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IntroductionMonosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation.MethodsThe expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line.ResultsMSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression.ConclusionsOur findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.

Little is known about the mechanisms that arrest FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. In our previous studies, we observed a loss of immunoreactivity of Abs directed against FcγRIIa following its cross-linking. In this study, we report on the mechanisms involved in this event. A stimulated internalization of FcγRIIa leading to the down-regulation of its surface expression was observed by flow cytometry and confocal microscopy. Immunoprecipitation of the receptor showed that FcγRIIa is ubiquitinated after stimulation. MG132 and clasto-lactacystin β-lactone inhibited the loss of immunoreactivity of FcγRIIa, suggesting that this receptor was down-regulated via the proteasomal pathway. The E3 ubiquitin ligase c-Cbl was found to translocate from the cytosol to the plasma membrane following receptor cross-linking. Furthermore, c-Cbl was recruited to the same subset of high-density, detergent-resistant membrane fractions as stimulated FcγRIIa itself. Silencing the expression of c-Cbl by small interfering RNA decreased FcγRIIa ubiquitination and prevented its degradation without affecting the internalisation process. It also prolonged the stimulation of the tyrosine phosphorylation response to the cross-linking of the receptor. We conclude that c-Cbl mediates the ubiquitination of stimulated FcγRIIa and thereby contributes to the termination of FcγRIIa signaling via its proteasomal degradation, thus leading to the down-regulation of neutrophil signalisation and function (phagocytosis) through this receptor.

Objective. Monosodium urate monohydrate (MSU) crystals are among the most potent proinflammatory stimuli, and an innate immune inflammatory response to the crystal surface is involved in the pathogenesis of gouty arthritis. Release of the crystals into the joint cavity promotes an acute inflammation characterized by massive infiltration of neutrophils, which leads to tissue damage. The aim of the present study was to assess the involvement of the tyrosine kinase Tec in MSU crystal-initiated transduction events in human neutrophils.Methods. Immunoprecipitation and immunoblotting techniques were used for the cellular signaling studies. Chemotaxis and enzyme-linked immunosorbent assay techniques were used for the functional studies. Silencing of Tec expression using specific small interfering RNA was also performed.Results. MSU crystals induced the phosphorylation and activation of Tec in a Src-dependent manner. This activation was necessary for the MSU crystalinduced secretion of interleukin-1␤ (IL-1␤) and IL-8 and for the generation of chemotactic activity in supernatants of MSU crystal-stimulated neutrophils. In addition, colchicine, an effective drug for the treatment of gout, inhibited the MSU crystal-induced tyrosine phosphorylation of Tec, thus modulating its kinase activity.Conclusion. Our findings show that Tec is the principal kinase of the Tec family that plays a major role in the responses of human neutrophils to MSU crystals, which are likely to be involved in the initiation and perpetuation of gout. Our results suggest that the specific inhibition of Tec during the acute phase of MSU crystal-induced inflammation may be considered for the treatment of gouty arthritis.

Human neutrophils constitutively express a unique combination of Fc␥Rs, namely Fc␥RIIa and Fc␥RIIIb. Numerous lines of evidence support the concept that these Fc␥Rs generate only partially characterized intracellular signals. However, despite the fact that both receptors are likely to be engaged simultaneously in a physiological setting, no recent publications have investigated the distinct, although partially convergent, results of their joint activation in IgG-dependent responses. To examine the significance of the co-expression of Fc␥RIIa and Fc␥RIIIb on human neutrophils, we analyzed the neutrophil responses to stimuli that engage these Fc␥Rs, namely the phagocytosis of human IgG-opsonized zymosan and the responses to heat-aggregated IgGs. Blocking antibodies to either Fc␥R significantly decreased the phagocytic index and the stimulated production of superoxide anions. Both receptors are required for optimal IgG-dependent responses by human neutrophils. On the other hand, only blocking antibodies to Fc␥RIIIb, but not to Fc␥RIIa, inhibited the mobilization of calcium in response to heat-aggregated IgGs. Furthermore, phagocytosis of IgG-opsonized zymosan by human neutrophils required an extracellular influx of calcium that was blocked only by antibodies against Fc␥RIIIb. We also observed that this calcium influx as well as the IgG-dependent phagocytosis were dependent on the integrity of the plasma membrane detergent-resistant microdomains to which both isoforms were recruited following stimulation by heat-aggregated IgGs. These data clarify the mechanisms that regulate the Fc␥Rs constitutively expressed on human neutrophils, describe a specific contribution of Fc␥RIIIb at the level of the mobilization of calcium, and provide evidence for a crucial role of detergent-resistant microdomains in this process.

We previously described a non-classical mechanism that arrests Fc␥RIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of Fc␥RIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated Fc␥RIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the Fc␥RIIa degradation process because the cholesterol-depleting agent methyl-␤-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented Fc␥RIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of Fc␥RIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate Fc␥RIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated Fc␥RIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of Fc␥RIIa signaling. Fc␥ receptors (Fc␥Rs)3 represent a family of membrane proteins involved in the recognition of the Fc portion of immunoglobulin G. They act as sensors for opsonized pathogens or immune complexes, and their engagement initiates intracellular signals that lead to multiple cell functions, such as degranulation, activation of the respiratory burst, and phagocytosis (1-3). Fc␥R isoforms either express an immunoreceptor tyrosine-based activating motif (ITAM) in their intracellular portion or associate with ITAM-containing accessory proteins with the exception of Fc␥RIIb, the unique inhibitory member of the Fc␥R family that transmits inhibitory signals through an immunoreceptor tyrosine-based inhibition motif (ITIM) (4, 5). In the case of antibody-mediated responses, such as IgG-dependent phagocytosis, neutrophils and other immune effectors, such as macrophages, have the capacity to trigger strong proinflammatory responses, which must be tightly controlled. On human neutrophils, only two Fc␥Rs, namely Fc␥RIIa (CD32a) and Fc␥RIIIb (CD16b), are constitutively expressed, neither of which possess an ITIM. The available data indicate that in contrast to other human phagocytes, including macrophages, human neutrophils express very little (6) or no (7) Fc␥RIIb.Several lines of evidence indicate that Fc␥RIIa is directly involve...

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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.