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ABSTRACT: We shed new light on the expression and function of the proteinase-activated receptor (PAR) family, associated with inflammation and hyperalgesia, in human granulocytes. Resting cells expressed constitutive levels of PAR-2 and PAR-3 mRNA but not PAR-1 or PAR-4. Based on flow cytometry, stimulation with opsonized bacteria (Bop) specifically up-regulated cell surface expression of PAR-2 in a concentration-dependent and time-dependent manner, independent of transcription or de novo protein synthesis. Primary granules were identified as a source of preformed PAR-2 that can readily be mobilized at the surface on fusion with the plasma membrane. Cellular response to PAR-2 activation, measured as changes in intracellular calcium concentration, was enhanced in PAR-2 up-regulated cells. Increase of cell-surface PAR-2 and of cell responsiveness were dependent specifically on the engagement of immunoglobulin (Ig)-binding receptors. Together, our results reveal that mobilization of intracellular granules, in response to Ig-receptor activation, up-regulates PAR-2 surface expression and makes neutrophils more responsive to proteinase activity. This enhanced response to PAR-2 activation indicates that molecular communication between pain and inflammation may be more important than previously believed.
The FASEB Journal 02/2010; 24(6):2116-25. · 5.71 Impact Factor
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ABSTRACT: The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine-phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals. The tyrosine-phosphorylation patterns induced by MSU crystals and by the ligation of CD16 were inhibited by piceatannol, which, conversely, only slightly delayed but did not diminish the peak of tyrosine phosphorylation induced by cross-linking CD32 or by the addition of fMet-Leu-Phe. Moreover, piceatannol inhibited the activity of Syk as monitored by in vitro kinase assays, by its in situ tyrosine phosphorylation, and by its activity toward exogenous substrates after stimulation by MSU crystals. We also measured the impact of piceatannol on the mobilization of calcium, the production of superoxide anions, and the activity of PLD stimulated by MSU crystals. We noted a distinct inhibition of all these responses by piceatannol. Finally, the morphological changes observed in neutrophils as characteristic of MSU crystal internalization were diminished significantly by piceatannol. The results obtained show that Syk plays a critical and central role in the signal-transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.
Journal of Leukocyte Biology 11/2001; 70(4):659-68. · 4.99 Impact Factor
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ABSTRACT: SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G(1) to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown. The present study was initiated to examine the role of SAM68 in the phagocytic responses of the terminally differentiated human neutrophils. The results obtained show that SAM68 is present in human neutrophils and that it is tyrosine phosphorylated in response to stimulation by monosodium urate crystals or by ligation of CD32. Stimulation of neutrophils by these agonists decreases the association of SAM68 with Sepharose-conjugated poly-U beads. Additionally, the amount of immunoprecipitable SAM68 was modulated differentially after stimulation by monosodium urate crystals or by CD32 engagement indicating that the posttranslational modifications and/or protein associations of SAM68 induced by these two agonists differed. The results of this study provide evidence for an involvement of SAM68 in signal transduction by phagocytic agonists in human neutrophils and indicate that SAM68 may play a role in linking the early events of signal transduction to the posttranscriptional modulation of RNA.
The Journal of Immunology 05/2001; 166(7):4664-71. · 5.79 Impact Factor
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ABSTRACT: ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D (PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GFP-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the ability of cytohesin-1 mAbs to immunoprecipitate cytohesin-1. Among the hybridomas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-1. Furthermore, mAb 2E11 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditions. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected both cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not GFP-cytohesin-1 under native conditions. Another antibody, mAb 10A12, also selectively immunoprecipitated GFP-ARNO under native conditions, but the epitope recognized by this mAb is unlikely to be linear as no signal was obtained by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal antibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin-1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in levels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulocytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be useful as a potential marker for granulocytic differentiation.
Journal of Immunological Methods 04/2001; 249(1-2):121-36. · 2.20 Impact Factor
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ABSTRACT: The activation of human polymorphonuclear neutrophil leukocytes (neutrophils) is associated with an increased synthesis of the highly phosphorylated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)). The aims of the present investigation were to determine whether the newly described, G protein-dependent phosphatidylinositol 3-kinase (PI3K), p110gamma, was involved in the responses to chemotactic factors interacting with G protein-coupled receptors. The presence of p110gamma in neutrophils was first established both at the protein and the mRNA level. Stimulation of the cells with fMet-Leu-Phe or interleukin-8 increased the PI3K activity in p110gamma, but not p85, immunoprecipitates. The time course of this effect (threshold within less than 5 s, maximal activation at 10-15 s) was consistent with that of the generation of PtdIns(3,4,5)P(3). Wortmannin, a PI3K inhibitor, abrogated the effects of fMet-Leu-Phe, which were also significantly inhibited by pertussis toxin. Finally, fMet-Leu-Phe also induced a significant translocation of p110gamma to a particulate fraction derived from these cells. These data indicate that p110gamma represent the major PI3K activated by fMet-Leu-Phe and interleukin-8 at very early time points following the stimulation of human neutrophils.
Journal of Biological Chemistry 09/2000; 275(31):23636-41. · 4.77 Impact Factor
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ABSTRACT: Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)-induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase Calpha (PKCalpha), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKCalpha, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKCalpha through Ado A2a receptor occupancy. (Blood. 2000;95:519-527)
Blood 02/2000; 95(2):519-27. · 9.90 Impact Factor
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ABSTRACT: The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10-6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubation (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered. Tyrosine phosphorylation of Cbl in response to CD32 cross-linking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphorylation on serine/threonine residues. However, tyrosine phosphorylation of Syk and of the receptor itself were not affected. Furthermore, the mobilization of intracellular calcium stimulated by CD32 cross-linking was totally abrogated by calyculin. Finally, the stimulation of superoxide production observed in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events regulate the signaling pathways activated by CD32 cross-linking in neutrophils and identify a novel mechanism of modulation of the functional responsiveness of human neutrophils to CD32 cross-linking.
The Journal of Immunology 01/2000; 164(2):1020-8. · 5.79 Impact Factor
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ABSTRACT: We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V4+-OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase Calpha (PKCalpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKCalpha translocation. Three tyrosine kinases inhibitors, ST-638, methyl 2,5-dihydroxycinnamate, and genistein reduced fMLP-stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP-induced increase of GTPgammaS-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKCalpha to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKCalpha in the signaling pathway leading to PLD activation by fMLP. RO 31-8220, a specific inhibitor of PKC, reduced PMA-induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP-stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31-8220 the levels of membrane-bound RhoA were increased. The levels of membrane-bound ARF and PKCalpha were unaffected by RO 31-8220 during PMA stimulation. In contrast, fMLP-induced recruitment of ARF and RhoA was insensitive to RO 31-8220 but PKCalpha translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATP-independent manner. Furthermore, increased fMLP-induced PKCalpha translocation in the presence of RO 31-8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31-8220 are used together in intact HL60 cells.
Journal of Leukocyte Biology 12/1999; 66(6):1021-30. · 4.99 Impact Factor
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ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates many of the biological activities of human neutrophils. The signaling pathways via which these effects are mediated are not fully understood. We have shown previously that GM-CSF treatment of human neutrophils activates the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway and, more specifically, Jak2, STAT3, and STAT5B in neutrophils. GM-CSF also stimulates the activity of the phosphatidylinositol 3-kinase (PI3-kinase) in a tyrosine kinase-dependent manner. Here we report that pretreating the cells with a Jak2 inhibitor (AG-490) abolishes tyrosine phosphorylation of the p85 subunit of PI3-kinase induced by GM-CSF. Furthermore, p85 was found to associate with Jak2, but not with Lyn, in stimulated cells in situ and with its autophosphorylated form in vitro; however, Jak2 did not bind to either of the two Src homology 2 (SH2) domains of the p85 subunit of PI3-kinase. Although STAT5B bound to the carboxyl-terminal SH2 domain of p85, it was absent from the complex containing PI3-kinase and Jak2. These results suggest that stimulation of the activity of PI3-kinase induced by GM-CSF is mediated by Jak2 and that the association between Jak2 and p85 depends on an adaptor protein yet to be identified.
Journal of Biological Chemistry 03/1999; 274(9):5333-8. · 4.77 Impact Factor
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ABSTRACT: Protein kinase Calpha (PKCalpha) and small GTPases of the Rho and ADP-ribosylation factor (Arf) family are implicated in the regulation of phospholipase D1 (PLD1) activity. Although they are involved in fMet-Leu-Phe (fMLP)-mediated PLD activation, their role in monosodium urate (MSU)-stimulated PLD1 activity in human neutrophils is not clear. The translocation of PKCalpha, RhoA and Arf from the cytosol to the membranes was monitored. fMLP induced a cytochalasin B (CB)-dependent recruitment of Arf, RhoA and PKCalpha to neutrophil membranes. CB also increased the activation of PLD 10-fold. In contrast with fMLP, MSU stimulated a sustained and time-dependent relocalization of Arf and PKCalpha, but not of RhoA, to the membrane fraction. MSU-stimulated PLD was activated with a time course preceding membrane recruitment of Arf and PKCalpha in the absence of CB. Furthermore, MSU-induced PLD activation and the membrane recruitment of PKCalpha, but not that of Arf, were inhibited by CB. An anti-FcgammaRIIIB antibody, VIFcRIII, prevented the membrane relocalization of Arf and PKCalpha and the stimulation of the levels of tyrosine phosphorylation and of PLD activity induced by MSU. Erbstatin and ST-638, two inhibitors of tyrosine kinases, inhibited the MSU-induced translocation of Arf and PKCalpha but not MSU-induced tyrosine phosphorylation and PLD activation. Furthermore MSU crystals did not cause the tyrosine phosphorylation of PLD1. The present study indicates that soluble and particulate agonists show selectivity in inducing the translocation of RhoA in neutrophils and that the ability of MSU to increase PLD activation was independent of the membrane relocalization of Arf and PKCalpha.
Biochemical Journal 02/1999; 337 ( Pt 2):185-92. · 4.90 Impact Factor
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ABSTRACT: Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX-2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM-CSF, TNF-alpha, and fMLP, induced COX-2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX-2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX-2 protein expression was initiated. In LPS-treated neutrophils, efficient induction of COX-2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX-2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX-2, raising the possibility that phospholipase D activation might take part in the process of COX-2 induction. Major COX-2-derived prostanoids synthesized by inflammatory neutrophils were identified by liquid-chromatography and tandem mass-spectrometry as TXA2 and PGE2. The agonist-induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX-2 inhibitor NS-398. These results show that COX-2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX-2 isoform is preeminent over COX-1 for the stimulated-production of prostanoids, and also suggest that neutrophil COX-2 displays a distinct profile of expression among circulatory cells.
The FASEB Journal 10/1998; 12(12):1109-23. · 5.71 Impact Factor
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ABSTRACT: Superantigens such as staphylococcal enterotoxin A and B (SEA and SEB) activate the immune system by stimulating a large proportion of T lymphocytes through specific Vbeta regions of the TCR and activating macrophages by binding to MHC class II molecules. While the mechanisms by which superantigens activate T lymphocytes have been elucidated, their role in the generation of local immune responses to bacterial invasion is still unclear. In this study we have examined the ability of the superantigens SEA and SEB to elicit an inflammatory reaction in vivo, in s.c. air pouches in the mouse. Upon injection into the s.c. air pouch, the two superantigens stimulated a time-dependent increase in the number of leukocytes appearing in the pouch exudate. The leukocytes migrating into the pouch exudate were predominantly neutrophils, with some mononuclear phagocytes and eosinophils present. No T lymphocytes were detected either in the pouch lining tissue or in the exudate cells. Injection of SEA resulted in increased ICAM-1 expression, as detected by immunohistochemistry, on endothelial cells in the tissue surrounding the air pouch and accumulation of TNF-alpha and the chemokines macrophage inflammatory protein-2 (MIP-2), MIP-1alpha, and JE in the pouch exudate. In addition, pretreatment of mice with Abs raised against ICAM-1, TNF-alpha, MIP-2, MIP-1alpha, KC, or JE inhibited leukocyte accumulation induced by SEA. These data demonstrate that bacterial superantigens may promote inflammation at extravascular sites in vivo, and that this response is secondary to the generation of inflammatory mediators, including chemokines.
The Journal of Immunology 09/1998; 161(3):1204-11. · 5.79 Impact Factor
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ABSTRACT: The formation and deposition of monosodium urate (MSU) microcrystals in articular and periarticular tissues is the causative agent of acute or chronic inflammatory responses known as gouty arthritis. Mononuclear phagocyte activation is involved in early triggering events of gout attacks. Because stimulated mononuclear phagocytes can constitute an important source of the inducible isoform of cyclooxygenase (COX-2), we evaluated the effects that proinflammatory microcrystals might have on COX-2 protein expression in crystal-stimulated monocytes. We found that MSU crystals, but not calcium pyrophosphate dihydrate (CPPD) crystals, induced COX-2, which correlated with the synthesis of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2). Crystal-induced de novo synthesis of COX-2 was dependent on transcriptional and translational events. Inhibition of tyrosine phosphorylation, by herbimycin A, blocked crystal-induced COX-2. Similarly, an inhibitor of the p38 mitogen-activated protein kinase, SB 203580, inhibited the stimulation of COX-2. Colchicine inhibited crystal-induced COX-2. In all cases, prostanoid synthesis was concomitantly inhibited. Taken together, these results implicate COX-2 in the development of MSU-induced inflammation.
Blood 04/1998; 91(5):1769-76. · 9.90 Impact Factor
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ABSTRACT: The inflammatory reaction associated with the deposition of monosodium urate (MSU) crystals in synovial spaces is known to be due to interactions with polymorphonuclear neutrophils mediated by presently unidentified surface structures. In this study, we have observed that antibodies directed against CD16 (VIFcRIII) and CD11b (VIM12) selectively and potently inhibit the activation of neutrophils by MSU crystals. The responses affected include the stimulation of tyrosine phosphorylation, activation of the tyrosine kinase syk, tyrosine phosphorylation of the proto-oncogene Cbl, mobilization of calcium, and stimulation of the activity of phospholipase D and of the production of superoxide anions. Tyrosine phosphorylation responses to MSU crystals develop during the Me2SO4-induced differentiation of HL-60 cells in parallel with the surface expression of CD16. These data strongly support the hypothesis that inflammatory microcrystals interact opportunistically with CD16 initially, and that the signal transduction pathways activated thereby depend on CD11b. An examination of the relevance of the hypothesis that an uncontrolled activation of CD16/CD11b may play a role in inflammatory reactions associated with a dysregulation of neutrophil function (other than crystal arthropathies) appears warranted on the basis of the present results.
The FASEB Journal 03/1998; 12(2):209-20. · 5.71 Impact Factor
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ABSTRACT: Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates many of the biological functions of human neutrophils. This includes the stimulation of protein synthesis and the tyrosine phosphorylation of various proteins among which is JAK2. The present study was aimed at characterizing in detail the pattern of activation by GM-CSF of the JAK/STAT pathway in human neutrophils. The results obtained show that the stimulation of human neutrophils by GM-CSF specifically led to tyrosine phosphorylation of JAK2 and had no effect on JAK1, JAK3, or TYK2. Furthermore, GM-CSF induced the tyrosine phosphorylation of STAT3 and STAT5 but not of STAT1, STAT2, STAT4, or STAT6. Tyrosine phosphorylation of STAT3 was transient reaching its maximum at 15 min. STAT5 presented a different pattern of tyrosine phosphorylation. The anti-STAT5 antibodies identified two proteins at 94 and 92 kDa. The 94-kDa STAT5 was constitutively tyrosine phosphorylated and showed no change upon GM-CSF stimulation. On the other hand, the 92-kDa STAT5 was tyrosine phosphorylated within 1 min of GM-CSF treatment and this was maintained for at least 30 min. By the use of specific antibodies, it was determined that only STAT5B, and not STAT5A, was tyrosine phosphorylated in GM-CSF-treated neutrophils. Furthermore, GM-CSF treatment induced an increase in the ability of STAT3 and STAT5B, but not STAT5A, to bind DNA probes. The specificity of the pattern of activation of the JAK/STAT pathway suggests that it may be directly linked to the modulation of the functions of mature nondividing, human neutrophils by GM-CSF.
Journal of Biological Chemistry 02/1998; 273(2):1058-63. · 4.77 Impact Factor
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ABSTRACT: Production of chemokines (chemotactic cytokines) by neutrophils is likely to be important in the regulation of inflammation and the control of infection. In this study we show that exposure of human neutrophils to various microbial pathogens leads to the production of both macrophage inflammatory protein 1alpha (MIP-1alpha) and IL-8. The bacterial microbes, Salmonella typhimurium and Pseudomonas aeruginosa, and Staphylococcus aureus all strongly induced both IL-8 and MIP-1alpha secretion, whereas Streptococcus pneumoniae, Staphylococcus epidermidis, and the opportunistic yeast Candida albicans were less potent. Saccharomyces cerevisiae and zymosan both induced IL-8 secretion but failed to stimulate that of MIP-1alpha. Coincubation of neutrophils with the proinflammatory cytokine TNF-alpha and the micro-organisms also led to differential expression of MIP-1alpha and IL-8. Significant enhancement of the induction of both MIP-1alpha and IL-8 by S. typhimurium, P. aeruginosa, and S. pneumoniae as well as by C. albicans was observed. In contrast, while IL-8 production in response to S. cerevisiae and zymosan was enhanced in the presence of TNF-alpha, no MIP-1alpha was produced. These combined results indicate that while neutrophils exposed to some micro-organisms alone or in the presence of inflammatory cytokines such as TNF-alpha will produce both MIP-1alpha and IL-8, resulting in generation of signals for the recruitment of mononuclear leukocytes and neutrophils, respectively, certain types of microorganisms can skew this response toward synthesis of IL-8.
The Journal of Immunology 01/1998; 160(1):449-54. · 5.79 Impact Factor
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ABSTRACT: The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcgammaRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.
Journal of Leukocyte Biology 01/1998; 62(6):901-10. · 4.99 Impact Factor
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ABSTRACT: In this study, we have evaluated the role of specific chemotactic cytokines in leukocyte recruitment to s.c. tissue in response to TNF-alpha in vivo. Injection of TNF-alpha into s.c. air pouches led to a rapid, transient accumulation of leukocytes. Maximal accumulation of leukocytes in the air pouch was observed at between 2 and 4 h after injection of TNF-alpha. The cellular exudate comprised predominantly neutrophils, with smaller numbers of eosinophils and mononuclear phagocytes also being recruited. However, lymphocyte recruitment was not observed. TNF-alpha injection induced a time-dependent increase in the levels of immunoreactive macrophage inflammatory protein (MIP)-2, MIP-1alpha, and JE in the pouch exudate as well as increased steady-state mRNA levels of KC, MIP-2, MIP-1alpha, and JE in the tissue lining the s.c. pouch and of MIP-2, MIP-1alpha, and JE in the exudate cell population. Passive immunization with specific Abs directed against each of these chemokines significantly inhibited the accumulation of neutrophils, mononuclear phagocytes, and eosinophils in response to TNF-alpha. Taken together, these data demonstrate the existence of a chemokine network in vivo involving at least four individual chemokines that regulates recruitment of the major peripheral blood granulocytes and mononuclear phagocytes to s.c. sites during acute inflammation. To our knowledge, these data are also the first demonstration that the C-C chemokine JE is involved in neutrophil recruitment in a physiologic system in vivo.
The Journal of Immunology 11/1997; 159(7):3595-602. · 5.79 Impact Factor
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ABSTRACT: The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.
Journal of Biological Chemistry 09/1997; 272(33):20660-4. · 4.77 Impact Factor
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ABSTRACT: Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases. Here we describe a technique that we found useful for preserving the tyrosine phosphorylation of cellular proteins. The technique depends on the denaturing lysis of neutrophils followed by the removal of the denaturing agents using Sephadex columns. Preparing neutrophil lysates by this technique has proven to be reliable in terms of maintaining the stability of the tyrosine phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification.
Journal of Immunological Methods 04/1997; 202(2):183-91. · 2.20 Impact Factor
