Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling

Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/β, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.     

Biochemical and biological characterization of a human Rac2 GTPase mutant associated with phagocytic immunodeficiency

The Rho GTPase, Rac2, is expressed only in hematopoietic cell lineages, suggesting a specific cellular function in these cells. Genetic targeting studies in mice showed that Rac2 is an essential regulator of neutrophil chemotaxis, L-selectin capture and rolling, and superoxide production. Recently, a dominant negative mutation of Rac2, D57N, has been reported to be associated with a human phagocytic immunodeficiency. To understand further the cellular phenotypes associated with this D57N Rac2 mutant we examined its biochemical characteristics and functional effects when expressed in primary murine bone marrow cells. When compared with wild type (WT) Rac2, D57N Rac2 displayed approximately 10% GTP binding ability resulting from a markedly enhanced rate of GTP dissociation and did not respond to the guanine nucleotide exchange factors. These results suggest that D57N Rac2 may act in a dominant negative fashion in cells by sequestering endogenous guanine nucleotide exchange factors. When expressed in hematopoietic cells, D57N Rac2 reduced endogenous activities of not only Rac2, but also Rac1 and decreased cell expansion in vitro in the presence of growth factors due to increased cell apoptosis. Unexpectedly, D57N expression had no effect on proliferation. In contrast, expansion of cells transduced with WT Rac2 and a dominant active mutant, Q61L, was associated with significantly increased proliferation. Transplantation of transduced bone marrow cells into lethally irradiated recipients showed that the percentage of D57N-containing peripheral blood cells decreased markedly from 40% at 1 month to <5% by 3 months postinjection. Neutrophils derived in vitro from the transduced progenitor cells containing D57N demonstrated markedly impaired migration and O(2)(-) responses to formyl-methionyl-leucyl-phenylalanine, reflecting the same cellular phenotype in these differentiated cells as those described previously in patient cells. These data suggest that the phenotypic abnormalities associated with D57N Rac2 may involve not only neutrophil cellular functions, but also abnormal cell survival in other hematopoietic cells and that overexpression of Rac leads to increased proliferation of normal cells in vitro, whereas deficiency of Rac leads to increased apoptosis.     

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.     

The actin regulatory protein HS1 interacts with Arp2/3 and mediates efficient neutrophil chemotaxis

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.     

Altered CXCR2 signaling in beta-arrestin-2-deficient mouse models

CXCR2 is a G-protein-coupled receptor (GPCR) that binds the CXC chemokines, CXCL1-3 and CXCL5-8, and induces intracellular signals associated with chemotaxis. Many adaptor proteins are actively involved in the sequestration, internalization, and trafficking of CXCR2 and transduction of agonist-induced intracellular signaling. We have previously shown that adaptor protein beta-arrestin-2 (betaarr2) plays a crucial role in transducing signals mediated through CXCR2. To further investigate the role of betaarr2 on CXCR2-mediated signaling during acute inflammation, zymosan-induced neutrophils were isolated from peritoneal cavities of betaarr2-deficient (betaarr2(-/-)) and their wild-type (betaarr2(+/+)) littermate mice, and neutrophil CXCR2 signaling activities were determined by measurement of Ca(2+) mobilization, receptor internalization, GTPase activity, and superoxide anion production. The results showed that the deletion of betaarr2 resulted in increased Ca(2+) mobilization, superoxide anion production, and GTPase activity in neutrophils, but decreased receptor internalization relative to wild-type mice. Two animal models, the dorsal air pouch model and the excisional wound healing model, were used to further study the in vivo effects of betaarr2 on CXCR2-mediated neutrophil chemotaxis and on cutaneous wound healing. Surprisingly, the recruitment of neutrophils was increased in response to CXCL1 in the air pouch model and in the excisional wound beds of betaarr2(-/-) mice. Wound re-epithelialization was also significantly faster in betaarr2(-/-) mice than in betaarr2(+/+) mice. Taken together, the data indicate that betaarr2 is a negative regulator for CXCR2 in vivo signaling.     

Requirement for Vav proteins in post-recruitment neutrophil cytotoxicity in IgG but not complement C3-dependent injury

The signals linking neutrophil opsonic receptors, FcgammaRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcgammaRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcgammaRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcgammaR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcgammaR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions.     

Protein O-GlcNAc modulates motility-associated signaling intermediates in neutrophils

The modification of serine/threonine residues on cytoplasmic and nuclear proteins by N-acetylglucosamine (O-GlcNAc) is suggested to play a role in the regulation of a variety of signal transduction pathways. We have previously shown that glucosamine (GlcNH(2)), a metabolic precursor of O-GlcNAcylation, increases (2)O-GlcNAc and enhances motility in neutrophils. Here, we extend this correlation by showing that a mechanistically distinct means of increasing O-GlcNAc, achieved by inhibition of O-GlcNAc removal with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), increases basal cellular motility and directional migration induced by the chemoattractant formyl-methionine-leucine-phenylalanine (fMLP). Furthermore, we demonstrate that O-GlcNAc modulates the activities of signaling intermediates known to regulate neutrophil movement. GlcNH(2) and PUGNAc increase both the basal and fMLP-induced activity of a central mediator of cellular motility, the small GTPase Rac. Phosphoinositide 3-kinase, an important regulator of Rac activity and neutrophil motility, is shown to regulate the signaling pathway on which GlcNH(2) and PUGNAc act. Rac is an important upstream regulatory element in p38 and p44/42 mitogen-activated protein kinase (MAPK) signaling in neutrophils, and these MAPKs are implicated in chemotactic signal transduction. We show that GlcNH(2) and PUGNAc treatment increases p42/44 and p38 MAPK activities and that these increases are associated with activation of upstream MAPK kinases. These data indicate that O-GlcNAcylation is an important signaling element in neutrophils that modulates the activities of several critical signaling intermediates involved in the regulation of cellular movement.     

DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation.     

Lithium down-regulates the expression of CXCR4 in human neutrophils

The CXC chemokine receptor CXCR4 and its unique ligand SDF-1 (stromal-derived factor-1) play critical roles for the retention of hematopoietic cells within the bone marrow (BM) and for their mobilization into the circulation. Lithium often produces neutrophilia in psychiatric patients, but the mechanism of mobilization related to neutrophilia has not been fully clarified. We showed here that lithium dose-dependently reduces the levels of surface CXCR4 protein and mRNA in neutrophils, but not in lymphocytes. The chemotactic migration of neutrophils in response to SDF-1 was reduced after a pre-incubation with lithium. We provide evidence that lithium down-regulates the CXCR4 expression of neutrophils and it attenuates their responsiveness to SDF-1. Our studies support the concept that down-regulation of CXCR4 is one of the mechanisms by which causes neutrophilia.     

Redox and Src family kinase signaling control leukocyte wound attraction and neutrophil reverse migration

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.     

Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions

Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To elucidate the specific role of Rac1 in neutrophils, we generated mice with a conditional Rac1 deficiency restricted to cells of the granulocyte/monocyte lineage. As observed in Rac2-deficient neutrophils, Rac1-deficient neutrophils demonstrated profound defects in inflammatory recruitment in vivo, migration to chemotactic stimuli, and chemoattractant-mediated actin assembly. In contrast, superoxide production is normal in Rac1-deficient neutrophils but markedly diminished in Rac2 null cells. These data demonstrate that although Rac1 and Rac2 are both required for actin-mediated functions, Rac2 is specifically required for activation of the neutrophil NADPH oxidase.     

The hemopoietic Rho/Rac guanine nucleotide exchange factor Vav1 regulates N-formyl-methionyl-leucyl-phenylalanine-activated neutrophil functions

Vav1 is a hemopoietic-specific Rho/Rac guanine nucleotide exchange factor that plays a prominent role in responses to multisubunit immune recognition receptors in lymphoid cells, but its contribution to regulation of neutrophil functions is unknown. Activated Rho family GTPases are critical participants in neutrophil signaling cascades initiated by binding of FMLP and other chemoattractants to their cognate G protein-coupled receptors. Therefore, we investigated whether Vav1 regulates chemoattractant-induced responses in neutrophils. We found that superoxide production elicited by FMLP in Vav1(-/-) murine neutrophils isolated from either bone marrow or from peritoneal exudates was substantially reduced compared with that of wild type. Filamentous actin generation in FMLP-stimulated Vav1(-/-) neutrophils was also markedly reduced, whereas it was normal in response to IL-8 or leukotriene B(4). FMLP induced tyrosine phosphorylation of Vav1, whereas IL-8 or leukotriene B(4) did not, correlating with the requirement for Vav1 in chemoattractant-stimulated filamentous actin generation. Neutrophil motility in vitro and neutrophil mobilization into peripheral blood in vivo elicited by FMLP were both decreased in Vav1(-/-) mice. Hence, this study defines a new role for Vav1 in regulating granulocytic leukocytes as well as linking Vav1 to specific cellular responses downstream of a seven transmembrane domain receptor.     

Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.     

Simvastatin regulates CXC chemokine formation in streptococcal M1 protein-induced neutrophil infiltration in the lung

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome and acute lung injury. Statins exert beneficial effects in septic patients although the mechanisms remain elusive. This study examined effects of simvastatin on M1 protein-provoked pulmonary inflammation and tissue injury. Male C57BL/6 mice were pretreated with simvastatin or a CXCR2 antagonist before M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for determination of neutrophil infiltration, formation of edema, and CXC chemokines. Flow cytometry was used to determine Mac-1 expression on neutrophils. Gene expression of CXC chemokines was determined in alveolar macrophages by using quantitative RT-PCR. M1 protein challenge caused massive infiltration of neutrophils, edema formation, and production of CXC chemokines in the lung as well as upregulation of Mac-1 on circulating neutrophils. Simvastatin reduced M1 protein-induced infiltration of neutrophils and edema in the lung. In addition, M1 protein-induced Mac-1 expression on neutrophils was abolished by simvastatin. Furthermore, simvastatin markedly decreased pulmonary formation of CXC chemokines and gene expression of CXC chemokines in alveolar macrophages. Moreover, the CXCR2 antagonist reduced M1 protein-induced neutrophil expression of Mac-1 and accumulation of neutrophils as well as edema formation in the lung. These novel findings indicate that simvastatin is a powerful inhibitor of neutrophil infiltration in acute lung damage triggered by streptococcal M1 protein. The inhibitory effect of simvastatin on M1 protein-induced neutrophil recruitment appears related to reduced pulmonary generation of CXC chemokines. Thus, simvastatin may be a useful tool to ameliorate acute lung injury in streptococcal infections.     

Targeting Rac1 Signaling Inhibits Streptococcal M1 Protein-Induced CXC Chemokine Formation,Neutrophil Infiltration and Lung Injury

Infections with Streptococcus pyogenes exhibit a wide spectrum of infections ranging from mild pharyngitis to severe Streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most commonly associated with STSS. In the present study, we hypothesized that Rac1 signaling might regulate M1 protein-induced lung injury. We studied the effect of a Rac1 inhibitor (NSC23766) on M1 protein-provoked pulmonary injury. Male C57BL/6 mice received NSC23766 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Treatment with NSC23766 decreased M1 protein-induced neutrophil infiltration, edema formation and tissue injury in the lung. M1 protein challenge markedly enhanced Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Rac1 activity had no effect on M1 protein-induced expression of Mac-1 on neutrophils. However, Rac1 inhibition markedly decreased M1 protein-evoked formation of CXC chemokines in the lung. Moreover, NSC23766 completely inhibited M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. We conclude that these novel results suggest that Rac1 signaling is a significant regulator of neutrophil infiltration and CXC chemokine production in the lung. Thus, targeting Rac1 activity might be a potent strategy to attenuate streptococcal M1 protein-triggered acute lung damage.     

Downregulation of CXCL12 signaling and altered hematopoietic stem and progenitor cell trafficking in a murine model of acute Anaplasma phagocytophilum infection

Infection with a variety of bacterial pathogens results in hematopoietic stem and progenitor cell (HSPC) mobilization. The mechanism and kinetics of HSPC mobilization during infection are largely unknown. Previously, we found altered HSPC activity in bone marrow, spleen and blood during infection with Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis. We hypothesized that altered CXCL12/CXCR4 signaling, a central pathway for HSPC homing to, and retention within, the bone marrow, plays a role in infection-induced alterations in HSPC number and trafficking. Mice were infected with A. phagocytophilum. Lineage-cKit+ HSPCs were enumerated and proliferation determined. CXCL12 and CXCR4 mRNA were quantified along with CXCL12 protein, and CXCR4 surface, intracellular and total protein expression in HSPCs was determined. Increased bone marrow proliferation of HSPCs began at 2 d post-infection followed by HSPC mobilization and splenic homing. Proliferation of resident HSPCs contributed to increased splenic HSPC numbers. Bone marrow CXCL12 mRNA and protein levels were decreased at 4-8 d post-infection concurrent with HSPC mobilization. CXCR4 protein parameters were decreased in bone marrow HSPCs throughout 2-6 d post-infection. Reduction of CXCL12/CXCR4 signaling simultaneously occurs with HSPC mobilization from bone marrow. Findings suggest that deranged CXCL12/CXCR4 signaling plays a causal role in HSPC mobilization during acute A. phagocytophilum infection.     

Rac GTPase isoform-specific regulation of NADPH oxidase and chemotaxis in murine neutrophils in vivo. Role of the C-terminal polybasic domain

The Rho family GTPase Rac acts as a molecular switch for signal transduction to regulate various cellular functions. Mice deficient in the hematopoietic-specific Rac2 isoform exhibit agonist-specific defects in neutrophil chemotaxis and superoxide production, despite expression of the highly homologous Rac1 isoform. To examine whether functional defects in rac2(-/-) neutrophils reflect effects of an overall decrease in total cellular Rac or an isoform-specific role for Rac2, retroviral vectors were used to express exogenous Rac1 or Rac2 at levels similar to endogenous. In rac2(-/-) neutrophils differentiated from transduced myeloid progenitors in vitro, increasing cellular Rac levels by expression of either exogenous Rac1 or Rac2 increased formylmethionylleucylphenylalanine- or phorbol ester-stimulated NADPH oxidase activity. Of note, placement of an epitope tag on the N terminus of Rac1 or Rac2 blunted reconstitution of responses in rac2(-/-) neutrophils. In rac2(-/-) neutrophils isolated from mice transplanted with Rac-transduced bone marrow cells, superoxide production and chemotaxis were fully reconstituted by expression of exogenous Rac2, but not Rac1. A chimeric Rac1 protein in which the Rac1 C-terminal polybasic domain, which contains six lysines or arginines, was replaced with that of the human Rac2 polybasic domain containing only three basic residues, also reconstituted superoxide production and chemotaxis, whereas expression of a Rac2 derivative in which the polybasic domain was replaced with that of Rac1 did not and resulted in disoriented cell motility. Thus, the composition of the polybasic domain is sufficient for determining Rac isoform specificity in the production of superoxide and chemotaxis in murine neutrophils in vivo.     

CXCR2 is essential for maximal neutrophil recruitment and methacholine responsiveness after ozone exposure

Ozone (O(3)), a common air pollutant, induces airway inflammation and airway hyperresponsiveness. In mice, the neutrophil chemokines KC and macrophage inflammatory protein-2 (MIP-2) are expressed in the lungs following O(3) exposure. The purpose of this study was to determine whether CXCR2, the receptor for these chemokines, is essential to O(3)-induced neutrophil recruitment, injury to lungs, and increases in respiratory system responsiveness to methacholine (MCh). O(3) exposure (1 ppm for 3 h) increased the number of neutrophils in the bronchoalveolar lavage fluid (BALF) of wild-type (BALB/c) and CXCR2-deficient mice. However, CXCR2-deficient mice had significantly fewer emigrated neutrophils than did wild-type mice. The numbers of neutrophils in the blood and concentrations of BALF KC and MIP-2 did not differ between genotypes. Together, these data suggest CXCR2 is essential for maximal chemokine-directed migration of neutrophils to the air spaces. In wild-type mice, O(3) exposure increased BALF epithelial cell numbers and total protein levels, two indirect measures of lung injury. In contrast, in CXCR2-deficient mice, the number of BALF epithelial cells was not increased by O(3) exposure. Responses to inhaled MCh were measured by whole body plethysmography using enhanced pause as the outcome indicator. O(3) exposure increased responses to inhaled MCh in both wild-type and CXCR2-deficient mice 3 h after O(3) exposure. However, at 24 h after exposure, responses to inhaled MCh were elevated in wild-type but not CXCR2-deficient mice. These results indicate CXCR2 is essential for maximal neutrophil recruitment, epithelial cell sloughing, and persistent increases in MCh responsiveness after an acute O(3) exposure.     

RhoG regulates the neutrophil NADPH oxidase

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG(-/-) neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG(-/-) neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.     

Rho signaling inhibition mitigates lung injury via targeting neutrophil recruitment and selectin-AKT signaling

Neutrophils, the early responders of the immune system, eliminate intruders, but their over-activation can also instigate tissue damage leading to various autoimmune and inflammatory disease conditions. As approaches causing neutropenia are associated with immunodeficiency, targeting aberrant neutrophil infiltration offers an attractive strategy in neutrophil-centered diseases including acute lung injury. Rho GTPase family proteins Rho, Rac and Cdc42 play important role as regulators of chemotaxis in diverse systems. Rho inhibitors protected against lung injuries, while genetic Rho-deficiency exhibited neutrophil hyperactivity and exacerbated lung injury. These differential outcomes might be due to distinct effects on different cell types or activation/ inhibition of specific signaling pathways responsible for neutrophil polarity, migration and functions. In this study, we explored neutrophil centric effects of Rho signaling mitigation. Consistent with previous reports, Rho signaling inhibitor Y-27632 provided protection against acute lung injury, but without regulating LPS mediated systemic increase of neutrophils in the circulation. Interestingly, the adoptive transfer approach identified a specific defect in neutrophil migration capacity after Rho signaling mitigation. These defects were associated with loss of polarity and altered actin dynamics identified using time-lapse in vitro studies. Further analysis revealed a rescue of stimulation-dependent L-selectin shedding on neutrophils with Rho signaling inhibitor. Surprisingly, functional blocking of L-selectin (CD62L) led to defective recruitment of neutrophils into inflamed lungs. Further, single-cell level analyses identified MAPK signaling as downstream mechanism of Rho signaling and L-selectin mediated effects. p-AKT levels were diminished in detergent resistance membrane-associated signalosome upon Rho signaling inhibition and blockade of selectin. Moreover, inhibition of AKT signaling as well as selectin blocking led to defects in neutrophil polarity. Together, this study identified Rho-dependent distinct L-selectin and AKT signaling mediated regulation of neutrophil recruitment to inflamed lung tissue.