Paul H. Naccache

Crystal-induced neutrophil activation

Monosodium urate (MSU) crystals are among the most potent pro‐inflammatory stimuli and an innate immune inflammatory response to the crystal surface is intimately involved in the pathology of gouty arthritis. The responses of human neutrophils to MSU crystals represent an integral part of this innate response and a key component of the acute inflammatory response associated with gout. A significant, though incomplete, body of information concerning the implication of human neutrophils in MSU crystal‐induced inflammation and the signal transduction pathways activated in response to these agonists in neutrophils has accumulated over the last few years. This review focuses on the current state of knowledge concerning the activation of human neutrophils by MSU crystals specifically in the context of acute gout, as recent data begin to draw a comprehensive picture of the events leading to the often excessive functional responses of neutrophils to these particulate agonists. A non‐exhaustive list of the most important questions that remain to be assessed to further describe the physio‐pathological mechanisms of gouty arthritis is presented here.

Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils

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‐α, 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.—Pouliot, M., Gilbert, C., Borgeat, P., Poubelle, P. E., Bourgoin, S., Créminon, C., Maclouf, J., McColl, S. R., Naccache, P. H. Expression and activity of prostaglandin endoperoxide synthase‐2 in agonist‐activated human neutrophils. FASEB J. 612, 1109–1123 (1998)

Crystal-induced neutrophil activation VI. Involvement of FcγRIIIB (CD16) and CD11b in response to inflammatory microcrystals

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.—Barabé, F., Gilbert, C., Liao, N., Bourgoin, S. G., Naccache, P. H. Crystal‐induced neutrophil activation: involvement of FcγRIIIB (CD16) in response to inflammatory microcrystals. FASEB J. 12, 209–220 (1998)

Granulocyte-Macrophage Colony-stimulating Factor-activated Signaling Pathways in Human Neutrophils INVOLVEMENT OF Jak2 IN THE STIMULATION OF PHOSPHATIDYLINOSITOL 3-KINASE

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.

Biosynthesis and biological activity of leukotriene B4

The leukotrienes are a family of biologically active molecules derived from arachidonic acid. While prostaglandins and thromboxanes are products of the cyclooxygenase pathway of arachidonic acid metabolism, the leukotrienes are formed by arachidonate 5-lipoxygenase, an enzyme present in phagocytes, mast cells, and basophils. Inflammatory stimuli, such as chemotactic peptides, platelet-activating factor, phagocytic particles, and immunological stimuli, which activate phagocytes and mast cells, stimulate leukotriene synthesis. Leukotriene B4, a dihydroxy derivative of arachidonic acid, has a unique stimulatory activity on important functional responses of phagocytes; leukotriene B4 exerts chemotactic and chemokinetic activity towards phagocytes in vitro and in vivo, and it is a putative mediator of inflammation.

Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops.

Interleukin 8 (IL‐8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL‐8 receptors and the α subunit of guanine nucleotide regulatory protein (Giα2) upon stimulation of human neutrophils by IL‐8. In the present study, we identified by site‐directed mutagenesis key residues within the three intracellular loops of the IL‐8RA receptor involved in the interaction with Giα2. We first systematically mutated, in groups of two to four, all the residues in the three intracellular loops of the IL‐8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 293 cells. Four residues in the second intracellular loop (Y136, L137, I139, V140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL‐8. Other residues in the second and third intracellular loops were also found to affect IL‐8RA‐mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL‐8 binding affinities to the mutated receptors. Mutagenesis of the residues in the first intracellular loop had only weak effects on the mobilization of calcium induced by IL‐8. We then used a coimmunoprecipitation protocol with anti‐Giα2 antibodies to determine the involvement of the two regions defined above in Giα2 coupling to IL‐8 type A receptors. Whereas the anti‐Giα2 antibodies coimmunoprecipitated IL‐8 receptors in the wild‐type cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for Giα2 coupling and induction of intracellular calcium mobilization were next introduced into cells expressing wild‐type IL‐8RA or IL‐8RB to assess their role in coupling Giα2 to both IL‐8 receptors. The results obtained in the latter experiments suggest that the same regions of the second intracellular loop (Y136, L137, I139, V140) and of the third intracellular loop (M241) are critically involved in the coupling of both IL‐8RA and IL‐8 RB to Giα2 as well as to a downstream effector (or effectors) involved in calcium mobilization.—Damaj, B. B., McColl, S. R., Neote, K., Songqing, N., Ogborn, K. T., Hébert, C. A., Naccache, P. H. Identification of G‐protein binding sites of the human interleukin‐8 receptors by functional mapping of the intracellular loops. FASEB J. 10, 1426‐1434 (1996)

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 p110γ or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110γ 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.

Up-Regulation of Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Human Endometriotic Cells by Macrophage Migration Inhibitory Factor: Involvement of Novel Kinase Signaling Pathways

Cyclooxygenase (COX) is the rate-limiting enzyme in the metabolic conversion of arachidonic acid to prostaglandins (PGs), including prostaglandin E(2) (PGE(2)), a major mediator of inflammation and angiogenesis. Herein, we report that macrophage migration inhibitory factor (MIF), a potent proinflammatory and growth-promoting factor found at elevated concentrations in the peritoneal fluid of women with endometriosis and active endometriosis lesions, acts directly on ectopic endometrial cells to stimulate the synthesis of COX-2, the inducible form of COX, and the release of PGE(2). MIF treatment strongly activated p38 and ERK MAPK, and specific inhibitors of both pathways completely blocked basal and MIF-induced PGE(2) synthesis. Whereas p38 inhibitors negatively affected the stimulated synthesis of COX-2 and that of PGE(2), ERK inhibitors only decreased the production of PGE(2). These findings show for the first time a direct role for MIF in the up-regulation of COX-2 synthesis and PGE(2) secretion in ectopic endometrial cells. They further indicate that whereas p38 and ERK MAPK signaling pathways both play a significant role in the regulation of basal and MIF-induced synthesis of PGE(2) by ectopic endometrial cells, only p38 kinase is involved in the regulation of COX-2 expression in these cells. This suggests that MIF acts at more than one level to stimulate the synthesis of PGE(2) and triggers the coordinate activation of multiple enzymes in the biosynthesis pathway. Our data provide evidence for a novel mechanism by which MIF can induce a proinflammatory phenotype in ectopic endometrial cells, and favor the establishment of endometriosis and its related clinical symptoms.

Evidence for the involvement of tyrosine kinases in the locomotory responses of human neutrophils

Erbstatin, a recently described inhibitor of tyrosine kinases, has been used to examine the potential role of tyrosine phosphorylation in human neutrophil locomotion. Preincubation of human neutrophils with erbstatin inhibited both spontaneous and directed migration induced by chemotactic factors such as formyl‐methionylleucylphenylalanine (fMet‐Leu‐Phe) and leukotriene B4. The decreased migratory responses were correlated with an inhibition of adherence of neutrophils to serum‐coated surfaces. Erbstatin did not, however, affect the adherence of human neutrophils to uncoated surfaces. These results indicated that the inhibitory effects of erbstatin were specific and not due to a generalized alteration of the surface of human neutrophils. To elucidate the mechanism of the inhibitory effect of erbstatin on adherence properties, the expression of the leukocyte integrin Mo1 was studied. Erbstatin induced a small but significant increase in the expression of Mo1, but decreased the stimulation of the expression of Mo1 elicited by fMet‐Leu‐Phe. These results suggest that mechanisms in addition to alteration of the number of surface integrins are involved in the inhibition of neutrophil adherence and locomotion by erbstatin. J. Leukoc. Biol. 51: 103–108; 1992.

Adenosine Up-Regulates Cyclooxygenase-2 in Human Granulocytes: Impact on the Balance of Eicosanoid Generation

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine’s anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE2 in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE2 on leukotriene B4 formation and involved the EP2 receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE2 generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.