Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.     

The Role of Phosphoinositide 3-Kinases in Neutrophil Migration in 3D Collagen Gels

The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.     

Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1

In human neutrophils, IL-8 induces chemotaxis, the respiratory burst, and granule release, and enhances cellular adhesion, a beta(2) integrin-dependent event. IL-8 stimulates neutrophil adhesion to purified fibrinogen in a Mac-1-dependent manner. Mitogen-activated protein kinase (MAPK) activation was detected in human neutrophil lysates after treatment with IL-8 and PMA, but not the activating mAb CBR LFA 1/2. IL-8-stimulated neutrophil adhesion to fibrinogen was blocked 50% by the MAPK/extracellular signal-related kinase-activating enzyme inhibitor PD098059. Adhesion was blocked approximately 75% by inhibition of the phosphatidylinositol-3 kinase (PI3K) pathway with LY294002, supporting that activation of both MAPK and PI3K may play a role in IL-8-dependent inside-out signals that activate Mac-1. Activation of MAPK was inhibited in IL-8-stimulated cells in the presence of PI3K inhibitors LY294002 or wortmannin, supporting a model in which PI3K is upstream of MAPK. IL-8-stimulated neutrophil adhesion was inhibited 50% by bisindolylmaleimide-I, implicating protein kinase C (PKC) in the intracellular signaling from the IL-8R to Mac-1. A 74-kDa molecular mass species was detected by an activation-specific Ab to PKC when cells were stimulated with PMA or IL-8, but not a beta(2)-activating Ab. Inhibition of either MAPK or PKC resulted in partial inhibition of IL-8-stimulated polymorphonuclear neutrophil adhesion, and treatment with both inhibitors simultaneously completely abolished IL-8-stimulated adhesion to ligand. Inhibition of PI3K blocked MAPK activation, but not PKC activation, suggesting a branch point that precedes PI3K activation. These data suggest that both MAPK and PKC are activated in response to IL-8 stimulation, and that these may represent independent pathways for beta(2) integrin activation in neutrophils.     

Collagen-binding motif peptide, a cleavage product of osteopontin, stimulates human neutrophil chemotaxis via pertussis toxin-sensitive G protein-mediated signaling

The collagen-binding motif (CBM) peptide, a cleavage product of osteopontin (OPN), stimulated intracellular calcium increase in human neutrophils. CBM peptide-stimulated calcium was inhibited by pertussis toxin (PTX), suggesting the influence of PTX-sensitive G-proteins. In addition CBM peptide stimulated the chemotactic migration of human neutrophils and human monocytes. CBM peptide-induced neutrophil chemotaxis was completely inhibited by PTX, once again indicating the influence of Gi proteins. CBM peptide was also found to induce mitogen activated protein kinase activation. CBM peptide-induced neutrophil chemotaxis was mediated by p38 kinase as well as an extracellular signal-regulated protein kinase. Taken together, the results suggest that a cleavage product of OPN, CBM peptide, initiates immune responses by inducing neutrophil trafficking via certain PTX-sensitive cell surface receptors.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

The major outer sheath protein of Treponema denticola selectively inhibits Rac1 activation in murine neutrophils

Treponema denticola major outer sheath protein (Msp) inhibits neutrophil chemotaxis in vitro , but key regulatory mechanisms have not been identified. Because the Rac small GTPases regulate directional migration in response to chemoattractants, the objective was to analyse the effects of Msp on formyl -methionyl-leucyl-phenylalanine (fMLP)-mediated neutrophil polarization and Rac activation in murine neutrophils. Msp pretreatment of neutrophils inhibited both polarization and chemotactic migration in response to fMLP. Activation of small GTPases was measured by p21 binding domain (PBD) pulldown assays, followed by Western analysis, using monoclonal anti-Rac1, anti-Rac2, anti-cdc42 and anti-RhoA antibodies. Enriched native Msp selectively inhibited fMLP-stimulated Rac1 activation in a concentration-dependent manner, but did not affect Rac2, cdc42 or RhoA activation. Murine neutrophils transfected with vectors expressing fluorescent probes PAK-PBD-YFP and PH-AKT-RFP were used to determine the effects of Msp on the localization of activated Rac and PI3 kinase products. Real-time confocal images showed that Msp inhibited the polarized accumulation of activated Rac and PI3-kinase products upon exposure to fMLP. The findings indicate that T. denticola Msp inhibition of neutrophil polarity may be due to the selective suppression of the Rac1 pathway.     

Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase,protein kinase C-delta,and NF-kappa B signaling pathways

Neutrophils are abundant, short-lived leukocytes with a key role in the defense against rapidly dividing bacteria. They enter apoptosis spontaneously within 24-48 h of leaving the bone marrow. However, their life span can be extended during inflammatory responses by several proinflammatory cytokines. Inappropriate survival of neutrophils contributes to chronic inflammation and tissue damage associated with diseases such as rheumatoid arthritis. We have previously reported that type I IFNs can inhibit both cytokine deprivation and Fas-induced apoptosis in activated T cells. IFN-beta locally produced by hyperplastic fibroblasts within the pannus tissue of patients with rheumatoid arthritis contributes to the inappropriately extended life span of infiltrating T cells. Type I IFNs are equally effective at delaying spontaneous apoptosis in human neutrophils. In the work presented here we investigated the signaling pathways involved in mediating this effect. The antiapoptotic actions of IFN-beta were targeted at an early stage of neutrophil apoptosis, occurring upstream of mitochondrial permeability transition, and were phosphatidylinositol 3-kinase (PI3K) dependent, as they were blocked by the PI3K inhibitor LY294002. Analysis of signaling pathways downstream of PI3K revealed that the antiapoptotic effect of type 1 IFN was inhibited by rottlerin, SN50, and cycloheximide, indicating requirements for activation of protein kinase C-delta, NF-kappaB, and de novo protein synthesis, respectively. Moreover, EMSA was used to show that the activation of NF-kappaB occurred downstream of PI3K and protein kinase C-delta activation. We conclude that type I IFNs inhibit neutrophil apoptosis in a PI3K-dependent manner, which requires activation of protein kinase C-delta and induction of NF-kappaB-regulated genes.     

Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. II. Effects of LFM-A13, a specific Btk inhibitor

Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils. LFM-A13 decreased the tyrosine phosphorylation induced by fMet-Leu-Phe and inhibited the production of superoxide anions and the stimulation of adhesion, chemotaxis, and phospholipase D activity. We observed a decreased accumulation of phosphatidylinositol-3,4,5-trisphosphate in response to fMet-Leu-Phe in LFM-A13-pretreated cells even though the inhibitor had no direct effect on the lipid kinase activity of the p110 gamma or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110 gamma by fMet-Leu-Phe. The phosphorylation of Akt and of extracellular signal-regulated kinases 1/2 and p38 were similarly inhibited by LFM-A13. LFM-A13 also negatively affected the translocation of Rac-2, RhoA, ADP ribosylation factor-1, Tec, Bmx, and Btk induced by fMet-Leu-Phe. The results of this study provide evidence for an involvement of Btk and possibly other Tec kinase family members in the regulation of the functional responsiveness of human neutrophils and link these events, in part at least, to the modulation of levels of phosphatidylinositol-3,4,5-trisphosphate.     

The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR.

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of beta-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O(2)(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O(2)(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O(2)(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I(A) PI3K, but not translocation of the p110gamma subunit of class I(B) PI3K, to the neutrophil membrane. Inhibition of protein kinase Czeta (PKCzeta) inhibited MBP-stimulated O(2)(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta (Thr(505)) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I(A) PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I(A) PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O(2)(-) production in response to MBP stimulation.     

Exogenous nitric oxide elicits chemotaxis of neutrophils in vitro

Nitric oxide (NO) has been shown to be both an intercellular and intracellular messenger. We propose here that exogenous NO induces chemotactic locomotion of human neutrophils. Indeed, when human neutrophils were placed in a gradient of a nitric oxide donor (S-nitroso-N-acetylpenicillamine; SNAP), a directed locomotion was induced, as evidenced by experiments of chemotaxis under agarose. Degraded SNAP (i.e., SNAP solution which had previously released NO) did not induce directed locomotion. Moreover, oxyhemoglobin, a scavenger of free NO, suppressed the chemotactic effect of SNAP, whereas LY-83583, a soluble guanylate cyclase inhibitor, inhibited the SNAP-mediated chemotaxis in a dose-response manner. Other unrelated NO donors, SIN-1 and S-nitroso-cysteine—a natural S-nitroso-compound, also induced a directed locomotion of neutrophils. Taken together, these in vitro experiments indicate that exogenous NO could mediate the chemotaxis of neutrophils and thus suggest that NO could contribute to neutrophil recruitment in vivo. © 1995 Wiley-Liss Inc.     

Receptor for advanced glycation end-products (RAGE) modulates neutrophil adhesion and migration on glycoxidated extracellular matrix

AGEs (advanced glycation end-products) accumulate in collagen molecules during uraemia and diabetes, two diseases associated with high susceptibility to bacterial infection. Because neutrophils bind to collagen during their locomotion in extravascular tissue towards the infected area we investigated whether glycoxidation of collagen (AGE-collagen) alters neutrophil migration. Type I collagen extracted from rat tail tendons was used for in vitro glycoxidation (AGE-collagen). Neutrophils were obtained from peripheral blood of healthy adult volunteers and were used for the in vitro study of adhesion and migration on AGE- or control collagen. Glycoxidation of collagen increased adhesion of neutrophils to collagen surfaces. Neutrophil adhesion to AGE-collagen was inhibited by a rabbit anti-RAGE (receptor for AGEs) antibody and by PI3K (phosphoinositide 3-kinase) inhibitors. No effect was observed with ERK (extracellular-signal-regulated kinase) or p38 MAPK (mitogen-activated protein kinase) inhibitors. AGE-collagen was able to: (i) induce PI3K activation in neutrophils, and (ii) inhibit chemotaxis and chemokinesis of chemoattractant-stimulated neutrophils. Finally, we found that blocking RAGE with anti-RAGE antibodies or inhibiting PI3K with PI3K inhibitors restored fMLP (N-formylmethionyl-leucyl-phenylalanine)-induced neutrophil migration on AGE-collagen. These results show that RAGE and PI3K modulate adhesion and migration rate of neutrophils on AGE-collagen. Modulation of adhesiveness may account for the change in neutrophil migration rate on AGE-collagen. As neutrophils rely on their ability to move to perform their function as the first line of defence against bacterial invasion, glycoxidation of collagen may participate in the suppression of normal host defence in patients with diabetes and uraemia.     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.     

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.     

Effects of Jarastatin, a Novel Snake Venom Disintegrin, on Neutrophil Migration and Actin Cytoskeleton Dynamics

A new disintegrin, an RGD-containing peptide of 6 kDa called jarastatin, was purified from Bothrops jararaca venom. It is a potent inhibitor of platelet aggregation induced by ADP, collagen, and thrombin. The effect of jarastatin on neutrophil migration in vivo and in vitro and on the actin cytoskeleton dynamics of these cells was investigated. Incubation in vitro with jarastatin significantly inhibited, in a concentration-dependent manner, the chemotaxis of human neutrophils toward fMLP, IL-8, and jarastatin itself. Despite this inhibitory effect, jarastatin induced neutrophil chemotaxis. A significant increase of F-actin content was observed in jarastatin-treated neutrophils. Furthermore, as demonstrated by confocal microscopy after FITC-phalloidin labeling, these cells accumulated F-actin at the plasmalemma, a distribution similar to that observed in fMLP-stimulated cells. Pretreatment of mice with jarastatin inhibited neutrophil migration into peritoneal cavities induced by carrageenan injection. The results suggest that binding of jarastatin to neutrophil integrins promotes cellular activation and triggers a dynamic alteration of the actin filament system and that this is one of the first event in integrin-mediated signaling.