reactive oxygen species (ROS) production that mediate tissue regeneration. To identify mechanisms that function downstream of redox signals that modulate regeneration, areporter of mesenchymal cells was generated by driving GFP from thepromoter in zebrafish. Early redox signaling mediatedreporter activity at the wound margin. Moreover, both ROS and vimentin were necessary for collagen production and reorganization into projections at the leading edge of the wound. Second harmonic generation time-lapse imaging revealed that the collagen projections were associated with dynamic epithelial extensions at the wound edge during wound repair. Perturbing collagen organization by burn wound disrupted epithelial projections and subsequent wound healing. Taken together our findings suggest that ROS and vimentin integrate early wound signals to orchestrate the formation of collagen-based projections that guide regenerative growth during efficient wound repair.

  • Huemer K, Squirrell JM, Swader R, Pelkey K, LeBert DC, Huttenlocher A, Eliceiri KW (2017) Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae. J Vis Exp (128): View Abstract · Pubmed Record

    The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

  • Barros-Becker F, Lam PY, Fisher R, Huttenlocher A (2017) Live imaging reveals distinct modes of neutrophil and macrophage migration within interstitial tissues. J. Cell. Sci. 130(22):3801-3808 (PMC5702045) View Abstract · Pubmed Record

    Cell motility is required for diverse processes during immunity and inflammation. Classically, leukocyte motility is defined as an amoeboid type of migration, however some leukocytes, like macrophages, also employ a more mesenchymal mode of migration. Here, we sought to characterize the mechanisms that regulate neutrophil and macrophage migrationby using real-time imaging of leukocyte motility within interstitial tissues in zebrafish larvae. Neutrophils displayed a rounded morphology and rapid protease-independent motility, lacked defined paxillin puncta, and had persistent rearward polarization of stable F-actin and the microtubule network. By contrast, macrophages displayed an elongated morphology with reduced speed and increased directional persistence and formed paxillin-containing puncta but had a less-defined polarization of the microtubule and actin networks. We also observed differential effects of protease inhibition, microtubule disruption and ROCK inhibition on the efficiency of neutrophil and macrophage motility. Taken together, our findings suggest that larval zebrafish neutrophils and macrophage display distinct modes of migration within interstitial tissues.

  • Vincent WJB, Harvie EA, Sauer JD, Huttenlocher A (2017) Neutrophil derived LTB4 induces macrophage aggregation in response to encapsulated Streptococcus iniae infection. PLoS ONE 12(6):e0179574 (PMC5489177) View Abstract · Pubmed Record

    Immune cells sense and react to a multitude of factors including both host and microbe-derived signals. Understanding how cells translate these cues into particular cellular behaviors is a complex yet critical area of study. We have previously shown that both neutrophils and macrophages are important for controlling the fish pathogen Streptococcus iniae. Here, we report both host and bacterial determinants leading to the formation of organized macrophage aggregates as part of the host inflammatory response in a subset of infected larvae. Streptococcal capsule was a required signal for aggregate formation. Macrophage aggregation coincided with NFκB activity, and the formation of these aggregates is mediated by leukotriene B4 (LTB4) produced by neutrophils. Depletion, inhibition, or genetic deletion of leukotriene A4 hydrolase (Lta4h), which catalyzes the last step in LTB4 synthesis, resulted in the absence of macrophage aggregation. Larvae with impaired neutrophil function also had impaired macrophage aggregation; however, aggregate formation was partially rescued with the addition of exogenous LTB4. Neutrophil-specific expression of lta4h was sufficient to rescue macrophage aggregation in Lta4h-deficient larvae and increased host survival following infection. In summary, our findings highlight a novel innate immune response to infection in which specific bacterial products drive neutrophils that modulate macrophage behavior through eicosanoid signaling.

  • Wiemann P, Perevitsky A, Lim FY, Shadkchan Y, Knox BP, Landero Figueora JA, Choera T, Niu M, Steinberger AJ, Wüthrich M, Idol RA, Klein BS, Dinauer MC, Huttenlocher A, Osherov N, Keller NP (2017) Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense. Cell Rep 19(10):2174-2176 View Abstract · Pubmed Record
  • Knox BP, Huttenlocher A, Keller NP (2017) Real-time visualization of immune cell clearance of Aspergillus fumigatus spores and hyphae. Fungal Genet. Biol. 105:52-54 (PMC5589445) View Abstract · Pubmed Record

    Invasive aspergillosis (IA) is a disease of the immunocompromised host and generally caused by the opportunistic fungal pathogen Aspergillus fumigatus. While both host and fungal factors contribute to disease severity and outcome, there are fundamental features of IA development including fungal morphological transition from infectious conidia to tissue-penetrating hyphae as well as host defenses rooted in mechanisms of innate phagocyte function. Here we address recent advances in the field and use real-time in vivo imaging in the larval zebrafish to visually highlight conserved vertebrate innate immune behaviors including macrophage phagocytosis of conidia and neutrophil responses post-germination.

  • Powell D, Tauzin S, Hind LE, Deng Q, Beebe DJ, Huttenlocher A (2017) Chemokine Signaling and the Regulation of Bidirectional Leukocyte Migration in Interstitial Tissues. Cell Rep 19(8):1572-1585 (PMC5505660) View Abstract · Pubmed Record

    Motile cells navigate through complex tissue environments that include both attractive and repulsive cues. In response to tissue wounding, neutrophils, primary cells of the innate immune response, exhibit bidirectional migration that is orchestrated by chemokines and their receptors. Although progress has been made in identifying signals that mediate the recruitment phase, the mechanisms that regulate neutrophil reverse migration remain largely unknown. Here, we visualize bidirectional neutrophil migration to sterile wounds in zebrafish larvae and identify specific roles for the chemokine receptors Cxcr1 and Cxcr2 in neutrophil recruitment to sterile injury and infection. Notably, we also identify Cxcl8a/Cxcr2 as a specific ligand-receptor pair that orchestrates neutrophil chemokinesis in interstitial tissues during neutrophil reverse migration and resolution of inflammation. Taken together, our findings identify distinct receptors that mediate bidirectional leukocyte motility during interstitial migration depending on the context and type of tissue damage in vivo.

  • Wiemann P, Perevitsky A, Lim FY, Shadkchan Y, Knox BP, Landero Figueora JA, Choera T, Niu M, Steinberger AJ, Wüthrich M, Idol RA, Klein BS, Dinauer MC, Huttenlocher A, Osherov N, Keller NP (2017) Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense. Cell Rep 19(5):1008-1021 (PMC5512462) View Abstract · Pubmed Record

    The Fenton-chemistry-generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an Aspergillus fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages (AMΦs), and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses.

  • Rosowski EE, Deng Q, Keller NP, Huttenlocher A (2016) Rac2 Functions in Both Neutrophils and Macrophages To Mediate Motility and Host Defense in Larval Zebrafish. J. Immunol. 197(12):4780-4790 (PMC5367389) View Abstract · Pubmed Record

    Leukocyte motility is required for host defense responses. Rac-family Rho GTPases are implicated in leukocyte function; however, the distinct roles of different Rac isoforms in host defense in vivo have remained unclear. In this study, we generated Rac2-deficient zebrafish using transcription activator-like effector nucleases to directly compare the role of Rac2 in vivo in neutrophils and macrophages in motility and the response to infection. This zebrafish larval model is highly amenable to live imaging of leukocyte behavior, and we report that in rac2-/- larvae both neutrophils and macrophages are defective in basic motility, leading to impaired responses to localized wounds or infections. rac2-/- larvae are highly susceptible to infection with Pseudomonas aeruginosa, which can be almost fully rescued by ectopic expression of either Rac2 or Rac1 specifically in neutrophils, indicating that these isoforms have partially overlapping functions in vivo. Rescue of Rac2 expression specifically in macrophages also confers resistance to Pseudomonas infection, highlighting an important role for Rac2 in this leukocyte population as well. Surprisingly, in contrast to neutrophils expressing a Rac2 dominant inhibitory human disease mutation, rac2-/- neutrophils do not have altered polarity or mobilization from hematopoietic tissue, suggesting that a different Rac isoform, such as Rac1, also contributes to these phenotypes in vivo.

  • Knox BP, Blachowicz A, Palmer JM, Romsdahl J, Huttenlocher A, Wang CC, Keller NP, Venkateswaran K (2016) Characterization of Aspergillus fumigatus Isolates from Air and Surfaces of the International Space Station. mSphere 1(5): (PMC5082629) View Abstract · Pubmed Record

    One mission of the Microbial Observatory Experiments on the International Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain a better understanding of how fungi may adapt to microgravity environments and how this may affect interactions with humans in a closed habitat. Here, we report an initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fumigatus collected from the ISS and a comparison to the experimentally established clinical isolates Af293 and CEA10. Whole-genome sequencing of ISSFT-021 and IF1SW-F4 showed 54,960 and 52,129 single nucleotide polymorphisms, respectively, compared to Af293, which is consistent with observed genetic heterogeneity among sequenced A. fumigatus isolates from diverse clinical and environmental sources. Assessment of in vitro growth characteristics, secondary metabolite production, and susceptibility to chemical stresses revealed no outstanding differences between ISS and clinical strains that would suggest special adaptation to life aboard the ISS. Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more lethal than Af293 and CEA10. Taken together, these genomic, in vitro, and in vivo analyses of two A. fumigatus strains isolated from the ISS provide a benchmark for future investigations of these strains and for continuing research on specific microbial isolates from manned space environments. IMPORTANCE As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.

  • Huemer K, Squirrell JM, Swader R, LeBert DC, Huttenlocher A, Eliceiri KW (2016) zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae. Zebrafish 14(1):42-50 (PMC5312606) View Abstract · Pubmed Record

    Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.

  • Johnson CJ, Cabezas-Olcoz J, Kernien JF, Wang SX, Beebe DJ, Huttenlocher A, Ansari H, Nett JE (2016) The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps. PLoS Pathog. 12(9):e1005884 (PMC5021349) View Abstract · Pubmed Record

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

  • Davis JM, Huang M, Botts MR, Hull CM, Huttenlocher A (2016) A Zebrafish Model of Cryptococcal Infection Reveals Roles for Macrophages, Endothelial Cells, and Neutrophils in the Establishment and Control of Sustained Fungemia. Infect. Immun. 84(10):3047-62 (PMC5038067) View Abstract · Pubmed Record

    Cryptococcal meningoencephalitis is a fungal infection that predominantly affects immunocompromised patients and is uniformly fatal if left untreated. Timely diagnosis is difficult, and screening or prophylactic measures have generally not been successful. Thus, we need a better understanding of early, asymptomatic pathogenesis. Inhaled cryptococci must survive the host immune response, escape the lung, and persist within the bloodstream in order to reach and invade the brain. Here we took advantage of the zebrafish larval infection model to assess the process of cryptococcal infection and disease development sequentially in a single host. Using yeast or spores as infecting particles, we discovered that both cell types survived and replicated intracellularly and that both ultimately established a sustained, low-level fungemia. We propose that the establishment and maintenance of this sustained fungemia is an important stage of disease progression that has been difficult to study in other model systems. Our data suggest that sustained fungemia resulted from a pattern of repeated escape from, and reuptake by, macrophages, but endothelial cells were also seen to play a role as a niche for cryptococcal survival. Circulating yeast collected preferentially in the brain vasculature and eventually invaded the central nervous system (CNS). As suggested previously in a mouse model, we show here that neutrophils can play a valuable role in limiting the sustained fungemia, which can lead to meningoencephalitis. This early stage of pathogenesis-a balanced interaction between cryptococcal cells, macrophages, endothelial cells, and neutrophils-could represent a window for timely detection and intervention strategies for cryptococcal meningoencephalitis.

  • Hind LE, Vincent WJ, Huttenlocher A (2016) Leading from the Back: The Role of the Uropod in Neutrophil Polarization and Migration. Dev. Cell 38(2):161-9 (PMC4982870) View Abstract · Pubmed Record

    Cell motility is required for diverse biological processes including development, homing of immune cells, wound healing, and cancer cell invasion. Motile neutrophils exhibit a polarized morphology characterized by the formation of leading-edge pseudopods and a highly contractile cell rear known as the uropod. Although it is known that perturbing uropod formation impairs neutrophil migration, the role of the uropod in cell polarization and motility remains incompletely understood. Here we discuss cell intrinsic mechanisms that regulate neutrophil polarization and motility, with a focus on the uropod, and examine how relationships among regulatory mechanisms change when cells change their direction of migration.

  • LeBert DC, Squirrell JM, Huttenlocher A, Eliceiri KW (2016) Second harmonic generation microscopy in zebrafish. Methods Cell Biol. 133:55-68 (PMC5489409) View Abstract · Pubmed Record

    Modern optical imaging has progressed rapidly with the ability to noninvasively image cellular and subcellular phenomena with high spatial and temporal resolution. In particular, emerging techniques such as second harmonic generation (SHG) microscopy can allow for the monitoring of intrinsic contrast, such as that from collagen, in live and fixed samples. When coupled with multiphoton fluorescence microscopy, SHG can be used to image interactions between cells and the surrounding extracellular environment. There is recent interest in using these approaches to study inflammation and wound healing in zebrafish, an important model for studying these processes. In this chapter we present the practical aspects of using second harmonic generation to image interactions between leukocytes and collagen during wound healing in zebrafish.

  • de Oliveira S, Rosowski EE, Huttenlocher A (2016) Neutrophil migration in infection and wound repair: going forward in reverse. Nat. Rev. Immunol. 16(6):378-91 (PMC5367630) View Abstract · Pubmed Record

    Neutrophil migration and its role during inflammation has been the focus of increased interest in the past decade. Advances in live imaging and the use of new model systems have helped to uncover the behaviour of neutrophils in injured and infected tissues. Although neutrophils were considered to be short-lived effector cells that undergo apoptosis in damaged tissues, recent evidence suggests that neutrophil behaviour is more complex and, in some settings, neutrophils might leave sites of tissue injury and migrate back into the vasculature. The role of reverse migration and its contribution to resolution of inflammation remains unclear. In this Review, we discuss the different cues within tissues that mediate neutrophil forward and reverse migration in response to injury or infection and the implications of these mechanisms to human disease.

  • Boateng LR, Bennin D, De Oliveira S, Huttenlocher A (2016) Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion. J. Biol. Chem. 291(27):13987-98 (PMC4933159) View Abstract · Pubmed Record

    Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion.

  • Moussavi-Harami SF, Mladinich KM, Sackmann EK, Shelef MA, Starnes TW, Guckenberger DJ, Huttenlocher A, Beebe DJ (2016) Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species. Integr Biol (Camb) 8(2):243-52 (PMC4776335) View Abstract · Pubmed Record

    Neutrophil extracellular traps (NETs) were first reported in 2004, and since their discovery, there has been an increasing interest in NETs, how they are formed, their role in controlling infections, and their contribution to disease pathogenesis. Despite this rapid expansion of our understanding of NETs, many details remain unclear including the role of reactive oxygen species (ROS) in the formation of NETs. Further, to study NETs, investigators typically require a large number of cells purified via a lengthy purification regimen. Here, we report a microfluidic device used to quantify both ROS and NET production over time in response to various stimulants, including live bacteria. This device enables ROS and NET analysis using a process that purifies primary human neutrophils in less than 10 minutes and requires only a few microliters of whole blood. Using this device we demonstrate the ability to identify distinct capabilities of neutrophil subsets (including ROS production and NET formation), the ability to use different stimulants/inhibitors, and the ability to effectively use samples stored for up to 8 hours. This device permits the study of ROS and NETs in a user-friendly format and has potential for widespread applications in the study of human disease.

  • Powell DR, Huttenlocher A (2015) Neutrophils in the Tumor Microenvironment. Trends Immunol. 37(1):41-52 (PMC4707100) View Abstract · Pubmed Record

    Neutrophils are the first responders to sites of acute tissue damage and infection. Recent studies suggest that in addition to neutrophil apoptosis, resolution of neutrophil inflammation at wounds can be mediated by reverse migration from tissues and transmigration back into the vasculature. In settings of chronic inflammation, neutrophils persist in tissues, and this persistence has been associated with cancer progression. However, the role of neutrophils in the tumor microenvironment remains controversial, with evidence for both pro- and anti-tumor roles. Here we review the mechanisms that regulate neutrophil recruitment and resolution at sites of tissue damage, with a specific focus on the tumor microenvironment. We discuss the current understanding as to how neutrophils alter the tumor microenvironment to support or hinder cancer progression, and in this context outline gaps in understanding and important areas of inquiry.

  • Vincent WJ, Freisinger CM, Lam PY, Huttenlocher A, Sauer JD (2015) Macrophages mediate flagellin induced inflammasome activation and host defense in zebrafish. Cell. Microbiol. 18(4):591-604 (PMC5027955) View Abstract · Pubmed Record

    The inflammasome is an innate immune complex whose rapid inflammatory outputs play a critical role in controlling infection; however, the host cells that mediate inflammasome responses in vivo are not well defined. Using zebrafish larvae, we examined the cellular immune responses to inflammasome activation during infection. We compared the host responses with two Listeria monocytogenes strains: wild type and Lm-pyro, a strain engineered to activate the inflammasome via ectopic expression of flagellin. Infection with Lm-pyro led to activation of the inflammasome, macrophage pyroptosis and ultimately attenuation of virulence. Depletion of caspase A, the zebrafish caspase-1 homolog, restored Lm-pyro virulence. Inflammasome activation specifically recruited macrophages to infection sites, whereas neutrophils were equally recruited to wild type and Lm-pyro infections. Similar to caspase A depletion, macrophage deficiency rescued Lm-pyro virulence to wild-type levels, while defective neutrophils had no specific effect. Neutrophils were, however, important for general clearance of L. monocytogenes, as both wild type and Lm-pyro were more virulent in larvae with defective neutrophils. This study characterizes a novel model for inflammasome studies in an intact host, establishes the importance of macrophages during inflammasome responses and adds importance to the role of neutrophils in controlling L. monocytogenes infections.

  • Harvie EA, Huttenlocher A (2015) Neutrophils in host defense: new insights from zebrafish. J. Leukoc. Biol. 98(4):523-37 (PMC4569048) View Abstract · Pubmed Record

    Neutrophils are highly motile phagocytic cells that play a critical role in the immune response to infection. Zebrafish (Danio rerio) are increasingly used to study neutrophil function and host-pathogen interactions. The generation of transgenic zebrafish lines with fluorescently labeled leukocytes has made it possible to visualize the neutrophil response to infection in real time by use of optically transparent zebrafish larvae. In addition, the genetic tractability of zebrafish has allowed for the generation of models of inherited neutrophil disorders. In this review, we discuss several zebrafish models of infectious disease, both in the context of immunocompetent, as well as neutrophil-deficient hosts and how these models have shed light on neutrophil behavior during infection.

  • Yamahashi Y, Cavnar PJ, Hind LE, Berthier E, Bennin DA, Beebe D, Huttenlocher A (2015) Integrin associated proteins differentially regulate neutrophil polarity and directed migration in 2D and 3D. Biomed Microdevices 17(5):100 (PMC4678772) View Abstract · Pubmed Record

    Directed neutrophil migration in blood vessels and tissues is critical for proper immune function; however, the mechanisms that regulate three-dimensional neutrophil chemotaxis remain unclear. It has been shown that integrins are dispensable for interstitial three-dimensional (3D) leukocyte migration; however, the role of integrin regulatory proteins during directed neutrophil migration is not known. Using a novel microfluidic gradient generator amenable to 2D and 3D analysis, we found that the integrin regulatory proteins Kindlin-3, RIAM, and talin-1 differentially regulate neutrophil polarization and directed migration to gradients of chemoattractant in 2D versus 3D. Both talin-1-deficient and RIAM-deficient neutrophil-like cells had impaired adhesion, polarization, and migration on 2D surfaces whereas in 3D the cells polarized but had impaired 3D chemotactic velocity. Kindlin-3 deficient cells were able to polarize and migrate on 2D surfaces but had impaired directionality. In a 3D environment, Kindlin-3 deficient cells displayed efficient chemotaxis. These findings demonstrate that the role of integrin regulatory proteins in cell polarity and directed migration can be different in 2D and 3D.

  • Huttenlocher A, Smith JA (2015) Neutrophils in pediatric autoimmune disease. Curr Opin Rheumatol 27(5):500-4 View Abstract · Pubmed Record

    As first immune responders, neutrophils are essential mediators of host defense, and also contribute to chronic pathologic inflammation at the crossroads of innate and adaptive immunity. In this review, we will highlight the current understanding of the role of neutrophils in pediatric rheumatology, with a focus on juvenile idiopathic arthritis (JIA) and lupus. In inflamed tissues, neutrophils extrude neutrophil extracellular traps containing autoantigen that potentially drives lupus and rheumatoid factor-positive JIA. However, the contribution of neutrophil extracellular traps to pathogenesis remains an area of intense investigation. In JIA joints, neutrophils are activated to such an extent that associated circulating levels of S100A proteins may serve as biomarkers, correlating with disease activity, predicting response to treatment and heralding flares. Beyond the effects of 'normal' activation, neutrophils in JIA and lupus display dysregulation in gene expression, subset activation, and apoptosis. The role of neutrophils in pediatric rheumatology is an understudied area, but garnering increasing attention. Although clearly implicated in JIA and lupus, the specific contributions of neutrophils to pathogenesis and the use of neutrophil activity surrogates as biomarkers require further study. Clarification of these outstanding issues will have implications for diagnosis and treatment of pediatric rheumatologic conditions.

  • Rosowski EE, Huttenlocher A (2015) Neutrophils, wounds, and cancer progression. Dev. Cell 34(2):134-6 View Abstract · Pubmed Record

    Chronic inflammation is associated with tumorigenesis, but how acute inflammation affects the tumor microenvironment is less known. Recently, Antonio et al. (2015) found that neutrophils attracted to an acute wound such as a biopsy drive cell proliferation of nearby pre-neoplastic cells, suggesting that acute wounds may promote cancer progression.

  • LeBert DC, Squirrell JM, Rindy J, Broadbridge E, Lui Y, Zakrzewska A, Eliceiri KW, Meijer AH, Huttenlocher A (2015) Matrix metalloproteinase 9 modulates collagen matrices and wound repair. Development 142(12):2136-46 (PMC4483770) View Abstract · Pubmed Record

    Acute and chronic injuries are characterized by leukocyte infiltration into tissues. Although matrix metalloproteinase 9 (Mmp9) has been implicated in both conditions, its role in wound repair remains unclear. We previously reported a zebrafish chronic inflammation mutant caused by an insertion in the hepatocyte growth factor activator inhibitor gene 1 (hai1; also known as spint1) that is characterized by epithelial extrusions and neutrophil infiltration into the fin. Here, we performed a microarray analysis and found increased inflammatory gene expression in the mutant larvae, including a marked increase in mmp9 expression. Depletion of mmp9 partially rescued the chronic inflammation and epithelial phenotypes, in addition to restoring collagen fiber organization, as detected by second-harmonic generation imaging. Additionally, we found that acute wounding induces epithelial cell mmp9 expression and is associated with a thickening of collagen fibers. Interestingly, depletion of mmp9 impaired this collagen fiber reorganization. Moreover, mmp9 depletion impaired tissue regeneration after tail transection, implicating Mmp9 in acute wound repair. Thus, Mmp9 regulates both acute and chronic tissue damage and plays an essential role in collagen reorganization during wound repair.

  • Kimble J, Bement WM, Chang Q, Cox BL, Drinkwater NR, Gourse RL, Hoskins AA, Huttenlocher A, Kreeger PK, Lambert PF, Mailick MR, Miyamoto S, Moss RL, O'Connor-Giles KM, Roopra A, Saha K, Seidel HS (2015) Strategies from UW-Madison for rescuing biomedical research in the US. Elife 4:e09305 (PMC4484056) View Abstract · Pubmed Record

    A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US.

  • Jing L, Tamplin OJ, Chen MJ, Deng Q, Patterson S, Kim PG, Durand EM, McNeil A, Green JM, Matsuura S, Ablain J, Brandt MK, Schlaeger TM, Huttenlocher A, Daley GQ, Ravid K, Zon LI (2015) Adenosine signaling promotes hematopoietic stem and progenitor cell emergence. J. Exp. Med. 212(5):649-63 (PMC4419349) View Abstract · Pubmed Record

    Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

  • Moussavi-Harami SF, Pezzi HM, Huttenlocher A, Beebe DJ (2015) Simple microfluidic device for studying chemotaxis in response to dual gradients. Biomed Microdevices 17(3):9955 (PMC4768479) View Abstract · Pubmed Record

    Chemotaxis is a fundamental biological process where complex chemotactic gradients are integrated and prioritized to guide cell migration toward specific locations. To understand the mechanisms of gradient dependent cell migration, it is important to develop in vitro models that recapitulate key attributes of the chemotactic cues present in vivo. Current in vitro tools for studying cell migration are not amenable to easily study the response of neutrophils to dual gradients. Many of these systems require external pumps and complex setups to establish and maintain the gradients. Here we report a simple yet innovative microfluidic device for studying cell migration in the presence of dual chemotactic gradients through a 3-dimensional substrate. The device is tested and validated by studying the migration of the neutrophil-like cell line PLB-985 to gradients of fMLP. Furthermore, the device is expanded and used with heparinised whole blood, whereupon neutrophils were observed to migrate from whole blood towards gradients of fMLP eliminating the need for any neutrophil purification or capture steps.

  • Harvie EA, Huttenlocher A (2015) Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection. J Vis Exp (98): (PMC4541586) View Abstract · Pubmed Record

    The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection. The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

  • Lam PY, Mangos S, Green JM, Reiser J, Huttenlocher A (2015) In vivo imaging and characterization of actin microridges. PLoS ONE 10(1):e0115639 (PMC4309568) View Abstract · Pubmed Record

    Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.

  • Tauzin S, Starnes TW, Becker FB, Lam PY, Huttenlocher A (2014) Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration. J. Cell Biol. 207(5):589-98 (PMC4259815) View Abstract · Pubmed Record

    Tissue damage induces early recruitment of neutrophils through redox-regulated Src family kinase (SFK) signaling in neutrophils. Redox-SFK signaling in epithelium is also necessary for wound resolution and tissue regeneration. How neutrophil-mediated inflammation resolves remains unclear. In this paper, we studied the interactions between macrophages and neutrophils in response to tissue damage in zebrafish and found that macrophages contact neutrophils and induce resolution via neutrophil reverse migration. We found that redox-SFK signaling through p22phox and Yes-related kinase is necessary for macrophage wound attraction and the subsequent reverse migration of neutrophils. Importantly, macrophage-specific reconstitution of p22phox revealed that macrophage redox signaling is necessary for neutrophil reverse migration. Thus, redox-SFK signaling in adjacent tissues is essential for coordinated leukocyte wound attraction and repulsion through pathways that involve contact-mediated guidance.

  • Freisinger CM, Huttenlocher A (2014) Live imaging and gene expression analysis in zebrafish identifies a link between neutrophils and epithelial to mesenchymal transition. PLoS ONE 9(11):e112183 (PMC4221567) View Abstract · Pubmed Record

    Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas(V12). Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas(V12) using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas(V12) transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas(V12) in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.

  • Knox BP, Deng Q, Rood M, Eickhoff JC, Keller NP, Huttenlocher A (2014) Distinct innate immune phagocyte responses to Aspergillus fumigatus conidia and hyphae in zebrafish larvae. Eukaryotic Cell 13(10):1266-77 (PMC4187654) View Abstract · Pubmed Record

    Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA.

  • Huttenlocher A, Sahai E (2014) Editorial overview: cell adhesion and migration. Curr. Opin. Cell Biol. 30:v-vi View Abstract · Pubmed Record
  • Shelef MA, Bennin DA, Yasmin N, Warner TF, Ludwig T, Beggs HE, Huttenlocher A (2014) Focal adhesion kinase is required for synovial fibroblast invasion, but not murine inflammatory arthritis. Arthritis Res. Ther. 16(5):464 (PMC4203874) View Abstract · Pubmed Record

    Synovial fibroblasts invade cartilage and bone, leading to joint destruction in rheumatoid arthritis. However, the mechanisms that regulate synovial fibroblast invasion are not well understood. Focal adhesion kinase (FAK) has been implicated in cellular invasion in several cell types, and FAK inhibitors are in clinical trials for cancer treatment. Little is known about the role of FAK in inflammatory arthritis, but, given its expression in synovial tissue, its known role in invasion in other cells and the potential clinical availability of FAK inhibitors, it is important to determine if FAK contributes to synovial fibroblast invasion and inflammatory arthritis. After treatment with FAK inhibitors, invasiveness of human rheumatoid synovial fibroblasts was determined with Matrigel invasion chambers. Migration and focal matrix degradation, two components of cellular invasion, were assessed in FAK-inhibited rheumatoid synovial fibroblasts by transwell assay and microscopic examination of fluorescent gelatin degradation, respectively. Using mice with tumor necrosis factor α (TNFα)-induced arthritis in which fak could be inducibly deleted, invasion and migration by FAK-deficient murine arthritic synovial fibroblasts were determined as described above and arthritis was clinically and pathologically scored in FAK-deficient mice. Inhibition of FAK in human rheumatoid synovial fibroblasts impaired cellular invasion and migration. Focal matrix degradation occurred both centrally and at focal adhesions, the latter being a novel site for matrix degradation in synovial fibroblasts, but degradation was unaltered with FAK inhibitors. Loss of FAK reduced invasion in murine arthritic synovial fibroblasts, but not migration or TNFα-induced arthritis severity and joint erosions. FAK inhibitors reduce synovial fibroblast invasion and migration, but synovial fibroblast migration and TNFα-induced arthritis do not rely on FAK itself. Thus, inhibition of FAK alone is unlikely to be sufficient to treat inflammatory arthritis, but current drugs that inhibit FAK may inhibit multiple factors, which could increase their efficacy in rheumatoid arthritis.

  • Shelef MA, Sokolove J, Robinson WH, Huttenlocher A (2014) Reply: To PMID 24497204. Arthritis Rheumatol 66(9):2644-5 View Abstract · Pubmed Record
  • Kuehn HS, Ouyang W, Lo B, Deenick EK, Niemela JE, Avery DT, Schickel JN, Tran DQ, Stoddard J, Zhang Y, Frucht DM, Dumitriu B, Scheinberg P, Folio LR, Frein CA, Price S, Koh C, Heller T, Seroogy CM, Huttenlocher A, Rao VK, Su HC, Kleiner D, Notarangelo LD, Rampertaap Y, Olivier KN, McElwee J, Hughes J, Pittaluga S, Oliveira JB, Meffre E, Fleisher TA, Holland SM, Lenardo MJ, Tangye SG, Uzel G (2014) Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4. Science 345(6204):1623-7 (PMC4371526) View Abstract · Pubmed Record

    Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory receptor found on immune cells. The consequences of mutations in CTLA4 in humans are unknown. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3(+) regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21(lo) B cells and accumulation of B cells in nonlymphoid organs. Inherited human CTLA4 haploinsufficiency demonstrates a critical quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis.

  • LeBert DC, Huttenlocher A (2014) Inflammation and wound repair. Semin. Immunol. 26(4):315-20 View Abstract · Pubmed Record

    Wound repair requires the integration of complex cellular networks to restore tissue homeostasis. Defects in wound repair are associated with human disease including pyoderma gangrenosum, a heterogeneous disorder that is characterized by unhealed wounds and chronic inflammation of unclear etiology. Despite its clinical importance, there remain significant gaps in understanding how different types of cells communicate to integrate inflammation and wound repair. Recent progress in wound and regenerative biology has been gained by studying genetically tractable model organisms, like zebrafish, that retain the ability to regenerate. The optical transparency and ease of genetic manipulation make zebrafish an ideal model system to dissect multi-cellular and tissue level interactions during wound repair. The focus of this review is on recent advances in understanding how inflammation and wound repair are orchestrated and integrated to achieve wound resolution and tissue regeneration using zebrafish.

  • Shelef MA, Sokolove J, Lahey LJ, Wagner CA, Sackmann EK, Warner TF, Wang Y, Beebe DJ, Robinson WH, Huttenlocher A (2014) Peptidylarginine deiminase 4 contributes to tumor necrosis factor α-induced inflammatory arthritis. Arthritis Rheumatol 66(6):1482-91 (PMC4148484) View Abstract · Pubmed Record

    Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that has multiple associations with inflammation. In rheumatoid arthritis, PAD4 and protein citrullination are increased in inflamed joints, and anti-citrullinated protein antibodies (ACPAs) form against citrullinated antigens are formed. ACPA immune complexes can deposit in the joint and induce the production of tumor necrosis factor α (TNFα), a critical inflammatory cytokine in the pathogenesis of rheumatoid arthritis. Further, in other settings, TNFα has been shown to induce PAD4 activity and modulate antibody formation. We undertook this study to investigate whether TNFα and PAD4 may synergistically exacerbate autoantibody production and inflammatory arthritis. To determine whether TNFα and PAD4 augment autoantibody production and inflammatory arthritis, we first used a multiplex assay to determine whether mice with chronic inflammatory arthritis due to overexpression of TNFα develop autoantibodies against native and citrullinated antigens. With TNF(+) PAD4(+/+) and TNF(+) PAD4(-/-) mice, we then compared serum autoantibody levels by multiplex array, lymphocyte activation by flow cytometry, total serum IgG levels by enzyme-linked immunosorbent assay, arthritis by clinical and histologic scoring, and systemic inflammation using microfluidic devices. TNFα-overexpressing mice had increased levels of autoantibodies reactive against native and citrullinated antigens. PAD4(-/-) mice with TNFα-induced arthritis had lower levels of autoantibodies reactive against native and citrullinated antigens, decreased T cell activation and total IgG levels, and reduced inflammation and arthritis compared to PAD4(+/+) TNFα-overexpressing mice. PAD4 mediates autoantibody production and inflammatory arthritis downstream of TNFα.

  • Rouster-Stevens KA, Ardoin SP, Cooper AM, Becker ML, Dragone LL, Huttenlocher A, Jones KB, Kolba KS, Moorthy LN, Nigrovic PA, Stinson JN, Ferguson PJ, Colbert R, Cron R, Dent P, Elder M, Goldsmith D, Hollister J, Ilowite N, Kimura Y, Klein-Gitelman M, Lawson E, Passo M, Petty R, Punaro M, Rabinovich E, Reiff A, Sherry D, Sundel R, Zemel L (2014) Choosing Wisely: the American College of Rheumatology's Top 5 for pediatric rheumatology. Arthritis Care Res (Hoboken) 66(5):649-57 View Abstract · Pubmed Record

    To create a pediatric rheumatology Top 5 list as part of the American Board of Internal Medicine Foundation's Choosing Wisely campaign. Delphi surveys of a core group of representative pediatric rheumatology providers from across North America generated candidate Top 5 items. Items with high content agreement and perceived to be of prevalent use and of high impact were included in a survey of all American College of Rheumatology (ACR) members who identified themselves as providing care to pediatric patients. Items with the highest ratings were subjected to literature review and further evaluation. A total of 121 candidate items were proposed in the initial Delphi survey and were reduced to 28 items in subsequent surveys. These 28 items were sent to 1,198 rheumatology providers who care for pediatric patients, and 397 (33%) responded. Based upon survey data and literature review, the Top 5 items were identified. These items focused on testing for antinuclear antibodies, autoantibody panels, Lyme disease, methotrexate toxicity monitoring, and use of routine radiographs. The ACR pediatric rheumatology Top 5 is one of the first pediatric subspecialty-specific Choosing Wisely Top 5 lists and provides an opportunity for patients and providers to discuss appropriate use of health care in pediatric rheumatology.

  • Starnes TW, Bennin DA, Bing X, Eickhoff JC, Grahf DC, Bellak JM, Seroogy CM, Ferguson PJ, Huttenlocher A (2014) The F-BAR protein PSTPIP1 controls extracellular matrix degradation and filopodia formation in macrophages. Blood 123(17):2703-14 (PMC3999755) View Abstract · Pubmed Record

    PSTPIP1 is a cytoskeletal adaptor and F-BAR protein that has been implicated in autoinflammatory disease, most notably in the PAPA syndrome: pyogenic sterile arthritis, pyoderma gangrenosum, and acne. However, the mechanism by which PSTPIP1 regulates the actin cytoskeleton and contributes to disease pathogenesis remains elusive. Here, we show that endogenous PSTPIP1 negatively regulates macrophage podosome organization and matrix degradation. We identify a novel PSTPIP1-R405C mutation in a patient presenting with aggressive pyoderma gangrenosum. Identification of this mutation reveals that PSTPIP1 regulates the balance of podosomes and filopodia in macrophages. The PSTPIP1-R405C mutation is in the SRC homology 3 (SH3) domain and impairs Wiskott-Aldrich syndrome protein (WASP) binding, but it does not affect interaction with protein-tyrosine phosphatase (PTP)-PEST. Accordingly, WASP inhibition reverses the elevated F-actin content, filopodia formation, and matrix degradation induced by PSTPIP1-R405C. Our results uncover a novel role for PSTPIP1 and WASP in orchestrating different types of actin-based protrusions. Our findings implicate the cytoskeletal regulatory functions of PSTPIP1 in the pathogenesis of pyoderma gangrenosum and suggest that the cytoskeleton is a rational target for therapeutic intervention in autoinflammatory disease.

  • Sackmann EK, Berthier E, Schwantes EA, Fichtinger PS, Evans MD, Dziadzio LL, Huttenlocher A, Mathur SK, Beebe DJ (2014) Characterizing asthma from a drop of blood using neutrophil chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 111(16):5813-8 (PMC4000787) View Abstract · Pubmed Record

    Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient's neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 μm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.

  • Muscal E, Moorthy LN, Riebschleger MP, Eberhard A, Huttenlocher A, Klein-Gitelman MS, Prahalad S, Stevens KR, Schneider R, Nigrovic PA (2014) A177: Program Evaluation of the ACR/CARRA Inter-Institutional Mentoring Program (AMIGO) in Pediatric Rheumatology. Arthritis Rheumatol 66 Suppl 1:S231 View Abstract · Pubmed Record

    In pediatric rheumatology, the small size of many academic programs translates into limited mentoring options for early career physicians. To address this "mentorship gap," in 2011 the American College of Rheumatology (ACR) and the Childhood Arthritis and Rheumatology Research Alliance (CARRA) joined together to develop AMIGO, the ACR/CARRA Mentoring Interest Group, that now includes greater than half of pediatric rheumatology fellows and junior faculty in the US and Canada. We report ongoing program evaluation encompassing pilot (2011) and full roll-out (2012, 2013) phases of the AMIGO project. Mentees and mentors participating in the AMIGO project were surveyed via online questionnaire to determine dynamics of contact and perceived benefit. The entire pediatric rheumatology community was surveyed in 2011 to determine the state of mentoring and career development, with a repeat survey planned for early 2014. We tabulated the active dyads as of January 2014. As recently reported, detailed evaluation of the pilot phase 17 months after initial roll-out found that 19 of 20 pilot AMIGO dyads were still functioning, with substantial benefit noted by mentees in career guidance, scholarship, and job satisfaction. Benefits reported by mentors included improvement of their mentoring skills and development of their academic portfolios. Both mentees and mentors reported improved connectedness to the wider pediatric rheumatology community. 83 additional dyads were matched prior to 2012 and 2013 ACR meetings. Comparison of these 17-month pilot results to a December 2013/January 2014 survey of all active AMIGO participants is ongoing. A questionnaire administered to the whole pediatric rheumatology community in 2011 (n = 135 respondents, including an estimated 64-70% response rate among fellows and junior faculty) found that approximately 60% of fellows and junior faculty had a career development mentor. We will assess whether the AMIGO program has had a global impact upon the mentoring culture in pediatric rheumatology by collecting community-wide survey data in 2014 and then comparing to 2011 data. The 2011 AMIGO pilot program has confirmed the feasibility of a North American inter-institutional mentoring program in pediatric rheumatology. Participants identified benefits to both mentees and mentors in multiple domains, most prominently in career guidance, a core goal of the program. Ongoing program evaluation will determine how much of this benefit has been sustained after participation was opened to the whole community, and whether the program has had a measureable impact on the overall state of mentoring in pediatric rheumatology.

  • Lam PY, Fischer RS, Shin WD, Waterman CM, Huttenlocher A (2014) Spinning disk confocal imaging of neutrophil migration in zebrafish. Methods Mol. Biol. 1124:219-33 (PMC4087032) View Abstract · Pubmed Record

    Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In this chapter, we describe the practical aspects of imaging neutrophils in zebrafish embryos using spinning disk confocal microscopy. Similar setups can also be applied to image other motile cell types and signaling processes in translucent animals or tissues.

  • Shelef MA, Tauzin S, Huttenlocher A (2013) Neutrophil migration: moving from zebrafish models to human autoimmunity. Immunol. Rev. 256(1):269-81 (PMC4117680) View Abstract · Pubmed Record

    There has been a resurgence of interest in the neutrophil's role in autoimmune disease. Classically considered an early responder that dies at the site of inflammation, new findings using live imaging of embryonic zebrafish and other modalities suggest that neutrophils can reverse migrate away from sites of inflammation. These 'inflammation-sensitized' neutrophils, as well as the neutrophil extracellular traps and other products made by neutrophils in general, may have many implications for autoimmunity. Here, we review what is known about the role of neutrophils in three different autoimmune diseases: rheumatoid arthritis, systemic lupus erythematosus, and small vessel vasculitis. We then highlight recent findings related to several cytoskeletal regulators that guide neutrophil recruitment including Lyn, Rac2, and SHIP. Finally, we discuss how our improved understanding of the molecules that control neutrophil chemotaxis may impact our knowledge of autoimmunity.

  • Lam PY, Harvie EA, Huttenlocher A (2013) Heat shock modulates neutrophil motility in zebrafish. PLoS ONE 8(12):e84436 (PMC3868611) View Abstract · Pubmed Record

    Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.

  • Lam PY, Huttenlocher A (2013) Interstitial leukocyte migration in vivo. Curr. Opin. Cell Biol. 25(5):650-8 (PMC3782709) View Abstract · Pubmed Record

    Rapid leukocyte motility is essential for immunity and host defense. There has been progress in understanding the molecular signals that regulate leukocyte motility both in vitro and in vivo. However, a gap remains in understanding how complex signals are prioritized to result in directed migration, which is critical for both adaptive and innate immune function. Here we focus on interstitial migration and how external cues are translated into intracellular signaling pathways that regulate leukocyte polarity, directional sensing and motility in three-dimensional spaces.

  • Wiemer AJ, Wernimont SA, Cung TD, Bennin DA, Beggs HE, Huttenlocher A (2013) The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation. Biochem. Pharmacol. 86(6):770-81 (PMC3762933) View Abstract · Pubmed Record

    The focal adhesion kinase inhibitor, PF-562,271, is currently in clinical development for cancer, however it is not known how PF-562,271 affects T cell function. Here, we demonstrate inhibitory effects of PF-562,271 on the activation of primary human and mouse T cells. PF-562,271 inhibits T cell receptor signaling-induced T cell adhesion to intercellular adhesion molecule-1 and T cell interactions with antigen-presenting cells. An additional focal adhesion kinase inhibitor, PF-573,228, and genetic depletion of focal adhesion kinase also impair T cell conjugation with antigen-presenting cells. PF-562,271 blocks phosphorylation of the signaling molecules zeta chain associate protein of 70 kDa, linker of activated T cells, and extracellular signal-regulated kinase, and impairs T cell proliferation. The effects observed on T cell proliferation cannot solely be attributed to focal adhesion kinase inhibition, as genetic depletion did not alter proliferation. The effect of PF-562,271 on T cell proliferation is not rescued when proximal T cell receptor signaling is bypassed by stimulation with phorbol-12-myristate-13-acetate and ionomycin. Taken together, our findings demonstrate that focal adhesion kinase regulates integrin-mediated T cell adhesion following T cell receptor activation. Moreover, our findings suggest that PF-562,271 may have immunomodulatory effects that could impact its therapeutic applications.

  • Mladinich KM, Huttenlocher A (2013) WRAMPing up calcium in migrating cells by localized ER transport. Dev. Cell 26(6):560-1 (PMC3875829) View Abstract · Pubmed Record

    Morphological plasticity and front-rear polarity are essential for directed cell migration. In this issue of Developmental Cell, Witze et al. (2013) demonstrate that Wnt5a-mediated signaling induces localization of the cortical endoplasmic reticulum to the trailing edge of melanoma cells and mediates calcium flux, rear detachment, and motility.

  • Mathis L, Wernimont S, Affentranger S, Huttenlocher A, Niggli V (2013) Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation. PeerJ 1:e131 (PMC3757496) View Abstract · Pubmed Record

    We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90(-/-) mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.

  • Langereis JD, Koenderman L, Huttenlocher A, Ulfman LH (2013) Gelsolin expression increases β1 -integrin affinity and L1210 cell adhesion. Cytoskeleton (Hoboken) 70(7):385-93 (PMC4074084) View Abstract · Pubmed Record

    Integrins are functionally regulated by "inside-out" signaling, in that stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs, but the mechanisms that regulate inside out signaling are not completely understood. Previously, we identified gelsolin in a proteomics screen to identify proteins involved in inside-out control of integrins using the lymphocytic leukemia cell line L1210. Furthermore, we showed that gelsolin was involved in affinity regulation of β1 -integrins in the leukemic cell line U937. Here, we examined how gelsolin regulates β1 -integrin affinity in the leukemia cell line L1210. We show that gelsolin is mainly expressed at the cell membrane and is present near β1 -integrins. The role for actin polymerization in integrin affinity regulation was examined using the actin modulating agent jasplakinolide, which decreased β1 -integrin affinity. Similarly, knock-down of gelsolin in L1210 cells also decreased β1 -integrin affinity and cell adhesion to collagen. These data suggest that increased actin polymerization through gelsolin regulates β1 -integrin affinity and cell adhesion.

  • Deng Q, Sarris M, Bennin DA, Green JM, Herbomel P, Huttenlocher A (2013) Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish. J. Leukoc. Biol. 93(5):761-9 (PMC4050646) View Abstract · Pubmed Record

    Neutrophils are the first line of defense against tissue damage and are rapidly mobilized to sites of bacterial infection. However, the signals that regulate neutrophil recruitment are not well defined. Here, using photolabel-enabled fate mapping in zebrafish larvae, we show that localized otic infection with Pseudomonas aeruginosa induces systemic activation and mobilization of neutrophils from the CHT through Cxcr2 signaling. We have cloned the zebrafish Cxcr1 and Cxcr2 receptors and show that Cxcr2 functions as a Cxcl8 receptor in live zebrafish. With the use of morpholino-mediated depletion, we show that infection-induced neutrophil mobilization from the CHT is mediated by Cxcr2 but not Cxcr1. By contrast, Cxcr2 depletion does not affect neutrophil recruitment to the chemoattractant LTB4. Taken together, our findings identify Cxcl8-Cxcr2 signaling as an infection-induced long-range cue that mediates neutrophil motility and mobilization from hematopoietic tissues, positioning Cxcr2 as a critical pathway that mediates infection-induced systemic activation of neutrophils.

  • Bischel LL, Mader BR, Green JM, Huttenlocher A, Beebe DJ (2013) Zebrafish Entrapment By Restriction Array (ZEBRA) device: a low-cost, agarose-free zebrafish mounting technique for automated imaging. Lab Chip 13(9):1732-6 View Abstract · Pubmed Record

    The zebrafish has emerged as a useful model system for a variety of studies, including the investigation of inflammation and immunity. However, current zebrafish imaging techniques, such as agraose mounting, can be time-consuming and detrimental for long-term imaging. Alternatively, automated sorting and imaging systems can be costly and/or complicated to assemble. Here we describe the Zebrafish Entrapment by Restriction Array (ZEBRA) device, a microfluidic device that can be used to quickly and repeatably position zebrafish embryos in a predictable array using only a pipette. This technique is well suited for use with automated microscope stages leading to decreased imaging time and increased throughput compared to traditional methods. The addition of access ports above the embryo can be used to administer treatments, and potentially wounding or injections. We demonstrate the effectiveness of this device for a neutrophil migration screening application using larvae 3 days post fertilization (dpf) Tg(mpx:dendra2). Larvae were loaded into ZEBRA devices and treated with a neutrophil attractant (LTB4) or LTB4 with and without a PI3K inhibitor, LY294002. Treatment with LY294002 impaired neutrophil motility into the fin induced by LTB4 treatment. The findings report the development of ZEBRA a device that can be used to screen for small molecules that affect leukocyte motility and inflammation using live zebrafish.

  • Dagliyan O, Shirvanyants D, Karginov AV, Ding F, Fee L, Chandrasekaran SN, Freisinger CM, Smolen GA, Huttenlocher A, Hahn KM, Dokholyan NV (2013) Rational design of a ligand-controlled protein conformational switch. Proc. Natl. Acad. Sci. U.S.A. 110(17):6800-4 (PMC3637791) View Abstract · Pubmed Record

    Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.

  • Berthier E, Lim FY, Deng Q, Guo CJ, Kontoyiannis DP, Wang CC, Rindy J, Beebe DJ, Huttenlocher A, Keller NP (2013) Low-volume toolbox for the discovery of immunosuppressive fungal secondary metabolites. PLoS Pathog. 9(4):e1003289 (PMC3623715) View Abstract · Pubmed Record

    The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new "function-omic" avenues downstream of the metabolomics, identification, and purification phases.

  • Berthier E, Guckenberger DJ, Cavnar P, Huttenlocher A, Keller NP, Beebe DJ (2013) Kit-On-A-Lid-Assays for accessible self-contained cell assays. Lab Chip 13(3):424-31 (PMC3562598) View Abstract · Pubmed Record

    Microscale methods for cell-based assays typically rely on macroscopic reagent handling and fluidic loading protocols that are technically challenging and do not scale with the number of assays favorably. Here, we demonstrate a microfluidic platform technology called "Kit-On-A-Lid-Assay" (KOALA), that enables the creation of self-contained microfluidic cell-based assays, integrating all the steps required to perform cell-based assays. The KOALA platform allows the pre-packaging of reagents, cryopreservation of cell suspensions, thawing of cell suspensions, culture of cells, and operation of whole cell-based assays. The operation of the KOALA platform is user-friendly and consists of bringing together a lid containing the microchannels, and a base containing the pre-packaged reagents, thereby causing fluidic exchange in all the channels simultaneously. We demonstrate that the KOALA cell-based assays can be simply operated from start to finish without any external laboratory equipment.

  • Huttenlocher A (2013) ECM: chemoattraction but not adhesion. Blood 121(9):1489-91 View Abstract · Pubmed Record

    The mechanisms that regulate 3-dimensional (3D) neutrophil chemotaxis are poorly understood. In this issue of Blood, Afonso et al demonstrate that the collagen receptor Discoidin domain receptor 2 (DDR2) promotes neutrophil chemotaxis in 3D by triggering matrix metalloproteinase (MMP) activity and the generation of chemotactic collagen peptides.

  • Harvie EA, Green JM, Neely MN, Huttenlocher A (2013) Innate immune response to Streptococcus iniae infection in zebrafish larvae. Infect. Immun. 81(1):110-21 (PMC3536132) View Abstract · Pubmed Record

    Streptococcus iniae causes systemic infection characterized by meningitis and sepsis. Here, we report a larval zebrafish model of S. iniae infection. Injection of wild-type S. iniae into the otic vesicle induced a lethal infection by 24 h postinfection. In contrast, an S. iniae mutant deficient in polysaccharide capsule (cpsA mutant) was not lethal, with greater than 90% survival at 24 h postinfection. Live imaging demonstrated that both neutrophils and macrophages were recruited to localized otic infection with mutant and wild-type S. iniae and were able to phagocytose bacteria. Depletion of neutrophils and macrophages impaired host survival following infection with wild-type S. iniae and the cpsA mutant, suggesting that leukocytes are critical for host survival in the presence of both the wild-type and mutant bacteria. However, zebrafish larvae with impaired neutrophil function but normal macrophage function had increased susceptibility to wild-type bacteria but not the cpsA mutant. Taking these findings together, we have developed a larval zebrafish model of S. iniae infection and have found that although neutrophils are important for controlling infection with wild-type S. iniae, neutrophils are not necessary for host defense against the cpsA mutant.

  • Yoo SK, Lam PY, Eichelberg MR, Zasadil L, Bement WM, Huttenlocher A (2012) The role of microtubules in neutrophil polarity and migration in live zebrafish. J. Cell. Sci. 125(Pt 23):5702-10 View Abstract · Pubmed Record

    Microtubules control cell motility by positively regulating polarization in many cell types. However, how microtubules regulate leukocyte migration is not well understood, particularly in living organisms. Here we exploited the zebrafish system to study the role of microtubules in neutrophil migration in vivo. The localization of microtubules was visualized in motile neutrophils using various bioprobes, revealing that, in contrast to what has been seen in studies in vitro, the microtubule organizing center is positioned in front of the nucleus (relative to the direction of migration) in motile neutrophils. Microtubule disassembly impaired attraction of neutrophils to wounds but enhanced the polarity of F-actin dynamics as measured by the distribution of stable and dynamic F-actin. Microtubule depolymerization inhibited polarized phosphoinositol 3-kinase (PI(3)K) activation at the leading edge and induced rapid PI(3)K independent motility. Finally, we show that microtubules exert their effects on neutrophil polarity and motility at least in part by the negative regulation of both Rho and Rac activity. These results provide new insight into the role of microtubules in neutrophil migration in a living vertebrate and show that the motility of these professional migratory cells are subject to distinctly different rules from those established for other cell types.

  • Lam PY, Yoo SK, Green JM, Huttenlocher A (2012) The SH2-domain-containing inositol 5-phosphatase (SHIP) limits the motility of neutrophils and their recruitment to wounds in zebrafish. J. Cell. Sci. 125(Pt 21):4973-8 View Abstract · Pubmed Record

    Neutrophil recruitment to sites of injury or infection is essential for host defense, but it needs to be tightly regulated to prevent tissue damage. Phosphoinositide 3-kinase (PI3K), which generates the phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)], is necessary for neutrophil motility in vivo; however, the role of SH2-domain-containing 5-inositol phosphatase (SHIP) enzymes, which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], is not well understood. Here we show that SHIP phosphatases limit neutrophil motility in live zebrafish. Using real-time imaging of bioprobes specific for PI(3,4,5)P(3) and PI(3,4)P(2) in neutrophils, we found that PI(3,4,5)P(3) and PI(3,4)P(2) accumulate at the leading edge while PI(3,4)P(2) also localizes to the trailing edge of migrating neutrophils in vivo. Depletion of SHIP phosphatases using morpholino oligonucleotides led to increased neutrophil 3D motility and neutrophil infiltration into wounds. The increase in neutrophil wound recruitment in SHIP morphants was rescued by treatment with low dose PI3Kγ inhibitor, suggesting that SHIP limits neutrophil motility by modulating PI3K signaling. Moreover, overexpression of the SHIP phosphatase domain in neutrophils impaired neutrophil 3D migration. Taken together, our findings suggest that SHIP phosphatases control neutrophil inflammation by limiting neutrophil motility in vivo.

  • Shelef MA, Bennin DA, Mosher DF, Huttenlocher A (2012) Citrullination of fibronectin modulates synovial fibroblast behavior. Arthritis Res. Ther. 14(6):R240 View Abstract · Pubmed Record

    ABSTRACT: INTRODUCTION: Rheumatoid arthritis is an autoimmune arthritis characterized by joint destruction. Anti-citrullinated protein antibodies are pathologic in rheumatoid arthritis, but the role of the citrullinated proteins themselves is much less clear. Citrullination is the conversion of the arginine residues of a protein to citrulline. In the inflamed rheumatoid joint there is increased protein citrullination. Several proteins are citrullinated in rheumatoid arthritis, including collagen type II, fibrinogen, and fibronectin. Fibronectin is thought to mediate the adhesion of joint-invading synovial fibroblasts to the rheumatoid cartilage in addition to regulating other synovial fibroblast functions. However, the effect of citrullinated fibronectin on synovial fibroblasts is unknown. METHODS: To investigate the effect of citrullinated fibronectin on synovial fibroblast behavior, we cultured normal murine, arthritic murine, and human rheumatoid synovial fibroblasts. We then compared several synovial fibroblast functions in the presence of fibronectin versus citrullinated fibronectin. We assessed adhesion with time-lapse microscopy, migration with transwell assays, focal adhesion kinase and paxillin phosphorylation by western blot, and focal matrix degradation by fluorescent gelatin degradation. RESULTS: Normal synovial fibroblasts have impaired adhesion, spreading, migration, and integrin-mediated phosphorylation of focal adhesion kinase and paxillin on citrullinated fibronectin. Murine arthritic and human rheumatoid synovial fibroblasts also have impaired adhesion and spreading on citrullinated fibronectin, but focal matrix degradation is unaffected by citrullinated fibronectin. CONCLUSION: Citrullination of fibronectin alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of rheumatoid arthritis.

  • Sackmann EK, Berthier E, Young EW, Shelef MA, Wernimont SA, Huttenlocher A, Beebe DJ (2012) Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays. Blood 120(14):e45-53 (PMC3466974) View Abstract · Pubmed Record

    Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays.

  • Boateng LR, Huttenlocher A (2012) Spatiotemporal regulation of Src and its substrates at invadosomes. Eur. J. Cell Biol. 91(11-12):878-88 (PMC3501139) View Abstract · Pubmed Record

    In the past decade, substantial progress has been made in understanding how Src family kinases regulate the formation and function of invadosomes. Invadosomes are organized actin-rich structures that contain an F-actin core surrounded by an adhesive ring and mediate invasive migration. Src kinases orchestrate, either directly or indirectly, each phase of the invadosome life cycle including invadosome assembly, maturation and matrix degradation and disassembly. Complex arrays of Src effector proteins are involved at different stages of invadosome maturation and their spatiotemporal activity must be tightly regulated to achieve effective invasive migration. In this review, we highlight some recent progress and the challenges of understanding how Src is regulated temporally and spatially to orchestrate the dynamics of invadosomes and mediate cell invasion.

  • Yoo SK, Freisinger CM, LeBert DC, Huttenlocher A (2012) Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish. J. Cell Biol. 199(2):225-34 (PMC3471241) View Abstract · Pubmed Record

    Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H(2)O(2) at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H(2)O(2). A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate "wound signals" that integrate early wound responses and late epimorphic regeneration.

  • Deng Q, Huttenlocher A (2012) Leukocyte migration from a fish eye's view. J. Cell. Sci. 125(Pt 17):3949-56 (PMC3482313) View Abstract · Pubmed Record

    In the last five years, the zebrafish (Danio rerio) has rapidly gained popularity as a model system for studying leukocyte migration and trafficking in vivo. The optical clarity of zebrafish embryos, as well as the potential for genetic manipulation and the development of tools for live imaging, have provided new insight into how leukocytes migrate in response to directional cues in live animals. This Commentary discusses recent progress in our understanding of how leukocytes migrate in vivo, including the role of intracellular signaling through phosphatidylinositol 3-kinase (PI3K) in both random and directed migration. The importance of leukocyte reverse migration in the resolution of inflammation will also be discussed. Finally, we will highlight how zebrafish models have helped to provide new insight into leukocyte migration and the way in which migration is altered in disease.

  • Cavnar PJ, Mogen K, Berthier E, Beebe DJ, Huttenlocher A (2012) The actin regulatory protein HS1 interacts with Arp2/3 and mediates efficient neutrophil chemotaxis. J. Biol. Chem. 287(30):25466-77 (PMC3408136) View Abstract · Pubmed Record

    HS1 is an actin regulatory protein and cortactin homolog that is expressed in hematopoietic cells. Antigen receptor stimulation induces HS1 phosphorylation, and HS1 is essential for T cell activation. HS1 is also expressed in neutrophils; however, the function of HS1 in neutrophils is not known. Here we show that HS1 localizes to the neutrophil leading edge, and is phosphorylated in response to the chemoattractant formyl-Met-Leu-Phe (fMLP) in adherent cells. Using live imaging in microchannels, we show that depletion of endogenous HS1 in the neutrophil-like PLB-985 cell line impairs chemotaxis. We also find that HS1 is necessary for chemoattractant-induced activation of Rac GTPase signaling and Vav1 phosphorylation, suggesting that HS1-mediated Rac activation is necessary for efficient neutrophil chemotaxis. We identify specific phosphorylation sites that mediate HS1-dependent neutrophil motility. Expression of HS1 Y378F, Y397F is sufficient to rescue migration of HS1-deficient neutrophils, however, a triple phospho-mutant Y222F, Y378F, Y397F did not rescue migration of HS1-deficient neutrophils. Moreover, HS1 phosphorylation on Y222, Y378, and Y397 regulates its interaction with Arp2/3. Collectively, our findings identify a novel role for HS1 and its phosphorylation during neutrophil directed migration.

  • Starnes TW, Huttenlocher A (2012) Neutrophil reverse migration becomes transparent with zebrafish. Adv Hematol 2012:398640 (PMC3401556) View Abstract · Pubmed Record

    The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo.

  • Springer A, Huttenlocher A, Prinz W (2012) Language-induced modulation during the prediction of others' actions. Psychol Res 76(4):456-66 View Abstract · Pubmed Record

    Processing of action words has been shown to influence the perception of the actions the words refer to. Specifically, the accuracy with which people predict the future course of actions observed in another individual seems to be affected by verbal primes. Two processes may be involved in action prediction; dynamic simulation (updating) and static matching. The present study examined this issue by testing the impact of action verb processing on action prediction performance using a masked priming paradigm. Evidence of dynamic updating was revealed after prime verbs expressing dynamic actions (e.g., 'to catch') but not those expressing static actions (e.g., 'to lean'). In contrast to previous work, the primes were masked and did not require any response at all. Hence, our results indicate that implicit action-related linguistic processing may trigger action simulation that in turn might affect action prediction (see also Liepelt, Dolk, & Prinz, Psychological Research, 2012, in this issue).

  • Deng Q, Harvie EA, Huttenlocher A (2012) Distinct signalling mechanisms mediate neutrophil attraction to bacterial infection and tissue injury. Cell. Microbiol. 14(4):517-28 (PMC3302966) View Abstract · Pubmed Record

    The signals that guide neutrophils to sites of tissue injury or infection remain elusive. H(2)O(2) has been implicated in neutrophil sensing of tissue injury and transformed cells; however, its role in neutrophil recruitment to infection has not been explored. Here, using a pharmacological inhibitor of NADPH oxidases, diphenyleneiodonium (DPI), and genetic depletion of an epithelial-specific NADPH oxidase, we show that H(2)O(2) is not required for neutrophil detection of localized infection with the Gram-negative bacterium Pseudomonas aeruginosa. In contrast, PI3K signalling is required for neutrophil responses to both wounding and infection. In vivo imaging using a H(2)O(2) probe detects dynamic H(2)O(2) generation at wounds but not at infected tissue. Moreover, DPI no longer inhibits neutrophil wound attraction when P. aeruginosa is present in the media. Finally, DPI also fails to inhibit neutrophil recruitment to localized infection with the Gram-positive bacterium, Streptococcus iniae. Our findings demonstrate that different signals are involved in sensitizing neutrophils to pathogen versus non-pathogen induced tissue damage, providing a potential target to preferentially suppress non-specific immune damage without affecting the response to infection.

  • Boateng LR, Cortesio CL, Huttenlocher A (2012) Src-mediated phosphorylation of mammalian Abp1 (DBNL) regulates podosome rosette formation in transformed fibroblasts. J. Cell. Sci. 125(Pt 5):1329-41 (PMC3324585) View Abstract · Pubmed Record

    Podosomes are dynamic actin-based structures that mediate adhesion to the extracellular matrix and localize matrix degradation to facilitate cell motility and invasion. Drebrin-like protein (DBNL), which is homologous to yeast mAbp1 and is therefore known as mammalian actin-binding protein 1 (mAbp1), has been implicated in receptor-mediated endocytosis, vesicle recycling and dorsal ruffle formation. However, it is not known whether mAbp1 regulates podosome formation or cell invasion. In this study, we found that mAbp1 localizes to podosomes and is necessary for the formation of podosome rosettes in Src-transformed fibroblasts. Despite their structural similarity, mAbp1 and cortactin play distinct roles in podosome regulation. Cortactin was necessary for the formation of podosome dots, whereas mAbp1 was necessary for the formation of organized podosome rosettes in Src-transformed cells. We identified specific Src phosphorylation sites, Tyr337 and Tyr347 of mAbp1, which mediate the formation of podosome rosettes and degradation of the ECM. In contrast to dorsal ruffles, the interaction of mAbp1 with WASP-interacting protein (WIP) was not necessary for the formation of podosome rosettes. Finally, we showed that depletion of mAbp1 increased invasive cell migration, suggesting that mAbp1 differentially regulates matrix degradation and cell invasion. Collectively, our findings identify a role for mAbp1 in podosome rosette formation and cell invasion downstream of Src.

  • Yoo SK, Starnes TW, Deng Q, Huttenlocher A (2011) Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature 480(7375):109-12 (PMC3228893) View Abstract · Pubmed Record

    Tissue wounding induces the rapid recruitment of leukocytes. Wounds and tumours--a type of 'unhealed wound'--generate hydrogen peroxide (H(2)O(2)) through an NADPH oxidase (NOX). This extracellular H(2)O(2) mediates recruitment of leukocytes, particularly the first responders of innate immunity, neutrophils, to injured tissue. However, the sensor that neutrophils use to detect the redox state at wounds is unknown. Here we identify the Src family kinase (SFK) Lyn as a redox sensor that mediates initial neutrophil recruitment to wounds in zebrafish larvae. Lyn activation in neutrophils is dependent on wound-derived H(2)O(2) after tissue injury, and inhibition of Lyn attenuates neutrophil wound recruitment. Inhibition of SFKs also disrupted H(2)O(2)-mediated chemotaxis of primary human neutrophils. In vitro analysis identified a single cysteine residue, C466, as being responsible for direct oxidation-mediated activation of Lyn. Furthermore, transgenic-tissue-specific reconstitution with wild-type Lyn and a cysteine mutant revealed that Lyn C466 is important for the neutrophil wound response and downstream signalling in vivo. This is the first identification, to our knowledge, of a physiological redox sensor that mediates leukocyte wound attraction in multicellular organisms.

  • Wernimont SA, Wiemer AJ, Bennin DA, Monkley SJ, Ludwig T, Critchley DR, Huttenlocher A (2011) Contact-dependent T cell activation and T cell stopping require talin1. J. Immunol. 187(12):6256-67 (PMC3237745) View Abstract · Pubmed Record

    T cell-APC contact initiates T cell activation and is maintained by the integrin LFA-1. Talin1, an LFA-1 regulator, localizes to the immune synapse (IS) with unknown roles in T cell activation. In this study, we show that talin1-deficient T cells have defects in contact-dependent T cell stopping and proliferation. Although talin1-deficient T cells did not form stable interactions with APCs, transient contacts were sufficient to induce signaling. In contrast to prior models, LFA-1 polarized to T cell-APC contacts in talin1-deficient T cells, but vinculin and F-actin polarization at the IS was impaired. These results indicate that T cell proliferation requires sustained, talin1-mediated T cell-APC interactions and that talin1 is necessary for F-actin polarization and the stability of the IS.

  • Deng Q, Yoo SK, Cavnar PJ, Green JM, Huttenlocher A (2011) Dual roles for Rac2 in neutrophil motility and active retention in zebrafish hematopoietic tissue. Dev. Cell 21(4):735-45 (PMC3199325) View Abstract · Pubmed Record

    Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Noninvasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling in vivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively active CXCR4 mutation in zebrafish warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.

  • Wiemer AJ, Hegde S, Gumperz JE, Huttenlocher A (2011) A live imaging cell motility screen identifies prostaglandin E2 as a T cell stop signal antagonist. J. Immunol. 187(7):3663-70 (PMC3178752) View Abstract · Pubmed Record

    The T cell migration stop signal is a central step in T cell activation and inflammation; however, its regulatory mechanisms remain largely unknown. Using a live-cell, imaging-based, high-throughput screen, we identified the PG, PGE(2), as a T cell stop signal antagonist. Src kinase inhibitors, microtubule inhibitors, and PGE(2) prevented the T cell stop signal, and impaired T cell-APC conjugation and T cell proliferation induced by primary human allogeneic dendritic cells. However, Src inhibition, but not PGE(2) or microtubule inhibition, impaired TCR-induced ZAP-70 signaling, demonstrating that T cell stop signal antagonists can function either upstream or downstream of proximal TCR signaling. Moreover, we found that PGE(2) abrogated TCR-induced activation of the small GTPase Rap1, suggesting that PGE(2) may modulate T cell adhesion and stopping through Rap1. These results identify a novel role for PGs in preventing T cell stop signals and limiting T cell activation induced by dendritic cells.

  • Wiemer AJ, Wernimont S, Huttenlocher A (2011) Live imaging of LFA-1-dependent T-cell motility and stop signals. Methods Mol. Biol. 757:191-204 View Abstract · Pubmed Record

    T-cell motility is critical for leukocyte trafficking both in normal host defense and in pathologic conditions including chronic inflammatory disease. Despite progress in understanding the mechanisms of T-cell polarity and motility, we have limited understanding of the mechanisms that contribute to antigen-induced T cell arrest. Here, we describe methods to analyze leukocyte function antigen-1-mediated T-cell motility and T-cell receptor-induced stop signals using in vitro assays on two-dimensional surfaces. Specifically, methods for live time-lapse imaging of T cell random migration and arrest on ICAM-1-coated surfaces are described. Additionally, we detail methods for live imaging of T-cell motility within 3D substrates to analyze T cell-antigen-presenting cell (APC) interactions and APC-mediated stop signals.

  • Huttenlocher A, Horwitz AR (2011) Integrins in cell migration. Cold Spring Harb Perspect Biol 3(9):a005074 View Abstract · Pubmed Record

    Integrin-based adhesion has served as a model for studying the central role of adhesion in migration. In this article, we outline modes of migration, both integrin-dependent and -independent in vitro and in vivo. We next discuss the roles of adhesion contacts as signaling centers and linkages between the ECM and actin that allows adhesions to serve as traction sites. This includes signaling complexes that regulate migration and the interplay among adhesion, signaling, and pliability of the substratum. Finally, we address mechanisms of adhesion assembly and disassembly and the role of adhesion in cellular polarity.

  • Starnes TW, Cortesio CL, Huttenlocher A (2011) Imaging podosome dynamics and matrix degradation. Methods Mol. Biol. 769:111-36 View Abstract · Pubmed Record

    Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips.

  • Cavnar PJ, Berthier E, Beebe DJ, Huttenlocher A (2011) Hax1 regulates neutrophil adhesion and motility through RhoA. J. Cell Biol. 193(3):465-73 (PMC3087009) View Abstract · Pubmed Record

    Kostmann disease is an inherited severe congenital neutropenia syndrome associated with loss-of-function mutations in an adaptor protein HS1-associated protein X-1 (Hax1). How Hax1 regulates neutrophil function remains largely unknown. In this paper, we use ribonucleic acid interference to deplete Hax1 in the neutrophil-like cell line PLB-985 and identify Hax1 as a negative regulator of integrin-mediated adhesion and chemotaxis. Using microfluidics, we show that depletion of Hax1 impairs neutrophil uropod detachment and directed migration. Hax1-deficient cells also display increased integrin-mediated adhesion and reduced RhoA activity. Moreover, depletion of RhoA induces increased neutrophil adhesion and impaired migration, suggesting that Hax1 regulates neutrophil adhesion and chemotaxis through RhoA. Accordingly, activation of RhoA is sufficient to rescue adhesion of Hax1-deficient neutrophils. Together, our findings identify Hax1 as a novel regulator of neutrophil uropod detachment and chemotaxis through RhoA.

  • Yoo SK, Huttenlocher A (2011) Spatiotemporal photolabeling of neutrophil trafficking during inflammation in live zebrafish. J. Leukoc. Biol. 89(5):661-7 (PMC3079246) View Abstract · Pubmed Record

    How neutrophils traffic during inflammation in vivo remains elusive. To visualize the origin and fate of neutrophils during induction and resolution of inflammation, we established a genetically encoded photolabeling system by generating transgenic zebrafish that express a photoconvertible fluorescent reporter Dendra2 in neutrophils. Spatiotemporal photolabeling of neutrophils in vivo demonstrates that they emerge from the hematopoietic tissue in close proximity to injured tissue and repeat forward and reverse migration between the wound and the vasculature. Subsequently, neutrophils disperse throughout the body as wound-healing proceeds, contributing to local resolution at injured tissue and systemic dissemination of wound-sensitized neutrophils. Tissue damage also alters the fate of neutrophils in the caudal hematopoietic tissue and promotes caudorostral mobilization of neutrophils via the circulation to the cephalic mesenchyme. This work provides new insight into neutrophil behaviors during inflammation and resolution within a multicellular organism.

  • Cortesio CL, Boateng LR, Piazza TM, Bennin DA, Huttenlocher A (2011) Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration. J. Biol. Chem. 286(12):9998-10006 (PMC3060554) View Abstract · Pubmed Record

    The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.

  • Young EW, Berthier E, Guckenberger DJ, Sackmann E, Lamers C, Meyvantsson I, Huttenlocher A, Beebe DJ (2011) Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays. Anal. Chem. 83(4):1408-17 (PMC3052265) View Abstract · Pubmed Record

    Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.

  • Accetta D, Syverson G, Bonacci B, Reddy S, Bengtson C, Surfus J, Harbeck R, Huttenlocher A, Grossman W, Routes J, Verbsky J (2011) Human phagocyte defect caused by a Rac2 mutation detected by means of neonatal screening for T-cell lymphopenia. J. Allergy Clin. Immunol. 127(2):535-538.e1-2 View Abstract · Pubmed Record
  • Beebe DJ, Huttenlocher A (2010) Introduction to the mechanisms of directed cell migration themed issue. Integr Biol (Camb) 2(11-12):559-60 View Abstract · Pubmed Record
  • Berthier E, Surfus J, Verbsky J, Huttenlocher A, Beebe D (2010) An arrayed high-content chemotaxis assay for patient diagnosis. Integr Biol (Camb) 2(11-12):630-8 View Abstract · Pubmed Record

    Chemotaxis assays are essential tools for the study of gradient sensing and directed cell migration, and have the potential to aid in the diagnosis and characterization of patients with immune disorders. Current methods are limited in their ability to meet the more demanding requirements for clinical applications. Because patient samples have a short lifespan and sometimes a limited volume (e.g. pediatrics), the operational requirements for an efficient chemotaxis assay are increased in the clinical setting. Here we describe a microscale assay platform for gradient generation that overcomes these limitations. Passive fluidic methods are leveraged to provide a reliable microfluidic gradient generation device, operable in only three pipetting steps. In addition, arrayed imaging and advanced cell tracking algorithms enabled a 50-fold increase in throughput over current methods. These methods were employed to aid in the diagnostic evaluation of an infant who presented with severe, recurrent bacterial infections. Analysis of the infant's neutrophils revealed impaired cell polarization and chemotaxis in a gradient of the chemoattractant fMLP. The patient was subsequently diagnosed with an inhibitory mutation in the Rho GTPase, Rac2. The approach also enabled a microenvironmental screen of human primary neutrophil chemotaxis on fibronectin, fibrinogen and laminin with results suggesting that fibronectin, although commonly used, may not be the most appropriate matrix protein for chemotaxis assays. Together, these findings demonstrate the use of arrayed micro-devices to aid in the diagnosis of a primary immunodeficiency disorder, and illustrate the capability for increased throughput microenvironmental studies and screening targeted to specific human diseases.

  • Wernimont SA, Legate KR, Simonson WT, Fassler R, Huttenlocher A (2010) PIPKI gamma 90 negatively regulates LFA-1-mediated adhesion and activation in antigen-induced CD4+ T cells. J. Immunol. 185(8):4714-23 (PMC3014605) View Abstract · Pubmed Record

    T cell activation requires the formation and maintenance of stable interactions between T cells and APCs. The formation of stable T cell-APC contacts depends on the activation of the integrin LFA-1 (CD11aCD18). Several positive regulators of LFA-1 activation downstream of proximal TCR signaling have been identified, including talin; however, negative regulators of LFA-1 activity remain largely unexplored. Extended isoform of phosphatidylinositol phosphate kinase type I γ (PIPKIγ90) is a member of the type I phosphatidylinositol phosphate kinase family that has been shown previously to modulate talin activation of integrins through production of phosphatidylinositol 4,5-bisphosphate and direct binding to talin. In this study, we show that PIPKIγ90 negatively regulates LFA-1-mediated adhesion and activation of T cells. Using CD4(+) T cells from PIPKIγ90-deficient mice, we show that CD4(+) T cells exhibit increased LFA-1-dependent adhesion to ICAM-1 and increased rates of T cell-APC conjugate formation with enhanced LFA-1 polarization at the synapse. In addition to increased adhesiveness, PIPKIγ90-deficient T cells exhibit increased proliferation both in vitro and in vivo and increased production of IFN-γ and IL-2. Together, these results demonstrate that PIPKIγ90 is a negative regulator of Ag-induced T cell adhesion and activation.

  • Walters KB, Green JM, Surfus JC, Yoo SK, Huttenlocher A (2010) Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome. Blood 116(15):2803-11 (PMC2974588) View Abstract · Pubmed Record

    CXCR4 is a G protein-coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

  • Cortesio CL, Wernimont SA, Kastner DL, Cooper KM, Huttenlocher A (2010) Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome. Arthritis Rheum. 62(8):2556-8 (PMC2921034) View Abstract · Pubmed Record
  • Wernimont SA, Simonson WT, Greer PA, Seroogy CM, Huttenlocher A (2010) Calpain 4 is not necessary for LFA-1-mediated function in CD4+ T cells. PLoS ONE 5(5):e10513 (PMC2866319) View Abstract · Pubmed Record

    T cell activation and immune synapse formation require the appropriate activation and clustering of the integrin, LFA-1. Previous work has reported that the calpain family of calcium-dependent proteases are important regulators of integrin activation and modulate T cell adhesion and migration. However, these studies have been limited by the use of calpain inhibitors, which have known off-target effects. Here, we used a LoxP/CRE system to specifically deplete calpain 4, a small regulatory calpain subunit required for expression and activity of ubiquitously expressed calpains 1 and 2, in CD4+ T cells. CD4+ and CD8+ T cells developed normally in Capn4(F/F):CD4-CRE mice and had severely diminished expression of Calpain 1 and 2, diminished talin proteolysis and impaired casein degradation. Calpain 4-deficient T cells showed no difference in adhesion or migration on the LFA-1 ligand ICAM-1 compared to control T cells. Moreover, there was no impairment in conjugation between Capn4(F/F):CD4-CRE T cells and antigen presenting cells, and the conjugates were still capable of polarizing LFA-1, PKC-theta and actin to the immune synapse. Furthermore, T cells from Capn4(F/F):CD4-CRE mice showed normal proliferation in response to either anti-CD3/CD28 coated beads or cognate antigen-loaded splenocytes. Finally, there were no differences in the rates of apoptosis following extrinsic and intrinsic apoptotic stimuli. Our findings demonstrate that calpain 4 is not necessary for LFA-1-mediated adhesion, conjugation or migration. These results challenge previous reports that implicate a central role for calpains in the regulation of T cell LFA-1 function.

  • Chan KT, Bennin DA, Huttenlocher A (2010) Regulation of adhesion dynamics by calpain-mediated proteolysis of focal adhesion kinase (FAK). J. Biol. Chem. 285(15):11418-26 (PMC2857020) View Abstract · Pubmed Record

    The coordinated and dynamic regulation of adhesions is required for cell migration. We demonstrated previously that limited proteolysis of talin1 by the calcium-dependent protease calpain 2 plays a critical role in adhesion disassembly in fibroblasts (Franco, S. J., Rodgers, M. A., Perrin, B. J., Han, J., Bennin, D. A., Critchley, D. R., and Huttenlocher, A. (2004) Nat. Cell Biol. 6, 977-983). However, little is known about the contribution of other calpain substrates to the regulation of adhesion dynamics. We now provide evidence that calpain 2-mediated proteolysis of focal adhesion kinase (FAK) regulates adhesion dynamics in motile cells. We mapped the preferred calpain cleavage site between the two C-terminal proline-rich regions after Ser-745, resulting in a C-terminal fragment similar in size to the FAK-related non-kinase (FRNK). We generated mutant FAK with a point mutation (V744G) that renders FAK resistant to calpain proteolysis but retains other biochemical properties of FAK. Using time-lapse microscopy, we show that the dynamics of green fluorescent protein-talin1 are impaired in FAK-deficient cells. Expression of wild-type but not calpain-resistant FAK rescues talin dynamics in FAK-deficient cells. Taken together, our findings suggest a novel role for calpain proteolysis of FAK in regulating adhesion dynamics in motile cells.

  • Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A (2010) Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev. Cell 18(2):226-36 (PMC2824622) View Abstract · Pubmed Record

    Cell polarity is crucial for directed migration. Here we show that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P(3)-PI(3,4)P(2) during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism.

  • Cortesio CL, Perrin BJ, Bennin DA, Huttenlocher A (2010) Actin-binding protein-1 interacts with WASp-interacting protein to regulate growth factor-induced dorsal ruffle formation. Mol. Biol. Cell 21(1):186-97 (PMC2801713) View Abstract · Pubmed Record

    Growth factor stimulation induces the formation of dynamic actin structures known as dorsal ruffles. Mammalian actin-binding protein-1 (mAbp1) is an actin-binding protein that has been implicated in regulating clathrin-mediated endocytosis; however, a role for mAbp1 in regulating the dynamics of growth factor-induced actin-based structures has not been defined. Here we show that mAbp1 localizes to dorsal ruffles and is necessary for platelet-derived growth factor (PDGF)-mediated dorsal ruffle formation. Despite their structural similarity, we find that mAbp1 and cortactin have nonredundant functions in the regulation of dorsal ruffle formation. mAbp1, like cortactin, is a calpain 2 substrate and the preferred cleavage site occurs between the actin-binding domain and the proline-rich region, generating a C-terminal mAbp1 fragment that inhibits dorsal ruffle formation. Furthermore, mAbp1 directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. Finally, we demonstrate that the interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1. Taken together, these findings identify a novel role for mAbp1 in growth factor-induced dorsal ruffle formation through its interaction with WIP.

  • Wiemer AJ, Lokuta MA, Surfus JC, Wernimont SA, Huttenlocher A (2010) Calpain inhibition impairs TNF-alpha-mediated neutrophil adhesion, arrest and oxidative burst. Mol. Immunol. 47(4):894-902 (PMC2814964) View Abstract · Pubmed Record

    Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-alpha induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF-alpha-induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing focal complexes. Calpain inhibition induces random migration in TNF-alpha-stimulated cells and prevents the generation of reactive oxygen species, but does not alter TNF-alpha-mediated activation of p38 MAPK and ERK MAPK. These findings suggest that the TNF-alpha-induced neutrophil arrest requires the activity of calpain independent of p38 MAPK and ERK signaling seen after TNF-alpha stimulation. Together, our data suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF-alpha-induced inflammatory responses.

  • Schartner JM, Simonson WT, Wernimont SA, Nettenstrom LM, Huttenlocher A, Seroogy CM (2009) Gene related to anergy in lymphocytes (GRAIL) expression in CD4+ T cells impairs actin cytoskeletal organization during T cell/antigen-presenting cell interactions. J. Biol. Chem. 284(50):34674-81 (PMC2787330) View Abstract · Pubmed Record

    GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

  • Quinn BJ, Welch EJ, Kim AC, Lokuta MA, Huttenlocher A, Khan AA, Kuchay SM, Chishti AH (2009) Erythrocyte scaffolding protein p55/MPP1 functions as an essential regulator of neutrophil polarity. Proc. Natl. Acad. Sci. U.S.A. 106(47):19842-7 (PMC2785254) View Abstract · Pubmed Record

    As mediators of innate immunity, neutrophils respond to chemoattractants by adopting a highly polarized morphology. Efficient chemotaxis requires the formation of one prominent pseudopod at the cell front characterized by actin polymerization, while local inhibition suppresses the formation of rear and lateral protrusions. This asymmetric control of signaling pathways is required for directional migration along a chemotactic gradient. Here, we identify the MAGUK protein p55/MPP1 as a mediator of the frontness signal required for neutrophil polarization. We developed a p55 knockout (p55(-/-)) mouse model, and demonstrate that p55(-/-) neutrophils form multiple transient pseudopods upon chemotactic stimulation, and do not migrate efficiently in vitro. Upon agonist stimulation, p55 is rapidly recruited to the leading edge of neutrophils in mice and humans. Total F-actin polymerization, along with Rac1 and RhoA activation, appear to be normal in p55(-/-) neutrophils. Importantly, phosphorylation of Akt is significantly decreased in p55(-/-) neutrophils upon chemotactic stimulation. The activity of immunoprecipitated phosphatidylinositol 3-kinase gamma (PI3Kgamma), responsible for chemoattractant-induced synthesis of PIP(3) and Akt phosphorylation, is unperturbed in p55(-/-) neutrophils. Although the total amount of PIP(3) is normal in p55(-/-) neutrophils, PIP(3) is diffusely localized and forms punctate aggregates in activated p55(-/-) neutrophils, as compared to its accumulation at the leading edge membrane in the wild type neutrophils. Together, these results show that p55 is required for neutrophil polarization by regulating Akt phosphorylation through a mechanism that is independent of PI3Kgamma activity.

  • Mathias JR, Dodd ME, Walters KB, Yoo SK, Ranheim EA, Huttenlocher A (2009) Characterization of zebrafish larval inflammatory macrophages. Dev. Comp. Immunol. 33(11):1212-7 (PMC2742687) View Abstract · Pubmed Record

    Zebrafish have emerged as a powerful model system to study leukocyte recruitment and inflammation. Here we characterize the morphology and function of inflammatory macrophages in zebrafish larvae. These macrophages can be distinguished from neutrophils by immunolabeling of L-Plastin without MPO co-expression and by an elongated morphology. Live imaging of transgenic zMPO:GFP larvae demonstrate that GFP(lo) macrophages migrate to wounds by extension of thin pseudopods and carry out phagocytosis of tissue debris, and FACS analysis of leukocyte markers indicates expression of CSF1R in these macrophages. These findings identify distinct functional and morphological characteristics of inflammatory macrophages in zebrafish larvae.

  • Mathias JR, Walters KB, Huttenlocher A (2009) Neutrophil motility in vivo using zebrafish. Methods Mol. Biol. 571:151-66 View Abstract · Pubmed Record

    Zebrafish have emerged as a powerful model organism to study neutrophil chemotaxis and inflammation in vivo. Studies of neutrophil chemotaxis in animal models have previously been hampered both by the limited number of specimens available for analysis and by the need for invasive procedures to perform intravital microscopy. Due to the transparency and cell permeability of zebrafish embryos these limitations are circumvented, and the zebrafish system is amenable to both live time-lapse imaging of neutrophil chemotaxis and for screening of the effects of chemical compounds on the inflammatory response in vivo. Here, we describe methods to analyze neutrophil-directed migration toward wounds using both fixed embryos by myeloperoxidase activity assay, and live embryos by time-lapse microscopy. Further, methods are described for the evaluation of the effects of chemical compounds on neutrophil motility and the innate immune responses in zebrafish embryos.

  • Dodd ME, Hatzold J, Mathias JR, Walters KB, Bennin DA, Rhodes J, Kanki JP, Look AT, Hammerschmidt M, Huttenlocher A (2009) The ENTH domain protein Clint1 is required for epidermal homeostasis in zebrafish. Development 136(15):2591-600 (PMC2709065) View Abstract · Pubmed Record

    Epidermal hyperproliferation and inflammation are hallmarks of the human condition psoriasis. Here, we report that a zebrafish line with a mutation in the cargo adaptor protein Clint1 exhibits psoriasis-like phenotypes including epithelial hyperproliferation and leukocyte infiltration. Clint1 is an ENTH domain-containing protein that binds SNARE proteins and functions in vesicle trafficking; however, its in vivo function in animal models has not been reported to date. The clint1 mutants exhibit chronic inflammation characterized by increased Interleukin 1beta expression, leukocyte infiltration, bidirectional trafficking and phagocytosis of cellular debris. The defects in clint1 mutants can be rescued by expression of zebrafish clint1 and can be phenocopied with clint1-specific morpholinos, supporting an essential role for Clint1 in epidermal development. Interaction studies suggest that Clint1 and Lethal giant larvae 2 function synergistically to regulate epidermal homeostasis. Accordingly, clint1 mutants show impaired hemidesmosome formation, loss of cell-cell contacts and increased motility suggestive of epithelial to mesenchymal transition. Taken together, our findings describe a novel function for the ENTH domain protein Clint1 in epidermal development and inflammation and suggest that its deficiency in zebrafish generates a phenotype that resembles the human condition psoriasis.

  • Yoo SK, Huttenlocher A (2009) Innate immunity: wounds burst H2O2 signals to leukocytes. Curr. Biol. 19(14):R553-5 View Abstract · Pubmed Record

    How leukocytes are attracted to wounds is poorly understood. Recent work using zebrafish reveals a novel mechanism of early leukocyte recruitment to wounds through a concentration gradient of hydrogen peroxide.

  • Kim D, Lokuta MA, Huttenlocher A, Beebe DJ (2009) Selective and tunable gradient device for cell culture and chemotaxis study. Lab Chip 9(12):1797-800 (PMC2804468) View Abstract · Pubmed Record

    This article describes a microfluidic device for cell culture and chemotaxis studies under various temporal and spatial concentration gradients of the medium or chemoattractant. Vertical membranes formed using in situ fabrication are employed to avoid fluid flow inside the cell observation chamber. Thus, the medium and chemoattractants are primarily provided by diffusion, maintaining cell-cell communication via secreted factors. Neutrophils were used to demonstrate the capability of the device for chemotaxis research. Experiments exhibited successful migration up a concentration gradient of interleukin 8.

  • Brannon MK, Davis JM, Mathias JR, Hall CJ, Emerson JC, Crosier PS, Huttenlocher A, Ramakrishnan L, Moskowitz SM (2009) Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cell. Microbiol. 11(5):755-68 (PMC2933946) View Abstract · Pubmed Record

    Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

  • Chan KT, Cortesio CL, Huttenlocher A (2009) FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion. J. Cell Biol. 185(2):357-70 (PMC2700377) View Abstract · Pubmed Record

    Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.

  • Szabady RL, Lokuta MA, Walters KB, Huttenlocher A, Welch RA (2009) Modulation of neutrophil function by a secreted mucinase of Escherichia coli O157:H7. PLoS Pathog. 5(2):e1000320 (PMC2642718) View Abstract · Pubmed Record

    Escherichia coli O157:H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157:H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157:H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation.

  • Simonson WT, Markovina S, Grossman WJ, Lokuta MA, Doan AT, Seroogy CM, Huttenlocher A (2009) Common variable immunodeficiency with regulatory T-cell deficiency treated with rapamycin. Ann. Allergy Asthma Immunol. 102(2):170-1 View Abstract · Pubmed Record
  • Walters KB, Dodd ME, Mathias JR, Gallagher AJ, Bennin DA, Rhodes J, Kanki JP, Look AT, Grinblat Y, Huttenlocher A (2009) Muscle degeneration and leukocyte infiltration caused by mutation of zebrafish Fad24. Dev. Dyn. 238(1):86-99 (PMC2843540) View Abstract · Pubmed Record

    Factor for adipocyte differentiation 24 (fad24) is a novel gene that has been implicated in adipocyte differentiation and DNA replication. In a screen for zebrafish mutants that have an abnormal tissue distribution of neutrophils, we identified an insertional allele of fad24, fad24hi1019. Homozygous fad24hi1019 larvae exhibit muscle degeneration accompanied by leukocyte infiltration. Muscle degeneration was extensive and included tissue apoptosis and disorganized, poorly striated muscle fibers. Blocking apoptosis using pan-caspase inhibitors resulted in decreased neutrophil recruitment into the body of the larva, suggesting a causative link between apoptosis and leukocyte infiltration. These findings suggest that zebrafish is a powerful genetic model system to address the interplay between muscle degeneration and leukocyte infiltration, and indicate that tissue apoptosis may contribute to neutrophil recruitment in some inflammatory states.

  • Undyala VV, Dembo M, Cembrola K, Perrin BJ, Huttenlocher A, Elce JS, Greer PA, Wang YL, Beningo KA (2008) The calpain small subunit regulates cell-substrate mechanical interactions during fibroblast migration. J. Cell. Sci. 121(Pt 21):3581-8 (PMC3081789) View Abstract · Pubmed Record

    Cell migration involves the dynamic formation and release of cell-substrate adhesions, where the exertion and detection of mechanical forces take place. Members of the calpain family of calcium-dependent proteases are believed to have a central role in these processes, possibly through the regulation of focal adhesion dynamics. The ubiquitous calpains, calpain 1 (mu-calpain) and calpain 2 (m-calpain), are heterodimers consisting of large catalytic subunits encoded by the Capn1 and Capn2 genes, respectively, and the small regulatory subunit encoded by Capn4. We have examined the role of the calpain regulatory small subunit in traction force production and mechanosensing during cell migration. Capn4-deficient or rescued cells were plated on flexible polyacrylamide substrates, for both the detection of traction forces and the application of mechanical stimuli. The total force output of Capn4-deficient cells was approximately 75% lower than that of rescued cells and the forces were more randomly distributed and less dynamic in Capn4-deficient cells than in rescued cells. Furthermore, Capn4-deficient cells were less adhesive than wild-type cells and they also failed to respond to mechanical stimulations by pushing or pulling the flexible substrate, or by engaging dorsal receptors to the extracellular matrix. Surprisingly, fibroblasts deficient in calpain 1 or calpain 2 upon siRNA-mediated knockdown of Capn1 or Capn2, respectively, did not show the same defects in force production or adhesion, although they also failed to respond to mechanical stimulation. Interestingly, stress fibers were aberrant and also contained fewer colocalised vinculin-containing adhesions in Capn4-deficient cells than Capn1- and Capn2-knockdown cells. Together, these results suggest that the calpain small subunit plays an important role in the production of mechanical forces and in mediating mechanosensing during fibroblast migration. Furthermore, the Capn4 gene product might perform functions secondary to, or independent of, its role as a regulatory subunit for calpain 1 and calpain 2.

  • Wang X, Chen X, Rodenkirch L, Simonson W, Wernimont S, Ndonye RM, Veerapen N, Gibson D, Howell AR, Besra GS, Painter GF, Huttenlocher A, Gumperz JE (2008) Natural killer T-cell autoreactivity leads to a specialized activation state. Blood 112(10):4128-38 (PMC2581981) View Abstract · Pubmed Record

    Natural killer T (NKT) cells are innate-like T cells that recognize specific microbial antigens and also display autoreactivity to self-antigens. The nature of NKT-cell autoreactive activation remains poorly understood. We show here that the mitogen-activated protein kinase (MAPK) pathway is operative during human NKT-cell autoreactive activation, but calcium signaling is severely impaired. This results in a response that is biased toward granulocyte macrophage colony-stimulating factor (GM-CSF) secretion because this cytokine requires extracellular signal-regulated kinase (ERK) signaling but is not highly calcium dependent, whereas interferon-gamma (IFN-gamma), interleukin (IL)-4, and IL-2 production are minimal. Autoreactive activation was associated with reduced migration velocity but did not induce arrest; thus, NKT cells retained the ability to survey antigen presenting cells (APCs). IL-12 and IL-18 stimulated autoreactively activated NKT cells to secrete IFN-gamma, and this was mediated by Janus kinase-signal transducers and activators of transcription (JAK-STAT)-dependent signaling without induction of calcium flux. This pathway did not require concurrent contact with CD1d(+) APCs but was strictly dependent on preceding autoreactive stimulation that induced ERK activation. In contrast, NKT-cell responses to the glycolipid antigen alpha-galactosyl ceramide (alpha-GalCer) were dampened by prior autoreactive activation. These results show that NKT-cell autoreactivity induces restricted cytokine secretion and leads to altered basal activation that potentiates innate responsiveness to costimulatory cytokines while modulating sensitivity to foreign antigens.

  • Ferguson PJ, Lokuta MA, El-Shanti HI, Muhle L, Bing X, Huttenlocher A (2008) Neutrophil dysfunction in a family with a SAPHO syndrome-like phenotype. Arthritis Rheum. 58(10):3264-9 View Abstract · Pubmed Record

    SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis) is an inflammatory disorder of the bone, skin, and joints. We describe a family with multiple affected members who segregate a SAPHO syndrome-like phenotype, and we report the results of neutrophil studies and candidate gene analysis. We obtained written informed consent and a family history and reviewed medical records. We collected DNA and sequenced candidate genes, and we performed functional studies on neutrophils isolated from the proband and her mother. The pedigree segregated chronic osteomyelitis and cutaneous inflammation in a pattern that suggested an autosomal-dominant disorder. No coding sequence mutations were detected in PSTPIP1, PSTPIP2, LPIN2, SH3BP2, or NCF4. Analysis of neutrophil function in the proband, including nitroblue tetrazolium tests, myeloperoxidase assays, neutrophil chemotaxis, and neutrophil chemotaxis assays, revealed no identifiable abnormalities. However, an abnormality in the luminol, but not the isoluminol, respiratory burst assays following stimulation with phorbol myristate acetate (PMA) was detected in neutrophils isolated from the affected proband. Internal oxidant production was also reduced in the proband and her mother when neutrophils were treated with fMLP with or without platelet-activating factor, PMA alone, or tumor necrosis factor alpha alone. This family segregates a disorder characterized by chronic inflammation of the skin and bone. Functional differences in neutrophils exist between affected individuals and controls. The biologic significance of this defect remains unknown. Identification of the gene defect will help identify an immunologic pathway that, when dysregulated, causes inflammation of the skin and bone.

  • Abhyankar VV, Toepke MW, Cortesio CL, Lokuta MA, Huttenlocher A, Beebe DJ (2008) A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment. Lab Chip 8(9):1507-15 (PMC2804469) View Abstract · Pubmed Record

    While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g. pipettes), that provides robust soluble factor control within a three-dimensional biological matrix. We demonstrate long-lasting linear and non-linear concentration profiles that were maintained for up to ten days using 34.5 muL solute volume. We also demonstrate the ability to superimpose local soluble factor pulses onto existing gradients via defined dosing windows. The combination of long-term and transient gradient characteristics within a three-dimensional environment opens the door for signaling studies that investigate the migratory behavior of cells within a biologically representative matrix. To this end, we apply temporally evolving and long-lasting gradients to study the chemotactic responses of human neutrophils and the invasion of metastatic rat mammary adenocarcinoma cells (MtLN3) within three-dimensional collagen matrices.

  • Wernimont SA, Cortesio CL, Simonson WT, Huttenlocher A (2008) Adhesions ring: a structural comparison between podosomes and the immune synapse. Eur. J. Cell Biol. 87(8-9):507-15 (PMC2570187) View Abstract · Pubmed Record

    Podosomes and the immune synapse are integrin-mediated adhesive structures that share a common ring-like morphology. Both podosomes and immune synapses have a central core surrounded by a peripheral ring containing talin, vinculin and paxillin. Recent progress suggests significant parallels between the regulatory mechanisms that contribute to the formation of these adhesive structures. In this review, we compare the structures, functions and regulation of podosomes and the immune synapse.

  • Cooper KM, Bennin DA, Huttenlocher A (2008) The PCH family member proline-serine-threonine phosphatase-interacting protein 1 targets to the leukocyte uropod and regulates directed cell migration. Mol. Biol. Cell 19(8):3180-91 (PMC2488309) View Abstract · Pubmed Record

    Pombe Cdc15 homology (PCH) family members have emerged as important regulators of membrane-cytoskeletal interactions. Here we show that PSTPIP1, a PCH family member expressed in hematopoietic cells, regulates the motility of neutrophil-like cells and is a novel component of the leukocyte uropod where it colocalizes with other uropod components, such as type I PIPKIgamma. Furthermore, we show that PSTPIP1 association with the regulator of endocytosis, dynamin 2, and PSTPIP1 expression impairs transferrin uptake and endocytosis. We also show that PSTPIP1 localizes at the rear of neutrophils with a subpopulation of F-actin that is specifically detected by the binding of an F-actin probe that detects a more stable population of actin. Finally, we show that actin polymerization, but not the microtubule network, is necessary for the polarized distribution of PSTPIP1 toward the rear of the cell. Together, our findings demonstrate that PSTPIP1 is a novel component of the leukocyte uropod that regulates endocytosis and cell migration.

  • Huttenlocher A, Poznansky MC (2008) Reverse leukocyte migration can be attractive or repulsive. Trends Cell Biol. 18(6):298-306 (PMC2435406) View Abstract · Pubmed Record

    The directional migration of cells within multicellular organisms is governed by gradients of both chemical attractants and repellents in diverse processes, including leukocyte trafficking and neuronal pathfinding in vivo. These complex extracellular environments direct the orchestrated bidirectional trafficking of leukocytes between the vasculature and tissues. Substantial progress has been made in dissecting the molecular mechanisms involved in orchestrating the directed movement of leukocytes into host tissues; however, less is known about the reverse migration of leukocytes from the tissues to the vasculature. In this article, we discuss the functional interplay between chemoattraction and chemorepulsion in the bidirectional movement of cells in complex in vivo environments, and we describe how these mechanisms influence both normal physiology and human disease.

  • Lokuta MA, Nuzzi PA, Huttenlocher A (2008) Analysis of neutrophil polarization and chemotaxis. Methods Mol. Biol. 412:211-29 View Abstract · Pubmed Record

    Neutrophil polarization and directed migration (chemotaxis) are critical for the inflammatory response. Neutrophil chemotaxis is achieved by the sensing of narrow gradients of chemoattractant and the subsequent polarization and directed migration toward the chemotactic source. Despite recent progress, the signaling mechanisms that regulate neutrophil polarization during chemotaxis have not been clearly defined. Here, we describe methods to analyze neutrophil polarization and asymmetric redistribution of signaling components induced by chemoattractant using immunofluorescence. Further, methods are described to dissect the role of specific signaling pathways during chemotaxis by the use of murine neutrophils from transgenic mouse models. Finally, methods for time-lapse microscopy and transwell assay for the analysis of neutrophil chemotaxis will also be discussed.

  • Cortesio CL, Chan KT, Perrin BJ, Burton NO, Zhang S, Zhang ZY, Huttenlocher A (2008) Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion. J. Cell Biol. 180(5):957-71 (PMC2265405) View Abstract · Pubmed Record

    Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.

  • Lokuta MA, Senetar MA, Bennin DA, Nuzzi PA, Chan KT, Ott VL, Huttenlocher A (2007) Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis. Mol. Biol. Cell 18(12):5069-80 (PMC2096574) View Abstract · Pubmed Record

    Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility. Here, we demonstrate that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma661), which generates PtdIns(4,5)P(2), is enriched in the uropod during chemotaxis of primary neutrophils and differentiated HL-60 cells (dHL-60). Using time-lapse microscopy, we show that enrichment of PIPKIgamma661 at the cell rear occurs early upon chemoattractant stimulation and is persistent during chemotaxis. Accordingly, we were able to detect enrichment of PtdIns(4,5)P(2) at the uropod during chemotaxis. Overexpression of kinase-dead PIPKIgamma661 compromised uropod formation and rear retraction similar to inhibition of ROCK signaling, suggesting that PtdIns(4,5)P(2) synthesis is important to elicit the backness response during chemotaxis. Together, our findings identify a previously unknown function for PIPKIgamma661 as a novel component of the backness signal that regulates rear retraction during chemotaxis.

  • Mathias JR, Dodd ME, Walters KB, Rhodes J, Kanki JP, Look AT, Huttenlocher A (2007) Live imaging of chronic inflammation caused by mutation of zebrafish Hai1. J. Cell. Sci. 120(Pt 19):3372-83 View Abstract · Pubmed Record

    The hallmark of chronic inflammation is the infiltration and persistence of leukocytes within inflamed tissue. Here, we describe the first zebrafish chronic inflammation mutant identified in an insertional mutagenesis screen for mutants that exhibit abnormal tissue distribution of neutrophils. We identified a mutant line with an insertion in the Hepatocyte growth factor activator inhibitor 1 gene (hai1; also known as Spint1) that showed accumulation of neutrophils in the fin. The mutant embryos exhibited inflammation in areas of epidermal hyperproliferation that was rescued by knock-down of the type II transmembrane serine protease Matriptase 1 (also known as St14), suggesting a novel role for Hai1-Matriptase 1 pathway in regulating inflammation. Using time-lapse microscopy of mutant embryos that express GFP from a neutrophil-specific promoter, we found that individual neutrophils in inflamed tissue displayed random motility characterized by periods of pausing alternating with periods of motility. During periods of persistent movement the cells were highly polarized, while the pausing modes were characterized by a loss of cell polarity. In contrast to responses to acute injury, neutrophils did not exhibit clear retrograde chemotaxis or resolution of inflammation in the mutant. These findings illustrate the utility of zebrafish as a new model system to study chronic inflammation and to visualize immune responses with high resolution in vivo.

  • Chan KT, Cortesio CL, Huttenlocher A (2007) Integrins in cell migration. Meth. Enzymol. 426:47-67 View Abstract · Pubmed Record

    Integrins are cell-surface adhesion receptors that play a central role in regulating cell migration by mediating interactions between the extracellular matrix and the actin cytoskeleton. Substantial progress has been made in understanding the mechanisms by which the formation and breakdown of adhesions are regulated. Here we describe general methods used to study integrin-mediated cell migration. Furthermore, we outline detailed procedures to examine focal adhesion assembly and disassembly using time-lapse fluorescent microscopy. Finally, we provide methods for the analysis of podosomes, which are highly dynamic adhesive structures that form in immune cells and invasive cancer cells.

  • Clay H, Davis JM, Beery D, Huttenlocher A, Lyons SE, Ramakrishnan L (2007) Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish. Cell Host Microbe 2(1):29-39 (PMC3115716) View Abstract · Pubmed Record

    In tuberculosis, infecting mycobacteria are phagocytosed by macrophages, which then migrate into deeper tissue and recruit additional cells to form the granulomas that eventually contain infection. Mycobacteria are exquisitely adapted macrophage pathogens, and observations in the mouse model of tuberculosis have suggested that mycobacterial growth is not inhibited in macrophages until adaptive immunity is induced. Using the optically transparent and genetically tractable zebrafish embryo-Mycobacterium marinum model of tuberculosis, we have directly examined early infection in the presence and absence of macrophages. The absence of macrophages led rapidly to higher bacterial burdens, suggesting that macrophages control infection early and are not an optimal growth niche. However, we show that macrophages play a critical role in tissue dissemination of mycobacteria. We propose that residence within macrophages represents an evolutionary trade-off for pathogenic mycobacteria that slows their early growth but provides a mechanism for tissue dissemination.

  • Doan AT, Huttenlocher A (2007) RACK1 regulates Src activity and modulates paxillin dynamics during cell migration. Exp. Cell Res. 313(12):2667-79 (PMC2679865) View Abstract · Pubmed Record

    Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions.

  • Nuzzi PA, Lokuta MA, Huttenlocher A (2007) Analysis of neutrophil chemotaxis. Methods Mol. Biol. 370:23-36 View Abstract · Pubmed Record

    Neutrophils are the initial responders to bacterial infection or other inflammatory stimuli and comprise a key component of the innate immune response. In addition to their unique morphology and antimicrobial activity, neutrophils are characterized by the ability to migrate rapidly up shallow gradients of attractants in vivo. The directed migration of neutrophils, referred to as chemotaxis, requires the temporal and spatial regulation of intracellular signaling pathways allowing the neutrophil to detect a gradient of attractant, polarize, and migrate rapidly toward the highest concentration of the chemoattractant. A challenge to understanding neutrophil chemotaxis is the inherent difficulty encountered when working with primary neutrophils, which are difficult to purify in the resting state, are not easily transfected, are terminally differentiated, and have a short life span after purification. Here we discuss neutrophil purification methods and chemotaxis assays and provide methodology for working with a neutrophil-like cell line, the HL-60 promyelocytic leukemia cell line. We also discuss methods for HL-60 transfection using retroviral approaches and chemotaxis assays used with differentiated HL-60 cells.

  • Nuzzi PA, Senetar MA, Huttenlocher A (2007) Asymmetric localization of calpain 2 during neutrophil chemotaxis. Mol. Biol. Cell 18(3):795-805 (PMC1805107) View Abstract · Pubmed Record

    Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis.

  • Huttenlocher A, Horwitz AR (2007) Wound healing with electric potential. N. Engl. J. Med. 356(3):303-4 View Abstract · Pubmed Record
  • Simonson WT, Franco SJ, Huttenlocher A (2006) Talin1 regulates TCR-mediated LFA-1 function. J. Immunol. 177(11):7707-14 View Abstract · Pubmed Record

    The leukocyte integrin LFA-1 plays a critical role in T cell trafficking and T cell adhesion to APCs. It is known that integrin-mediated adhesion is regulated by changes in integrin ligand-binding affinity and valency through inside-out signaling. However, the molecular mechanisms involved in TCR-mediated LFA-1 regulation are not well understood. In this study, we show that the cytoskeletal protein talin1 is required for TCR-mediated activation of LFA-1 through regulation of LFA-1 affinity and clustering. Depletion of talin1 from human T cells by small interfering RNAs impairs TCR-induced adhesion to ICAM-1 and T cell-APC conjugation. TCR-induced LFA-1 polarization, but not actin polarization, is defective in talin1-deficient T cells. Although LFA-1 affinity is also reduced in talin1-deficient T cells, rescue of LFA-1 affinity alone is not sufficient to restore LFA-1 adhesive function. Together, our findings indicate that TCR-induced up-regulation of LFA-1-dependent adhesiveness and resulting T cell-APC conjugation require talin1.

  • Mathias JR, Perrin BJ, Liu TX, Kanki J, Look AT, Huttenlocher A (2006) Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J. Leukoc. Biol. 80(6):1281-8 View Abstract · Pubmed Record

    Neutrophil chemotaxis to sites of inflammation is a critical process during normal immune responses to tissue injury and infection and pathological immune responses leading to chronic inflammation. Although progress has been made in understanding the mechanisms that promote neutrophil recruitment to inflamed tissue, the mechanisms that regulate the resolution phase of the inflammatory response have remained relatively elusive. To define the mechanisms that regulate neutrophil-mediated inflammation in vivo, we have developed a novel transgenic zebrafish in which the neutrophils express GFP under control of the myeloperoxidase promoter (zMPO:GFP). Tissue injury induces a robust, inflammatory response, which is characterized by the rapid chemotaxis of neutrophils to the wound site. In vivo time-lapse imaging shows that neutrophils subsequently display directed retrograde chemotaxis back toward the vasculature. These findings implicate retrograde chemotaxis as a novel mechanism that regulates the resolution phase of the inflammatory response. The zMPO:GFP zebrafish provides unique insight into the mechanisms of neutrophil-mediated inflammation and thereby offers opportunities to identify new regulators of the inflammatory response in vivo.

  • Franco SJ, Senetar MA, Simonson WT, Huttenlocher A, McCann RO (2006) The conserved C-terminal I/LWEQ module targets Talin1 to focal adhesions. Cell Motil. Cytoskeleton 63(9):563-81 View Abstract · Pubmed Record

    The cytoskeletal protein Talin1 is a critical link between integrins and the actin cytoskeleton, where it is required for the structural and signaling functions of integrin-containing adhesion complexes. However, the elements in Talin1 that are responsible for localizing it to adhesion complexes are not known. In this report we have used a series of constructs based on the modular structure of Talin1 to determine the structural elements that specify the subcellular localization of Talin1. We show that the conserved actin-binding I/LWEQ module at the C-terminus of Talin1 is necessary and sufficient for targeting to focal adhesion complexes. We also used truncation and site-directed mutagenesis to demonstrate that this novel targeting function correlates with, but is separable from, the actin-binding properties of the Talin1 I/LWEQ module. In addition, we have shown that focal adhesion targeting, unlike actin binding, is not conserved among I/LWEQ module proteins. Finally, we have demonstrated that the subcellular localization of the Talin1 I/LWEQ module is regulated by an intrasteric interaction with an upstream alpha-helix, suggesting that both the actin binding and adhesion-targeting elements are masked in full-length Talin1. Our results define a novel role for the I/LWEQ module as the primary adhesion-complex targeting determinant of Talin1 and suggest that pathways that can relieve inhibition of I/LWEQ module function will be important for regulating the structural and signaling properties of adhesion complexes.

  • Su LT, Agapito MA, Li M, Simonson WT, Huttenlocher A, Habas R, Yue L, Runnels LW (2006) TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain. J. Biol. Chem. 281(16):11260-70 (PMC3225339) View Abstract · Pubmed Record

    m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes.

  • Abhyankar VV, Lokuta MA, Huttenlocher A, Beebe DJ (2006) Characterization of a membrane-based gradient generator for use in cell-signaling studies. Lab Chip 6(3):389-93 View Abstract · Pubmed Record

    This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel. The high fluidic resistance of the membrane limits fluid flow caused by pressure differences in the system, but allows diffusive transport of a chemical species through the membrane and into the channel. The large volume sink region at the end of the microfluidic channel helps to maintain spatial and temporal stability of the gradient. The chemical gradient in a 0.5 mm region near the sink region experiences a maximum of 10 percent change between the 6 and 24 h data points. We present the theory, design, and characterization of this device and provide an example of neutrophil chemotaxis as proof of concept for future quantitative cell-signaling applications.

  • Perrin BJ, Amann KJ, Huttenlocher A (2006) Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration. Mol. Biol. Cell 17(1):239-50 (PMC1345662) View Abstract · Pubmed Record

    Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.

  • Lokuta MA, Cooper KM, Aksentijevich I, Kastner DL, Huttenlocher A (2005) Neutrophil chemotaxis in a patient with neonatal-onset multisystem inflammatory disease and Muckle-Wells syndrome. Ann. Allergy Asthma Immunol. 95(4):394-9 View Abstract · Pubmed Record

    Neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic, cutaneous, and articular syndrome is an autoinflammatory disease characterized by urticarial rash, arthropathy, and central nervous system inflammation. To describe a 13-year-old girl with overlapping symptoms of NOMID and Muckle-Wells syndrome who has a mutation in cryopyrin (NALP3). We examined neutrophil migration using transwell assay and time-lapse videomicroscopy. We also examined p38 mitogen-activated protein kinase (MAPK) activation in patient and control neutrophils using Western blot analysis. Neutrophil defects in chemotactic migration were found to a variety of chemoattractants, including interleukin 8, N-formyl-methionyl-leucyl-phenylalanine, complement C5a, and leukotriene B4. Her neutrophils exhibited elevated basal and stimulated p38 MAPK activation in response to interleukin 8, N-formyl-methionyl-leucyl-phenylalanine, complement C5a, and leukotriene B4. This study is the first, to our knowledge, to demonstrate defects in neutrophil chemotaxis and p38 MAPK signaling in a patient with NOMID and Muckle-Wells syndrome and a cryopyrin mutation.

  • Franco SJ, Huttenlocher A (2005) Regulating cell migration: calpains make the cut. J. Cell. Sci. 118(Pt 17):3829-38 View Abstract · Pubmed Record

    The calpain family of proteases has been implicated in cellular processes such as apoptosis, proliferation and cell migration. Calpains are involved in several key aspects of migration, including: adhesion and spreading; detachment of the rear; integrin- and growth-factor-mediated signaling; and membrane protrusion. Our understanding of how calpains are activated and regulated during cell migration has increased as studies have identified roles for calcium and phospholipid binding, autolysis, phosphorylation and inhibition by calpastatin in the modulation of calpain activity. Knockout and knockdown approaches have also contributed significantly to our knowledge of calpain biology, particularly with respect to the specific functions of different calpain isoforms. The mechanisms by which calpain-mediated proteolysis of individual substrates contributes to cell motility have begun to be addressed, and these efforts have revealed roles for proteolysis of specific substrates in integrin activation, adhesion complex turnover and membrane protrusion dynamics. Understanding these mechanisms should provide avenues for novel therapeutic strategies to treat pathological processes such as tumor metastasis and chronic inflammatory disease.

  • Wells A, Huttenlocher A, Lauffenburger DA (2005) Calpain proteases in cell adhesion and motility. Int. Rev. Cytol. 245:1-16 View Abstract · Pubmed Record

    Cell adhesion and its role during cell spreading and motility are central to normal development and homeostasis, including its effects on immune response and wound repair and tissue regeneration. Disruption of cell adhesion impacts not only the healing process but promotes tumor invasion and metastasis. A family of intracellular, limited proteases, the calpains, has recently been shown to be a key molecular control point in attachment of cells to the surrounding matrix. Herein, the two main and ubiquitously expressed calpain isoforms will be introduced as to their modes of regulation and the current status of research will be discussed as to how these calpains might function in the biophysical process of adhesion and biological cellular responses of spreading and motility.

  • Lokuta MA, Huttenlocher A (2005) TNF-alpha promotes a stop signal that inhibits neutrophil polarization and migration via a p38 MAPK pathway. J. Leukoc. Biol. 78(1):210-9 View Abstract · Pubmed Record

    Neutrophils are a major component of the inflammatory response in patients with asthma and other inflammatory conditions. Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in the airway of patients with severe asthma and have been implicated in the recruitment of neutrophils into areas of inflammation. Here, we show that TNF-alpha induces a stop signal that promotes firm neutrophil adhesion and inhibits neutrophil polarization and chemotaxis to chemoattractants including interleukin-8 and C5a. TNF-alpha treatment of neutrophils plated on a fibrinogen-coated surface promotes firm neutrophil adhesion and the formation of vinculin-containing focal complexes. TNF-alpha induces a more than tenfold increase in p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase phosphorylation. Inhibition of p38 MAPK in neutrophils treated with TNF-alpha causes neutrophil polarization and motility. These findings suggest that TNF-alpha initiates a stop signal through a p38 MAPK pathway, which may promote the retention of neutrophils in inflammatory sites. Together, our data suggest that inhibition of p38 MAPK may be an attractive target to limit inflammatory responses that are mediated by TNF-alpha.

  • Huttenlocher A (2005) Cell polarization mechanisms during directed cell migration. Nat. Cell Biol. 7(4):336-7 View Abstract · Pubmed Record
  • Lehman TJ, Schechter SJ, Sundel RP, Oliveira SK, Huttenlocher A, Onel KB (2004) Thalidomide for severe systemic onset juvenile rheumatoid arthritis: A multicenter study. J. Pediatr. 145(6):856-7 View Abstract · Pubmed Record

    Thirteen children with difficult systemic onset juvenile rheumatoid arthritis were treated with thalidomide. At 6 months, 11 of the 13 were able to reduce their use of prednisone ( P < .002), with a concurrent improvement in erythrocyte sedimentation rate ( P < .0001) and an increase in hemoglobin level ( P < 0.005). Juvenile rheumatoid arthritis improvement scores >/=50% were obtained by 10 of the 13 children.

  • Franco SJ, Rodgers MA, Perrin BJ, Han J, Bennin DA, Critchley DR, Huttenlocher A (2004) Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat. Cell Biol. 6(10):977-83 View Abstract · Pubmed Record

    Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.

  • Panetti TS, Hannah DF, Avraamides C, Gaughan JP, Marcinkiewicz C, Huttenlocher A, Mosher DF (2004) Extracellular matrix molecules regulate endothelial cell migration stimulated by lysophosphatidic acid. J. Thromb. Haemost. 2(9):1645-56 View Abstract · Pubmed Record

    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are lipids that bind G-protein coupled receptors and differentially promote transmigration of endothelial cells. To determine if endothelial cell transmigration stimulated by LPA, not S1P, is dependent on the extracellular matrix. Bovine pulmonary artery (BPAE) endothelial cell transmigration and locomotion were measured using a modified-Boyden chamber and video microscopy, respectively. Results were related to strength of adhesion and characteristics of cell adhesive contacts. BPAEs responded to LPA by transmigration through gelatin- or collagen-coated filters, but not through fibronectin-, vitronectin-, or fibrinogen-coated filters. Fewer cells adhered to collagen or gelatin than to fibronectin in a static cell adhesion assay or after application of a g-force to detach cells. Video microscopy revealed that S1P stimulates large lamellipodia on two-dimensional fibronectin substrate. LPA stimulated lamellipodia on fibronectin, but the trailing edge remained attached, resulting in sting ray-shaped cells in video microscopy. LPA-treated cells on gelatin released the trailing edge. To understand how the extracellular matrix may regulate endothelial cell shape during movement, we surveyed changes in focal adhesion proteins. More Hic-5, a paxillin homolog, was detected in the detergent insoluble fraction of BPAEs attached to gelatin than fibronectin. No such difference was found in paxillin. In BPAEs, Hic-5 was localized to smaller punctate structures on fibronectin and longer, thinner focal adhesions on gelatin. These results indicated that localization of Hic-5 and strength of adhesion correlate with endothelial cell transmigration stimulated by LPA, but not with transmigration stimulated by S1P.

  • Franco S, Perrin B, Huttenlocher A (2004) Isoform specific function of calpain 2 in regulating membrane protrusion. Exp. Cell Res. 299(1):179-87 View Abstract · Pubmed Record

    Previous studies have demonstrated a role for calpains in cell migration through their capacity to regulate focal adhesion dynamics and rear retraction. In this study, we provide evidence that calpains also modulate membrane protrusion activity in fibroblasts. We find that an immortalized Capn4(-/-) fibroblast line displays an altered morphology, characterized by numerous thin membrane projections and increased transient membrane activity. Furthermore, we show that protrusion kinetics of lamellipodia at the leading edge are improperly regulated in Capn4(-/-) cells, leading to impaired net forward lamellipodial extension. To address the isoform specific functions of calpain 1 and calpain 2 during cell protrusion, we stably introduced small interfering RNAs (siRNAs) targeting each isoform into a fibroblast cell line. Despite a loss in calpain 1 activity, calpain 1 knockdown cells show normal morphology and membrane protrusion dynamics. However, cells in which calpain 2 is knocked down are characterized by a protrusive morphology, increased transient membrane activity and altered protrusion kinetics, similar to the Capn4(-/-) fibroblasts. Additionally, we find that calpain 2, but not calpain 1, is required for proteolysis of the cytoskeletal and focal adhesion proteins FAK, paxillin, spectrin, and talin. Together, our findings support a novel role for calpain 2 in limiting membrane protrusions and in regulating lamellipodial dynamics at the leading edge of migrating cells.

  • Robles E, Huttenlocher A, Gomez TM (2003) Filopodial calcium transients regulate growth cone motility and guidance through local activation of calpain. Neuron 38(4):597-609 View Abstract · Pubmed Record

    Spontaneous intracellular calcium ([Ca2+](i)) transients in growth cone filopodia reduce filopodial motility, slow neurite outgrowth, and promote turning when generated asymmetrically; however, the downstream effectors of these Ca2+ -dependent behaviors are unknown. We report that Ca2+ transients in filopodia activate the intracellular protease calpain, which slows neurite outgrowth and promotes repulsive growth cone turning upon local activation. Active calpain alters the balance between tyrosine kinase and phosphatase activities in filopodia, resulting in a net decrease in tyrosine phosphorylation, which mediates both filopodial stabilization and reduced lamellipodial protrusion. Our findings indicate that locally generated Ca2+ signals repel axon outgrowth through calpain-dependent regulation of phosphotyrosine signaling at integrin-mediated adhesion sites.

  • Holub A, Byrnes J, Anderson S, Dzaidzio L, Hogg N, Huttenlocher A (2003) Ligand density modulates eosinophil signaling and migration. J. Leukoc. Biol. 73(5):657-64 View Abstract · Pubmed Record

    Eosinophils are a major component of the inflammatory response in persistent airway inflammation in asthma. The factors that determine the retention of eosinophils in the airway remain poorly understood. Elevated levels of fibronectin have been observed in the airway of patients with asthma, and the levels correlate with eosinophil numbers. To determine if fibronectin density modulates eosinophil function, we investigated the effect of fibronectin and vascular cell adhesion molecule 1 (VCAM-1) density on eosinophil migration and signaling via the p38 and extracellular regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) signaling pathways. There was a dose-dependent inhibition of eosinophil spreading and migration on increasing concentrations of fibronectin but not VCAM-1. In addition, activation of p38 MAPK was inhibited at high fibronectin but not high VCAM-1 concentrations, and ERK activity was slightly reduced at high VCAM-1 and fibronectin concentrations. Together, the results demonstrate that fibronectin but not VCAM-1 inhibits eosinophil migration and signaling.

  • Dziadzio L, Kelly EA, Panzer SE, Jarjour N, Huttenlocher A (2003) Cytokine abnormalities in a patient with eosinophilic fasciitis. Ann. Allergy Asthma Immunol. 90(4):452-5 View Abstract · Pubmed Record

    Eosinophilic fasciitis (EF) and peripheral eosinophilia are rare in children. To describe a case of EF in a 3-year-old child who presented with acute painless induration of her forearm. Cytokine profiles were obtained and compared with patients with atopic disease during the acute presentation and after treatment with low-dose prednisone. The patient's serum showed elevation of transforming growth factor beta1 and interleukin-5, which improved after treatment with low-dose prednisone. The case suggests that patients with EF have cytokine abnormalities similar to atopic patients, but with a striking elevation of transforming growth factor beta1. The responsiveness of the clinical symptoms and cytokine profile to low dose prednisone supports treatment with low dose immunosuppressive therapy in this disorder.

  • Lokuta MA, Nuzzi PA, Huttenlocher A (2003) Calpain regulates neutrophil chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 100(7):4006-11 (PMC153038) View Abstract · Pubmed Record

    Cell polarization is required for directed cell migration. We investigated the role of the calcium-dependent protease calpain during neutrophil chemotaxis and found that calpain inhibition induced neutrophil adhesion, polarization, and rapid chemokinesis in the absence of exogenous activators. Resting neutrophils display constitutive calpain activity with mu-calpain being the predominant active isoform. Our findings suggest that constitutive calpain activity in resting neutrophils may function as a negative regulator of protrusion and migration. Specific inhibition of mu-calpain, but not m-calpain, induced neutrophil polarization and chemokinesis. In contrast to IL-8-induced chemokinesis, the chemokinesis induced by calpain inhibition was not reduced in the presence of pertussis toxin, suggesting that calpain functions downstream of G protein-coupled receptors. Further, both calpain inhibition and stimulation with IL-8 and formyl-Met-Leu-Phe (fMLP) induced an increase in Cdc42 and Rac activation. These findings are consistent with the involvement of calpain in chemotaxis pathways. Accordingly, calpain inhibition decreased neutrophil chemotaxis and directional persistence in a gradient of IL-8 and fMLP. Together, these data reveal a previously uncharacterized function for calpain in neutrophils and suggest that localized modulation of calpain activity may regulate neutrophil chemotaxis downstream of G-protein-coupled receptors.

  • Bhatt AK, Huttenlocher A (2003) Dynamic imaging of cell-substrate contacts. Meth. Enzymol. 361:337-52 View Abstract · Pubmed Record
  • Cox EA, Bennin D, Doan AT, O'Toole T, Huttenlocher A (2003) RACK1 regulates integrin-mediated adhesion, protrusion, and chemotactic cell migration via its Src-binding site. Mol. Biol. Cell 14(2):658-69 (PMC149999) View Abstract · Pubmed Record

    Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the beta integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.

  • Bhatt A, Kaverina I, Otey C, Huttenlocher A (2002) Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain. J. Cell. Sci. 115(Pt 17):3415-25 View Abstract · Pubmed Record

    Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.

  • Perrin BJ, Huttenlocher A (2002) Calpain. Int. J. Biochem. Cell Biol. 34(7):722-5 View Abstract · Pubmed Record

    The calcium-dependent thiol proteases, calpains, are widely expressed with ubiquitous and tissue specific isoforms. Calpains have been implicated in basic cellular processes including cell proliferation, apoptosis and differentiation. The focus of the current review is to summarize recent findings implicating calpains in cytoskeletal rearrangements and cell migration. Calpain cleaves many cytosolic proteins and therefore to be effective and limited in its scope, calpain activity has to be tightly regulated both temporally and spatially. Some mechanisms of regulation include calcium, growth factor-mediated phosphorylation and membrane targeting. Calpain inhibition reduces migration rates and inhibits cell invasiveness. Two putative mechanisms of calpain action during migration include its role as a signaling intermediate, acting upstream of Rho, and its effects on focal adhesion structure and disassembly. Therefore, calpains and downstream signaling molecules may be future targets for therapeutic interventions to treat cancer or chronic inflammation.

  • Ziegner UH, Kobayashi RH, Cunningham-Rundles C, EspaƱol T, Fasth A, Huttenlocher A, Krogstad P, Marthinsen L, Notarangelo LD, Pasic S, Rieger CH, Rudge P, Sankar R, Shigeoka AO, Stiehm ER, Sullivan KE, Webster AD, Ochs HD (2002) Progressive neurodegeneration in patients with primary immunodeficiency disease on IVIG treatment. Clin. Immunol. 102(1):19-24 View Abstract · Pubmed Record

    We have identified 14 patients with diverse primary immunodeficiencies who have developed progressive neurodegeneration of unknown etiology. All patients had received immunoglobulin replacement therapy for a mean duration of 6.5 years (range of 0.5-13.5 years) at the time of first neurological symptoms. Diagnostic tests of blood and cerebrospinal fluid analyses included chemistry, cultures, PCR for viral genomes, and cytology. In addition, neuroimaging and electrophysiologic studies were performed. Brain tissue histology (n = 5) revealed nonspecific encephalitis with microglial infiltration and neuronal loss. Twelve patients died 6 months to 15 years (median 4.3 years) after onset of neurologic findings. No evidence of any infectious disease that could have explained our patients' progressive encephalopathy was found either during their lifetimes or postmortem. These patients may have had an unusual manifestation of primary immunodeficiency diseases, an autoimmune reaction against neuronal tissue, a yet undefined infectious agent, or a complication of IVIG therapy. To help determine the etiology of this rare complication, an international surveillance system for primary immunodeficiency patients who develop progressive neurodegeneration of unknown cause is recommended.

  • Calderwood DA, Huttenlocher A, Kiosses WB, Rose DM, Woodside DG, Schwartz MA, Ginsberg MH (2001) Increased filamin binding to beta-integrin cytoplasmic domains inhibits cell migration. Nat. Cell Biol. 3(12):1060-8 View Abstract · Pubmed Record

    Multicellular animal development depends on integrins. These adhesion receptors link to the actin cytoskeleton, transmitting biochemical signals and force during cell migration and interactions with the extracellular matrix. Many integrin-cytoskeleton connections are formed by filamins and talin. The beta7 integrin tail binds strongly to filamin and supports less migration, fibronectin matrix assembly and focal adhesion formation than either the beta1D tail, which binds strongly to talin, or the beta1A tail, which binds modestly to both filamin and talin. To probe the role of filamin binding, we mapped the filamin-binding site of integrin tails and identified amino acid substitutions that led to selective loss of filamin binding to the beta7 tail and gain of filamin binding to the beta1A tail. These changes affected cell migration and membrane protrusions but not fibronectin matrix assembly or focal adhesion formation. Thus, tight filamin binding restricts integrin-dependent cell migration by inhibiting transient membrane protrusion and cell polarization.

  • Dourdin N, Bhatt AK, Dutt P, Greer PA, Arthur JS, Elce JS, Huttenlocher A (2001) Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts. J. Biol. Chem. 276(51):48382-8 View Abstract · Pubmed Record

    The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.

  • Cox EA, Sastry SK, Huttenlocher A (2001) Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases. Mol. Biol. Cell 12(2):265-77 (PMC30942) View Abstract · Pubmed Record

    Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.

  • Sastry SK, Lakonishok M, Wu S, Truong TQ, Huttenlocher A, Turner CE, Horwitz AF (1999) Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal. J. Cell Biol. 144(6):1295-309 (PMC2150582) View Abstract · Pubmed Record

    We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.

  • Cox EA, Huttenlocher A (1998) Regulation of integrin-mediated adhesion during cell migration. Microsc. Res. Tech. 43(5):412-9 View Abstract · Pubmed Record

    Migrating cells form dynamic and highly regulated adhesive interactions with their environment. In particular, integrin-mediated adhesions to the extracellular matrix (ECM) play a central role in cell migration. This review focuses on recent advances in understanding the adhesive mechanisms that regulate cell detachment at the rear of migrating fibroblasts and neutrophils. The contribution of several key adhesive regulators is discussed, including myosin mediated cell contractility, tyrosine phosphorylation, rho, calcium fluxes, and calpain. A challenge for future investigation will be to determine how adhesive events are spatially and temporally coordinated to promote productive directional cell movements.

  • Huttenlocher A, Lakonishok M, Kinder M, Wu S, Truong T, Knudsen KA, Horwitz AF (1998) Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J. Cell Biol. 141(2):515-26 (PMC2148455) View Abstract · Pubmed Record

    Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169-184), ectopic expression of the alpha5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing alpha5 integrin (alpha5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of beta1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the alpha5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.

  • Palecek SP, Huttenlocher A, Horwitz AF, Lauffenburger DA (1998) Physical and biochemical regulation of integrin release during rear detachment of migrating cells. J. Cell. Sci. 111 ( Pt 7:929-40 View Abstract · Pubmed Record

    Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.

  • Huttenlocher A, Palecek SP, Lu Q, Zhang W, Mellgren RL, Lauffenburger DA, Ginsberg MH, Horwitz AF (1997) Regulation of cell migration by the calcium-dependent protease calpain. J. Biol. Chem. 272(52):32719-22 View Abstract · Pubmed Record

    Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both beta1 and beta3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment. Furthermore, these inhibitors alter the fate of integrin receptors at the rear of the cell during migration. A Chinese hamster ovary cell line expressing low levels of calpain I also shows reduced migration rates with similar morphological changes, further implicating calpain in this process. Taken together, the data suggest that calpain inhibition modulates cell migration by stabilizing cytoskeletal linkages and decreasing the rate of retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may therefore be a potential therapeutic approach to control pathological cell migration such as tumor metastasis.

  • Huttenlocher A, Newman TB (1997) Evaluation of the erythrocyte sedimentation rate in children presenting with limp, fever, or abdominal pain. Clin Pediatr (Phila) 36(6):339-44 View Abstract · Pubmed Record

    An erythrocyte sedimentation rate (ESR) is commonly ordered as part of the evaluation of patients with nonspecific but potentially serious symptoms. To investigate the performance of ESR in this setting, we used a computerized database and medical chart review to identify children (n=299) with ESR done for a previously undiagnosed condition. Medical records were reviewed to determine symptoms at presentation, referral status, and subsequent diagnoses, which were classified as serious (n=93) or benign (n=206). We found that serious underlying disease was about 7 times as likely in patients with ESR>50 mm/hr (57/102) than in patients with ESR<20 mm/hr (7/89). Although the prevalence of serious disease was higher among referral patients, the likelihood ratios were similar for referral and primary-care patients. An erythrocyte sedimentation rate greater than 50 mm/hr was most informative in patients presenting with limp (likelihood ratio [LR] =8.2) and abdominal pain (LR=6.0) and least informative in patients presenting with fever (LR=2.5). On the other hand, an ESR<20 mm/hr is reassuring in patients presenting with fever (LR=0) or limp (LR=0.3), but not in patients presenting with abdominal pain (LR=0.8). An ESR between 20 and 50 mm/hr (23% of the patients) provided little information (LR 1.2-1.5) in each of the three groups. These results suggest that the ESR often provides useful information about the likelihood of serious illness among children presenting with worrisome but nonspecific symptoms, in particular in patients presenting with limp.

  • Huttenlocher A, Werb Z, Tremble P, Huhtala P, Rosenberg L, Damsky CH (1996) Decorin regulates collagenase gene expression in fibroblasts adhering to vitronectin. Matrix Biol. 15(4):239-50 View Abstract · Pubmed Record

    Vitronectin, a principal cell adhesion molecule in plasma and extracellular matrix, mediates cell adhesion and spreading via the alpha V family of integrins. In this study we demonstrate that decorin, a small dermatan sulfate proteoglycan, regulates extracellular matrix remodeling in rabbit synovial fibroblasts adhering to vitronectin. Decorin induced the expression of the matrix metalloproteinase collagenase (MMP-1) when present on the substrate with vitronectin, or with the 120-kDa cell-binding domain of fibronectin, but not when present with intact fibronectin or Type I collagen. Secreted collagenase was detected within 8 h of adhesion, there was no associated alteration in cell shape or focal contact formation in cells adhering to decorin plus vitronectin, whereas cell rounding was observed in cells adhering to decorin plus the 120-kDa fragment of fibronectin. The core protein of decorin, but not the glycosaminoglycan moiety, was sufficient to induce collagenase expression on both substrates; however, the glycosaminoglycan moiety of decorin as well as the core were required for cell rounding observed in cells adhering to the 120-kDa domain of fibronectin. The collagenase-inducing effect of decorin seems to be independent of its effects on transforming growth factor-beta, as function-blocking antibodies against transforming growth factor-beta did not interfere with the collagenase-inducing effects of decorin. These data indicate that decorin has specific gene regulatory effects in cells when present in the matrix with vitronectin or the 120-kDa fragment of fibronectin, polypeptides that are present in actively remodeling tissues. Thus, in combination, these adhesion regulatory molecules transduce novel signals that may contribute to the tissue remodeling process in morphogenesis, wound healing and disease states.

  • Huttenlocher A, Ginsberg MH, Horwitz AF (1996) Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity. J. Cell Biol. 134(6):1551-62 (PMC2121008) View Abstract · Pubmed Record

    Integrin cell surface adhesion receptors play a central role in mediating cell migration. We have developed a model system consisting of CHO cells ectopically expressing the alpha IIb beta 3 integrin to study integrin affinity and cytoskeletal interactions during cell migration. The alpha IIb beta 3 integrins are suited for study of integrin receptors during cell migration because they are well characterized with respect to ligand binding, cytoskeletal interactions, and signal transduction, and mutants with altered receptor function are available. The alpha IIb beta 3 receptor specifically mediates migration of alpha IIb beta 3-transfected CHO cells. The migration of transfected CHO cells was studied on a fibrinogen substrate both by time lapse videomicroscopy and by random and haptotactic transwell assays. Haptotactic and random transwell assays measured distinct aspects of migration, with the random transwell assay correlating most closely with time lapse videomicroscopy. Mutations in the cytoplasmic domains that increase ligand affinity or activation of the alpha IIb beta 3 receptor into a high affinity state by the LIBS6 antibody decreased the migration rate. Likewise, mutations that increase cytoskeletal organization without affecting affinity also decreased the migration rate. In contrast, truncation of the beta chain, which alters cytoskeletal associations as assayed by absence of focal adhesions, decreased haptotactic migration while increasing random migration. These effects on the migration rate were partially compensated for by altering substrate concentration, demonstrating optimum substrate concentrations that supported maximal migration. For example, cells expressing integrins locked in the high affinity state showed maximal migration at lower substrate concentrations than cells expressing low affinity receptor. Together, these results implicate the strength of adhesion between cell and substrate, as modulated by receptor affinity, organization of adhesive complexes, and substrate concentration, as important regulators of cell migration rate. Further, we demonstrate a dominant effect of high affinity integrin in inhibiting migration regardless of the organization of adhesive complexes. These observations have potential implications for tumor metastasis and its therapy.

  • Huttenlocher A, Sandborg RR, Horwitz AF (1995) Adhesion in cell migration. Curr. Opin. Cell Biol. 7(5):697-706 View Abstract · Pubmed Record

    Adhesive interactions play a central role in cell migration. The regulation of these interactions requires the coordination of a multiplicity of signals, both spatially and temporally. The role of the integrin family has received considerable recent attention. Progress has been made in the elucidation of the mechanisms by which growth factors and other motogenic factors stimulate migration. Major advances have also been made in understanding the mechanisms by which the formation and breakdown of adhesive complexes are regulated, including the participation of members of the rho family. Despite these advances, many important questions remain, and the field seems well positioned to answer them.

  • Huttenlocher A, Frieden IJ, Emery H (1995) Neonatal onset multisystem inflammatory disease. J. Rheumatol. 22(6):1171-3 View Abstract · Pubmed Record

    Neonatal onset multisystem inflammatory disease (NOMID) is a rare disorder involving a triad of arthropathy, rash, and central nervous system (CNS) involvement. We describe a girl with NOMID who presented with typical neonatal rash, arthropathy, fever, and failure to thrive, but has not developed evidence of ocular or CNS involvement. This case illustrates the spectrum of involvement seen in NOMID. Histopathology of the skin demonstrated neutrophilic eccrine hidradenitis, a unique finding, which may serve as a diagnostic clue in patients with this rare disorder.

  • Loskutoff DJ, Roegner K, Erickson LA, Schleef RR, Huttenlocher A, Coleman PL, Gelehrter TD (1986) The dexamethasone-induced inhibitor of plasminogen activator in hepatoma cells is antigenically-related to an inhibitor produced by bovine aortic endothelial cells. Thromb. Haemost. 55(1):8-11 View Abstract · Pubmed Record

    Glucocorticoids decrease plasminogen activator (PA) activity in HTC rat hepatoma cells by inducing a specific inhibitor of PA activity (PAI). This inhibitor is similar in several biochemical properties to the PAI purified from bovine aortic endothelial cells (BAEs). We have used reverse fibrin autography and antiserum against BAE PAI to establish more fully the biochemical and immunological relationship of these inhibitors. Both inhibitors migrated with an apparent Mr of approximately 50,000, and the activity of both PAIs was stimulated by treatment with SDS suggesting that each of these molecules exists in both an active and a latent form. Antiserum to the BAE PAI immunoprecipitated all of the HTC PAI demonstrable by reverse fibrin autography. Finally, using this antiserum in a functional immunoassay, we have demonstrated that dexamethasone increases both active and latent PAI made by HTC cells. These results indicate that HTC PAI and BAE PAI are antigenically as well as biochemically related molecules.