Regulation of the Expression, Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region.

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.

The Inhibitory Receptor CLEC12A Regulates PI3K-Akt Signaling to Inhibit Neutrophil Activation and Cytokine Release.

The myeloid inhibitory C-type lectin receptor CLEC12A limits neutrophil activation, pro-inflammatory pathways and disease in mouse models of inflammatory arthritis by a molecular mechanism that remains poorly understood. We addressed how CLEC12A-mediated inhibitory signaling counteracts activating signaling by cross-linking CLEC12A in human neutrophils. CLEC12A cross-linking induced its translocation to flotillin-rich membrane domains where its ITIM was phosphorylated in a Src-dependent manner. Phosphoproteomic analysis identified candidate signaling molecules regulated by CLEC12A that include MAPKs, phosphoinositol kinases and members of the JAK-STAT pathway. Stimulating neutrophils with uric acid crystals, the etiological agent of gout, drove the hyperphosphorylation of p38 and Akt. Ultimately, one of the pathways through which CLEC12A regulates uric acid crystal-stimulated release of IL-8 by neutrophils is through a p38/PI3K-Akt signaling pathway. In summary this work defines early molecular events that underpin CLEC12A signaling in human neutrophils to modulate cytokine synthesis. Targeting this pathway could be useful therapeutically to dampen inflammation.

The development of a targeted and more potent, anti-Inflammatory derivative of colchicine: Implications for gout.

BACKGROUND: Colchicine is routinely used for its anti-inflammatory properties to treat gout and Familial Mediterranean fever. More recently, it was also shown to be of therapeutic benefit for another group of diseases in which inflammation is a key component, namely, cardiovascular disease. Whilst there is considerable interest in repurposing this alkaloid, it has a narrow therapeutic index and is associated with undesirable side effects and drug interactions. We, therefore, developed a derivatives of colchicine that preferentially target leukocytes to increase their potency and diminish their side effects. The anti-inflammatory activity of the colchicine derivatives was tested in experimental models of neutrophil activation by the etiological agent of gout, monosodium urate crystals (MSU). METHODS: Using a rational drug design approach, the structure of colchicine was modified to increase its affinity for ßVI-tubulin, a colchicine ligand preferentially expressed by immune cells. The ability of the colchicine analogues with the predicted highest affinity for ßVI-tubulin to dampen neutrophil responses to MSU was determined with in vitro assays that measure MSU-induced production of ROS, release of IL-1 and CXCL8/IL-8, and the increase in the concentration of cytoplasmic calcium. The anti-inflammatory property of the derivatives was assessed in the air pouch model of MSU-induced inflammation in mice. RESULTS: The most effective compound generated, CCI, is more potent than colchicine in all the in vitro assays. It inhibits neutrophil responses to MSU in vitro at concentrations 10-100-fold lower than colchicine. Similarly, in vivo, CCI inhibits the MSU-induced recruitment of leukocytes at a 10-fold lower concentration than colchicine when administered prior to or after MSU. CONCLUSIONS: We provide evidence that colchicine can be rendered more potent atinhibiting MSU-induced neutrophil activation and inflammation using a rational drug design approach. The development of compounds such as CCI will provide more efficacious drugs that will not only alleviate gout patients of their painful inflammatory episodes at significantly lower doses than colchicine, but also be of potential therapeutic benefit for patients with other diseases treated with colchicine.

The Role of Inhibitory Receptors in Monosodium Urate Crystal-Induced Inflammation.

Inhibitory receptors are key regulators of immune responses. Aberrant inhibitory receptor function can either lead to an exacerbated or defective immune response. Several regulatory mechanisms involved in the inflammatory reaction induced by monosodium urate crystals (MSU) during acute gout have been identified. One of these mechanisms involves inhibitory receptors. The engagement of the inhibitory receptors Clec12A and SIRL-1 has opposing effects on the responses of neutrophils to MSU. We review the general concepts of inhibitory receptor biology and apply them to understand and compare the modulation of MSU-induced inflammation by Clec12A and SIRL-1. We also discuss gaps in our knowledge of the contribution of inhibitory receptors to the pathogenesis of gout and propose future avenues of research.

S100A9 potentiates the activation of neutrophils by the etiological agent of gout, monosodium urate crystals.

Gout is one of the most painful types of arthritis that arises when the body mounts an acute inflammatory reaction against a crystallized form of uric acid known as monosodium urate crystals (MSUs). Although MSUs are known to activate neutrophils, the most abundant leukocyte in the synovial fluid of patients with gout, few studies have investigated the effect on neutrophils of the simultaneous stimulation with MSU and proinflammatory mediators in the inflamed joint. Herein, we focused on a protein that is highly expressed in the synovium in gout, S100A9. The predominant expression of S100A9 in and around blood vessels suggests it may prime neutrophils during their migration toward the inflamed joint. Using a combination of functional and signaling assays, we found that S100A9 enhances the production of radical oxygen species as well as IL-1 and IL-8 release by human neutrophils activated with MSU. Moreover, upstream and downstream signaling events activated by MSUs in human neutrophils were also potentiated by S100A9, including the mobilization of intracellular calcium stores, tyrosine phosphorylation, the serine phosphorylation of PKC substrates, Akt, and p38. We also show that S100A9 alone increases glycolysis in human neutrophils, which is suggestive of an additional mechanism through which neutrophils can be primed. Together, our observations indicate a novel way in which S100A9 may contribute to the pathogenesis of gout, by priming neutrophils to respond to MSUs.

Signal Inhibitory Receptor on Leukocytes-1 Limits the Formation of Neutrophil Extracellular Traps, but Preserves Intracellular Bacterial Killing.

In response to microbial invasion, neutrophils release neutrophil extracellular traps (NETs) to trap and kill extracellular microbes. Alternatively, NET formation can result in tissue damage in inflammatory conditions and may perpetuate autoimmune disease. Intervention strategies that are aimed at modifying pathogenic NET formation should ideally preserve other neutrophil antimicrobial functions. We now show that signal inhibitory receptor on leukocytes-1 (SIRL-1) attenuates NET release by human neutrophils in response to distinct triggers, including opsonized Staphylococcus aureus and inflammatory danger signals. NET release has different kinetics depending on the stimulus, and rapid NET formation is independent of NADPH oxidase activity. In line with this, we show that NET release and reactive oxygen species production upon challenge with opsonized S. aureus require different signaling events. Importantly, engagement of SIRL-1 does not affect bacterially induced production of reactive oxygen species, and intracellular bacterial killing by neutrophils remains intact. Thus, our studies define SIRL-1 as an intervention point of benefit to suppress NET formation in disease while preserving intracellular antimicrobial defense.

Challenges in the characterization of neutrophil extracellular traps: The truth is in the details.

Neutrophil extracellular traps play a key role in defense against extracellular pathogens. The release of these chromatin structures, that contain a combination of cytoplasmic and granule proteins, is known as NETosis, a regulated cell death modality typical of neutrophils. NETosis is induced by pathogens as well as other stimuli such as activated platelets. Our understanding of the molecular events underlying this phenomenon remains incomplete. The currently used experimental approaches to study NETs are semi-quantitative, subjective in nature, and low throughput, rendering it difficult to compare results between laboratories. This is highlighted in two articles published in this issue of the European Journal of Immunology which present what appear to be contradicting results on NET formation. Considering the extensive research on NETosis and the importance of this phenomenon in the immune response, we find it timely to briefly review the lacunae in the most commonly used methods to investigate NETosis. The impact these technical difficulties have on the advancement of our knowledge in this field as well as potential solutions are also discussed.

A straight neutrophil path to healthy aging?

In this issue of Blood, Sapey et al. report that the human polymorphonuclear neutrophil leukocyte (or neutrophil) undergoes an age-related loss of its ability to migrate up chemotactic gradients, a functional defect that seems causally related to alterations in the polyphosphoinositide pathway.

Cross-linking of IgGs bound on circulating neutrophils leads to an activation of endothelial cells: possible role of rheumatoid factors in rheumatoid arthritis-associated vascular dysfunction.

BACKGROUND: Rheumatoid arthritis is characterized by the presence of circulating auto-antibodies, including rheumatoid factors, which recognize the Fc portion of IgGs. The neutrophil is the most abundant circulating leukocyte and it expresses high levels of FcγRs on its surface. The aim of the present study was to examine the capacity of circulating human neutrophils to be activated by rheumatoid factors and the consequences of these events on endothelium. METHODS: Neutrophil-bound IgGs were cross-linked with anti-human IgGs to mimick the presence of circulating rheumatoid factors and FcγRs-dependent signalling events and functions were examined. The IgG and IgM composition of rheumatoid factors isolated from the serum of RA patients was characterized. Adhesion of neutrophils to endothelial cells was quantified in response to the addition of rheumatoid factors. RESULTS: Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme. Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells. Finally, rheumatoid factors enhance neutrophil adhesion to endothelial cells. CONCLUSIONS: Our results show that activation of neutrophils' FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

Modulation of monosodium urate crystal-induced responses in neutrophils by the myeloid inhibitory C-type lectin-like receptor: potential therapeutic implications.

INTRODUCTION: Monosodium 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. METHODS: The 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. RESULTS: MSU 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. CONCLUSIONS: Our 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.

Inflammatory gout: observations over a half-century.

This is a discussion of acute gouty arthritis, seen for over 50 years of engagement. It addresses the evolution of our current understanding of the interaction between urate crystals and key cellular components of the gouty inflammatory paroxysm, with new material on pathogenesis.

CIN85 modulates the down-regulation of Fc gammaRIIa expression and function by c-Cbl in a PKC-dependent manner in human neutrophils.

We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa 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 FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-ß-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa 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 FcγRIIa signaling.

Fc gammaRIIIb triggers raft-dependent calcium influx in IgG-mediated responses in human neutrophils.

Human neutrophils constitutively express a unique combination of FcγRs, namely FcγRIIa and FcγRIIIb. Numerous lines of evidence support the concept that these FcγRs 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 FcγRIIa and FcγRIIIb on human neutrophils, we analyzed the neutrophil responses to stimuli that engage these FcγRs, namely the phagocytosis of human IgG-opsonized zymosan and the responses to heat-aggregated IgGs. Blocking antibodies to either FcγR 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 FcγRIIIb, but not to FcγRIIa, 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 FcγRIIIb. 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 FcγRs constitutively expressed on human neutrophils, describe a specific contribution of FcγRIIIb at the level of the mobilization of calcium, and provide evidence for a crucial role of detergent-resistant microdomains in this process.

Macrophage migration inhibitory factor elicits an angiogenic phenotype in human ectopic endometrial cells and triggers the production of major angiogenic factors via CD44, CD74, and MAPK signaling pathways.

CONTEXT: An active angiogenesis is required for ectopic endometrial tissue growth. Our previous studies led to the identification of macrophage migration inhibitory factor (MIF), which is markedly elevated in active, vascularized, and early-stage endometriotic lesions, as a potent mitogenic factor for endothelial cells. OBJECTIVE: Our objective was to study the mechanisms by which MIF may stimulate angiogenesis in ectopic endometrial implantation sites. DESIGN: Primary cultures of ectopic endometrial cells were exposed to MIF, and the release of major angiogenic factors with targeted disruption of MIF signaling pathways was assessed. PATIENTS: Patients were women found to have endometriosis during laparoscopy. SETTING: The study was conducted at a hospital and reproduction research laboratory. INTERVENTIONS: Biopsies were removed from endometriotic lesions. MAIN OUTCOME MEASURES: Vascular endothelial cell growth factor (VEGF), IL-8, and monocyte chemotactic protein-1 (MCP-1) mRNA and protein levels and expression and small interfering RNA silencing of MIF CD74/CD44 receptor complex and phosphorylation of ERK and p38 MAPKs were evaluated. RESULTS: MIF markedly up-regulated VEGF, IL-8, and MCP-1 expression in endometriotic cells. Such an effect was abolished by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), a specific inhibitor of MIF, and significantly down-regulated after specific small interfering RNA silencing of CD44 or CD74. MIF treatment strongly activated ERK and p38 MAPKs, and specific inhibitors of both pathways completely blocked basal and MIF-induced VEGF, IL-8, and MCP-1 synthesis. CONCLUSIONS: These results show for the first time that MIF exerts a potent indirect angiogenic effect by interacting with ectopic endometrial cells and inducing the secretion of major angiogenic factors via CD44, CD74, and MAPK signaling pathways and provide evidence for a possible new mechanism underlying endometriosis development and pathophysiology.

Proteinase-activated receptor-2 up-regulation by Fcgamma-receptor activation in human neutrophils.

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.

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.

Crystal-induced neutrophil activation: XI. Implication and novel roles of classical protein kinase C.

Monosodium urate (MSU) crystals are among the most potent proinflammatory stimuli, and an innate immune inflammatory response to the crystal surface is involved in the pathology of gouty arthritis. Furthermore, MSU crystals have recently been identified as danger signals able to induce the maturation of dendritic cells. Release of the crystals into the joint cavity promotes an acute inflammation characterized by a massive infiltration of neutrophils that leads to tissue damage. Protein kinase C (PKC) represents a family of serine/threonine kinases that play central signaling roles in multiple cellular responses. This family of kinases is divided into three subfamilies based on second messenger requirements: conventional (or classical), novel, and atypical. Despite their role in signal transduction, very little is known about the involvement of the PKC family in the inflammatory reaction induced by MSU crystals. In the present study, we show that MSU crystals activate conventional PKC isoforms, and that this activation is necessary for the MSU crystal-induced degranulation and generation of a chemotactic activity in the supernatants of MSU crystal-stimulated human neutrophils. Evidence is also obtained that the tyrosine kinase Syk is a substrate of PKC and that the PKC-mediated serine phosphorylation of Syk is necessary to its interaction with the regulatory subunit of PI3K kinases (p85) and thus to the subsequent activation of these lipid kinases. These results suggest novel means of modulating neutrophil responses (through the specific regulation of PKC) during the acute phase of MSU crystal-induced inflammation.

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.

The ubiquitin ligase c-Cbl down-regulates FcgammaRIIa activation in human neutrophils.

Little is known about the mechanisms that arrest FcgammaRIIa 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 FcgammaRIIa following its cross-linking. In this study, we report on the mechanisms involved in this event. A stimulated internalization of FcgammaRIIa leading to the down-regulation of its surface expression was observed by flow cytometry and confocal microscopy. Immunoprecipitation of the receptor showed that FcgammaRIIa is ubiquitinated after stimulation. MG132 and clasto-lactacystin beta-lactone inhibited the loss of immunoreactivity of FcgammaRIIa, 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 FcgammaRIIa itself. Silencing the expression of c-Cbl by small interfering RNA decreased FcgammaRIIa 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 FcgammaRIIa and thereby contributes to the termination of FcgammaRIIa signaling via its proteasomal degradation, thus leading to the down-regulation of neutrophil signalisation and function (phagocytosis) through this receptor.

Crystal-induced neutrophil activation: X. Proinflammatory role of the tyrosine kinase Tec.

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 crystal-induced secretion of interleukin-1beta (IL-1beta) 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.