Generation of biologically active C-reactive protein peptides by a neutral protease on the membrane of phorbol myristate acetate-stimulated neutrophils

The association of human C-reactive protein (CRP) with nonstimulated and PMA-stimulated human neutrophils and the concomitant degradation of CRP (monitored by TCA-soluble peptides and SDS-PAGE analysis) has been studied. Maximum association of 125I-labeled CRP with neutrophils and 125I-labeled CRP degradation during association with these cells was achieved by stimulating the neutrophils with PMA at 10 ng/ml; a concentration in which azurophil granule release was not significant. For PMA-stimulated neutrophils, the association of 125I-labeled CRP was 1.8 times higher and PMA-stimulated neutrophil-mediated degradation of the ligand was three times faster than that for nonstimulated cells. The neutrophil-associated 125I-labeled CRP in the absence and presence of PMA proved on SDS-PAGE analysis to be approximately 50% degraded. There was a positive correlation between the extent of CRP degradation and the association of 125I-labeled CRP with neutrophils. In addition to generation of neutrophil associated CRP intermediates, small soluble CRP peptides were generated during association of CRP with neutrophils. These peptides inhibited superoxide production from opsonized zymosan-activated neutrophils by approximately 40% at 10 micrograms/ml. 125I-labeled CRP degradation mediated by nonstimulated neutrophils, and neutrophil-conditioned medium (from both non-stimulated and PMA-stimulated cells) was inhibitable by alpha 1-antitrypsin and approximately seven times less at 1 h than that occurring during 125I-labeled CRP-association with PMA-stimulated neutrophils. The degradation of 125I-labeled CRP mediated by PMA-stimulated neutrophils was not fully inhibitable by alpha 1-antitrypsin. The data point to the involvement of a membrane-associated serine protease, which is maximally activated by PMA, in the degradation of 125I-labeled CRP during association with neutrophils. Our results indicate that at an inflammatory site CRP-derived peptides can be produced that inhibit the action of activated neutrophils.     

C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement.

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.     

Human neutrophil defensins increase neutrophil uptake of influenza A virus and bacteria and modify virus-induced respiratory burst responses

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.     

The role of the neutrophil in inflammatory diseases of the lung

Under certain circumstances, the neutrophil has been implicated in causing disease by damaging normal host tissue. This may occur in the adult respiratory distress syndrome (ARDS). The neutrophil has been implicated since a) substances that activate neutrophils are produced in association with the predisposing risks that lead to ARDS; b) activated neutrophils migrate into the alveolar spaces and their toxic products can be found in lung lavage fluid and in the breath of patients with ARDS; and c) the magnitude of the physiologic alterations correlate with the number of neutrophils in the alveolar space. Additionally, the neutrophils may be primed by substances which are released by activated platelets within the confines of the lung. Both platelet adenine nucleotides and the platelet-derived extracellular matrix protein (ECM), thrombospondin, can prime the neutrophil for subsequent O2- generation following activation of the cells with the chemotactic peptide, F-met-leu-phe (FMLP). Furthermore, neutrophils can be primed or O2- generation by the basement membrane ECM protein, laminin. Since neutrophils express receptors for both laminin and thrombospondin, these constituents may serve to modulate neutrophil behavior for subsequent oxidative metabolism and contribute to exacerbating pulmonary disease.     

Modulation of human neutrophil function by C-reactive protein

Investigation of the effect of C-reactive protein (CRP), an acute-phase reactant, on the functional capacities of human neutrophils was carried out as the basis for elucidating the biological function of C-reactive protein. An initial stimulation at low concentrations, followed by inhibition of superoxide production, and secretion of vitamin-B12-binding protein in the presence of two stimulants, phorbol myristate acetate and concanavalin A, and of neutrophil chemotaxis with increasing concentration of CRP was observed. Correlation between modulation of cell function, at least at relatively high CRP concentrations (greater than 50 micrograms/ml) and an increase in the intracellular level of cAMP is suggested. CRP was also found to enhance neutrophil phagocytosis of particles not containing phosphorylcholine, the native CRP ligand. The proposed role of CRP in neutrophil function is one of regulation and as a negative feedback for potential cytotoxic neutrophil functions.     

Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta

Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.     

Dihydroxyoctadecamonoenoate esters inhibit the neutrophil respiratory burst

The leukotoxins [9(10)-and 12(13)-EpOME] are produced by activated inflammatory leukocytes such as neutrophils. High EpOME levels are observed in disorders such as acute respiratory distress syndrome and in patients with extensive burns. Although the physiological significance of the EpOMEs remains poorly understood, in some systems, the EpOMEs act as a protoxin, with their corresponding epoxide hydrolase metabolites, 9,10-and 12,13-DiHOME, specifically exerting toxicity. Both the EpOMEs and the DiHOMEs were also recently shown to have neutrophil chemotactic activity. We evaluated whether the neutrophil respiratory burst, a surge of oxidant production thought to play an important role in limiting certain bacterial and fungal infections, is modulated by members of the EpOME metabolic pathway. We present evidence that the DiHOMEs suppress the neutrophil respiratory burst by a mechanism distinct from that of respiratory burst inhibitors such as cyclosporin H or lipoxin A4, which inhibit multiple aspects of neutrophil activation.     

Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases

The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose (Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and hydroxyl radical generated by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H2O2 induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOCl induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H2O2-induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOCl to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation pathway of HNC involving hydroxyl radical.     

Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin

Chemoattractants differ in their capacity to stimulate neutrophils to adhere to and to migrate through matrices containing fibrin. Formyl methionyl leucyl phenylalanine (fMLP) stimulates neutrophils to adhere closely to, but not to migrate into, fibrin gels. Leukotriene B4 (LTB4) stimulates neutrophils to adhere loosely to and to migrate through fibrin gels. We report that alpha5beta1 integrins regulate the different migratory behaviors on fibrin gels of neutrophils in response to these chemoattractants. fMLP, but not LTB4, activated neutrophil beta1 integrins, as measured by binding of mAb 15/7 to an activation epitope on the beta1 integrins. Antibodies or peptides that block alpha5beta1 integrins prevented fMLP-stimulated neutrophils from forming zones of close apposition on fibrin and reversed fMLP's inhibitory effect on neutrophil chemotaxis through fibrin. In contrast, neither peptides nor antibodies that block beta1 integrins affected the capacity of LTB4-stimulated neutrophils to form zones of loose apposition or to migrate through fibrin gels. These results suggest that chemoattractants generate at least two different messages that direct neutrophils, and perhaps other leukocytes, to accumulate at specific anatomic sites: a general message that induces neutrophils to crawl and a specific message that prepares neutrophils to stop when they contact appropriate matrix proteins for activated beta1 integrins.     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins     

Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death

Neutrophil interaction with activated endothelial cells (EC) is required for transmigration. We examined consequences of this interaction on NETosis. Co-culture of activated EC with neutrophils induced neutrophil extracellular trap (NET) formation, which was partially dependent on production of IL-8 by activated EC. Extended neutophil/EC co-culture resulted in EC damage, which could be abrogated by inclusion of either diphenyleneiodonium to inhibit the NAPDH oxidase pathway required for NETosis, or DNAse to disrupt NETs. These findings offer new insight into mechanisms whereby NETs trigger damage to the endothelium in sepsis, small vessel vasculitis and possibly the villous trophoblast in preeclampsia.     

Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation

We attempted to apply the particle counting method that employs laser-light scattering technique to quantify the change in numbers of neutrophil homotypic aggregates of 3 graded-sizes (small, medium and large). Ex vivo activation of human neutrophils by a chemotactic peptide, fMLP, predominantly produced small-sized aggregates (< 15 cells), and also, transiently, medium-sized aggregates (16-130 cells). Co-treatment of neutrophils with fMLP and cytochalasin B mainly produced medium-sized aggregates, with very few large-sized aggregates (> 130 cells). Interestingly, when protein kinase C was activated with phorbol 12-myristate 13-acetate small-, medium- and even large-sized aggregates of neutrophils were formed. Presence of extracellular calcium was required to produce these neutrophil aggregations. Both prostaglandin E2 (PGE2) and wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI-3K), inhibited neutrophil aggregation, whereas dbcAMP, a cell permeable analog of cyclic AMP, did not, confirming that PGE2 causes neutrophil aggregation probably through PI-3K inhibition rather than activation of adenylate cyclase. These results suggest that the application of the light scattering technique to characterize human neutrophil aggregates by both size and numbers, has advantages over conventional optical turbulent aggregometry, in that it discriminates neutrophil aggregations produced by different mechanisms of intracellular signaling.     

Pharmacologic characterization of the rabbit neutrophil receptor for platelet-activating factor

The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd) for PAF of 0.41 +/- 0.045 nM and a Bmax of 0.32 +/- 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 micrograms of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652-731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 +/- 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 +/- 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.     

Inflammatory response and neutrophil functions in players after a futsal match

Inflammatory response and neutrophil functions in players after a futsal match. J Strength Cond Res 26(9): 2507-2514, 2012-Futsal players suffer injuries resulting from muscle fatigue and contact or collision among players. Muscle lesions can be detected by measuring muscle lesion markers such as creatine kinase (CK) and lactate dehydrogenase (LDH) in plasma. After an initial lesion, there is an increase in the plasma levels of C-reactive protein (CRP) and proinflammatory cytokines. These mediators may activate neutrophils and contribute to tissue damage and increase susceptibility to invasive microorganisms. In this study, we investigated the effect of a futsal match on muscle lesion markers, cytokines, and CRP in elite players. The basal and stimulated neutrophil responsiveness after a match was also evaluated based on measurements of neutrophil necrosis, apoptosis, phagocytic capacity, reactive oxygen species (ROS) production, and cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-8, IL-1β, IL-10, and IL-1ra) production. Blood samples were taken from 16 players (26.4 ± 3.2 years, 70.2 ± 6.9 kg, 59.7 ± 5.1 ml·kg·min, sports experience of 4.4 ± 0.9 years) before and immediately after a match. Exercise increased the serum activities of CK (2.5-fold) and LDH (1.3-fold). Playing futsal also increased the serum concentrations of IL-6 (1.6-fold) and CRP (1.6-fold). The TNF-α, IL-1β, IL-8, IL-1ra, and IL-10 serum levels were not modified in the conditions studied. The futsal match induced neutrophil apoptosis, as indicated by phosphatidylserine externalization (6.0-fold). The exercise induced priming of neutrophils by increasing ROS (1.3-fold), TNF-α (5.8-fold), and IL-1β (4.8-fold) released in nonstimulated cells. However, in the stimulated condition, the exercise decreased neutrophil function, diminishing the release of ROS by phorbol myristate acetate-stimulated neutrophils (1.5-fold), and the phagocytic capacity (1.6-fold). We concluded that playing futsal induces inflammation, primes and activates neutrophils, and reduces the efficiency of neutrophil phagocytosis immediately after a match.     

Influence of opioid peptides on human neutrophil apoptosis and activation in vitro

BACKGROUND: It has been shown that cells of the immune system release opioid peptides and possess receptors for them. The concentrations of opioid peptides in the peripheral circulation rapidly increase during inflammation and acute stress response. AIMS: The effect of opioid peptides Met-enkephalin (M-ENK) and beta-endorphin (beta-END) on the oxidative metabolism of normal human neutrophils and their death by apoptosis in vitro was investigated. METHODS: Isolated from peripheral blood, neutrophils were incubated in the presence or absence of 10(-6) to 10(-10) M of M-ENK and beta-END for 12 and 18 h. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V-FITC protein binding to the cell surface. The MTT-reduction assay was employed to estimate the oxidative metabolism of neutrophils. RESULTS: Treatment with M-ENK caused a significant increase in apoptotic cells after 18 h of culture: *0 M (control) versus 10(-10) M, p < or = 0.02; **10(-10) M versus 10(-10) M, p < or = 0.02. Treatment with beta-END caused a significant increase in apoptotic cells after 12 h of culture: 0 M versus 10(-8) M, p < or = 0.03; **0 M versus 10(-10) M, p < or = 0.04. We found the significant increase in MTT reduction by neutrophils in the presence of M-ENK and beta-END both before and after the culture. However, the ability of neutrophils to reduce the MTT salt to formazan decreased significantly after the culture. CONCLUSIONS: We observed that the in vitro effect of opioid peptides on the neutrophil survival and their functional state was time and dose dependent. The presence of antioxidants in the culture medium modifies neutrophil survival.     

A cell attachment peptide from human C-reactive protein.

The serum acute phase reactant, C-reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27-38 present in each of the five identical subunits of CRP mediated cell attachment activity in vitro. Although the CRP-derived peptide contains a Tuftsin (TKPR)-like sequence at its amino-terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell-attachment activity to the CRP-derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP-derived peptide cell attachment activity. However, the addition of the RGDS and RGDSPASSLP cell-binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell-binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P-component (SAP), displayed nearly identical cell-attachment activity. Several monoclonal antibodies (mAb) specific for the CRP-derived cell-binding peptide neutralized its cell-attachment activity. These mAbs reacted with intact CRP and neutralized the cell-binding activity of CRP itself. The findings suggest that a peptide with cell-binding activity could be generated from the breakdown of CRP and then contribute directly to cellular events leading to tissue repair.     

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses

There is renewed interest in the use of maggots (Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H(2)O(2) production by fMLP-activated neutrophils; maximal inhibition was seen with 5-50 microg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 microg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca(2+)](i) in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.     

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.     

Adiponectin inhibits neutrophil apoptosis via activation of AMP kinase, PKB and ERK 1/2 MAP kinase

Neutrophils are abundant, short-lived leukocytes that play a key role in the immune defense against microbial infections. These cells die by apoptosis following activation and uptake of microbes and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Adiponectin exerts anti-inflammatory effects on neutrophil antimicrobial functions, but whether this abundant adipokine influences neutrophil apoptosis is unknown. Here we report that adiponectin in the physiological range (1–10 μg/ml) reduced apoptosis in resting neutrophils, decreasing caspase-3 cleavage and maintaining Mcl-1 expression by stabilizing this anti-apoptotic protein. We show that adiponectin induced phosphorylation of AMP-activated kinase (AMPK), protein kinase B (PKB), extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen activated protein kinase (MAPK). Pharmacological inhibition of AMPK, PKB and ERK 1/2 ablated the pro-survival effects of adiponectin and treatment of neutrophils with an AMPK specific activator (AICAR) and AMPK inhibitor (compound C) respectively decreased and increased apoptosis. Finally, activation of AMPK by AICAR or adiponectin also decreased ceramide accumulation in the neutrophil cell membrane, a process involved in the early stages of spontaneous apoptosis, giving another possible mechanism downstream of AMPK activation for the inhibition of neutrophil apoptosis.     

NADPH-oxidase activation in murine neutrophils via formyl peptide receptors

Neutrophils play a key role at inflammatory sites where, in addition to destroying infecting microorganisms, they may also have deleterious effects on host tissues. Both activities involve activation of the NADPH-oxidase that produces bactericidal and tissue-destructive reactive oxygen species (ROS). We activated the murine NADPH-oxidase using different types of neutrophil activators and characterized the oxidative responses with respect to magnitude, localization, and kinetics. We show that agonist-induced activation of murine neutrophils results exclusively in extracellular release of ROS and no intracellular production could be detected. We also show that the formylated peptide, formyl-Met-Leu-Phe (fMLF), is a much less potent activator of the murine NADPH-oxidase than of the human analogue. Nevertheless, fMLF responses can be primed by pretreating the murine neutrophils with either cytochalasin B or bacterial lipopolysaccharide. Finally, we show that a synthetic hexapeptide, WKYMVM, is a more potent stimulus than fMLF for murine neutrophils and that these two agonists probably act via nonidentical high-affinity receptors.