Modulation of monosodium urate crystal-induced responses in neutrophils by the myeloid inhibitory C-type lectin-like receptor: potential therapeutic implications
- Valérie Gagné†1,
- Louis Marois†1,
- Jean-Michel Levesque1,
- Hugo Galarneau1,
- Mireille H Lahoud2,
- Irina Caminschi2,
- Paul H Naccache1,
- Philippe Tessier1 and
- Maria JG Fernandes1Email author
© BioMed Central Ltd. 2013
Received: 6 October 2012
Accepted: 9 July 2013
Published: 9 July 2013
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.
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.
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.
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.
KeywordsCytokine production out Immunoreceptor tyrosine-based inhibitory motif Monosodium urate crystals Neutrophil Signaling
Gout is one of the most painful types of arthritis, and its prevalence is on the rise worldwide [1, 2]. The inflammatory reaction typical of an acute gout attack is initiated by the crystallized form of a by-product of purine metabolism, monosodium urate crystals (MSU). A gout attack can be viewed in three phases: the initiation phase, the effector phase and the subsequent resolution phase. According to the current understanding of the pathogenesis of acute gout, MSU activate resident articular cells (for example, macrophages) during the initiation phase, most commonly in the metatarsophalangeal joint . The activation of resident cells by MSU induces the synthesis of several inflammatory mediators, including active interleukin 1β (IL-1β), a cytokine that plays a pivotal role in the pathogenesis of gout, implicating nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome in this inflammatory disorder. IL-1β contributes to the initiation and perpetuation of the effector phase by virtue of its ability to stimulate endothelial cells of the vasculature to express potent chemokines (for example, IL-8) and adhesion molecules responsible for the massive recruitment of neutrophils to the joint.
The recruitment of a large number of neutrophils to the affected joint during the effector phase is the pathological hallmark of gouty arthritis . The presence of activated phagocytes in the joint is one of the main causes of tissue destruction and pain in gout. When activated by MSU, neutrophils release a panoply of inflammatory molecules, including cytokines (for example, IL-1β, IL-8, S100) and degradative enzymes, that perpetuate the inflammatory reaction as well as oxygen radicals that cause damage to the surrounding tissues (recently reviewed by Popa-Nita and Naccache ). At the molecular level, the signaling molecules driving MSU-induced neutrophil responses are just beginning to be identified. They include activated Src family kinases (for example, Lyn), Syk, protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and Tec . Since the activation of Src kinases is an early signaling event, the majority of MSU-induced effector functions depend on these kinases.
Animal studies and clinical observations underscore the pivotal role of the neutrophil in gout. A significant decrease in MSU-induced inflammation was reported in neutropenic mice. Moreover, medications used to treat gout, such as colchicine, downregulate MSU-induced neutrophil effector functions . It is thus of interest to characterize molecular mechanisms that regulate MSU-induced neutrophil activation.
Leukocyte activation is regulated in part by phosphatases that block early signaling events of activating receptors when recruited to the plasma membrane. Phosphatase recruitment occurs via immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the cytoplasmic portion of inhibitory receptors expressed on the surface of leukocytes . Inhibitory receptors can be classified into two main groups on the basis of their structure, namely, the immunoglobulin or the C-type lectin superfamily. Proteins of the latter contain at least one C-type lectin-like domain (CTLD). Myeloid inhibitory C-type lectin receptors are poorly characterized in comparison to their counterparts in natural killer cells (recently reviewed by Pyz et al. ). Findings from the few myeloid inhibitory receptors studied suggest, however, that these proteins are able to suppress several phagocyte effector functions, including phagocytosis, migration and cytokine production .
The myeloid inhibitory C-type lectin-like receptor (MICL) is a type II transmembrane protein comprising one CTLD in its extracellular domain and an ITIM in its cytoplasmic domain [8–12]. It is expressed by monocytes, macrophages, neutrophils, myeloid and plasmacytoid dendritic cells. The natural ligands of human MICL remain to be identified. Although unequivocal evidence of a negative regulatory function of MICL is still lacking in the published literature, the majority of the evidence suggests that MICL does indeed have inhibitory activity. The recruitment of the SH2-containing tyrosine phosphatases SHP-1 and SHP-2 to the ITIM of MICL was observed in a macrophage cell line . Moreover, in response to a variety of Toll-like receptor (TLR) ligands, the cell surface expression of MICL diminishes in human monocytes and macrophages, suggestive of a negative regulatory role for this receptor . In support of this hypothesis, the cell surface expression of MICL was shown to be diminished in neutrophils recruited to a site of inflammation using an abrasion model of the skin in human volunteers . The antibody-induced internalization of MICL in monocyte-derived dendritic cells, however, showed that the loss of cell surface MICL can either positively or negatively regulate cytokine production. The internalization of MICL prior to stimulation with lipopolysaccharide (LPS) suppresses the production of IL-12p40 and IL-12p70 . In contrast, the internalization of MICL prior to stimulation with the CD40 ligand enhances the production of tumor necrosis factor α (TNF-α), IL-12p40, IL-12p70, IL-6 and IL-10 . A role for MICL in antibody responses has also been reported. Targeting antigens to MICL can induce antibody responses .
Since MICL regulates the synthesis of cytokines in dendritic cells and its expression is modulated by proinflammatory stimuli, the objective of this study was to investigate the role of MICL in MSU-induced activation of neutrophils and to determine whether MSU modulate MICL expression. Herein we report for the first time that MSU diminish MICL expression in human neutrophils. A decrease in MICL expression in neutrophils selectively enhances MSU-induced cytokine release by neutrophils and also potentiates early signaling events. We also provide direct evidence that colchicine inhibits the MSU-induced loss of cell surface MICL, suggesting that the regulation of MICL expression may be a new mechanism through which this drug dampens inflammation.
Antibodies and chemicals
Two different antibodies against MICL were used in this study. Monoclonal antibodies 50C1  and HB3 which was kindly provided by Dr G Brown  recognize extracellular epitopes of MICL. FITC-conjugated F(ab')2 fragment goat anti-mouse IgG antibody (Fc fragment-specific, catalog no. 115-096-071) was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Horseradish peroxidase-labeled sheep anti-mouse immunoglobulins (IgGs) (no. NXA931) were obtained from GE Healthcare (Uppsala, Sweden). The antiphosphotyrosine (clone 4G10, no. 05-321) and the anti-p85 (no. 06-195) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Monoclonal anti-flotillin-1 antibody (no. 610820) was purchased from BD Transduction Laboratories (Mississauga, ON, Canada). The mouse IgG2a isotype antibody (no. 0572) was purchased from Beckman Coulter (Mississauga, ON, Canada). Anti-CD11b antibody (OKM1, no. 88012702) was purchased from Sigma-Aldrich Canada (Oakville, ON, Canada), and the anti-FPRL1 antibody was obtained from R&D Systems (Minneapolis, MN, USA). Sodium orthovanadate (Na3VO4), N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP)), colchicine and Dextran T500 were obtained from Sigma-Aldrich Canada. 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid (CHAPS), aprotinin and leupeptin were purchased from Roche Applied Science (Laval, QC, Canada). The Western Lightning Chemiluminescence Plus ECL kit was obtained from PerkinElmer (Boston, MA, USA). Ficoll-Paque and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from Wisent (St-Bruno, QC, Canada). Protein A Sepharose was purchased from GE Healthcare. Gelatin was obtained from Fisher Scientific (Nepean, ON, Canada). Fura-2-acetoxymethyl ester (Fura-2AM) was obtained from Invitrogen (Burlington, ON, Canada), and the triclinic MSU crystals were synthesized and characterized as previously described by Naccache et al. . Endotoxin contamination was ruled out by Limulus amebocyte lysate assay. siGENOME SMARTpool MICL (no. D-021369-01) and siGENOME nontargeting small interfering RNA (siRNA) pool 1 (negative control, no. D-001206-13-05) were purchased from Dharmacon Inc (Lafayette, CO, USA).
The Institutional Review Board of Laval University (Quebec, QC, Canada) approved the study, and volunteers signed a consent form. Neutrophils were collected from healthy adult volunteers and isolated as previously described . They were resuspended in Mg2+-free Hanks' balanced salt solution (HBSS) containing 1.6 mM CaCl2. The myeloid cell line PLB-985 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany) and grown in RPMI 1640 medium containing 10% decomplemented fetal bovine serum (FBS), 10 mM HEPES, 1 mM Na+ pyruvate at 37°C in a 5% CO2 humidified atmosphere. The cells were maintained in culture for 12 passages before new batches were thawed. To induce differentiation to a neutrophil-like phenotype, PLB-985 cells were cultured in medium supplemented with 0.3 mM dibutyryl cAMP for 3 days prior to each experiment.
Transfection of dibutyryl cAMP-differentiated PLB-985 cells
One day following the initiation of differentiation with 0.3 mM dibutyryl cAMP, PLB-985 cells were transiently transfected using a nucleofection system obtained from Amaxa Biosystems (Cologne, Germany). Briefly, 2 × 106 cells were centrifuged and resuspended in 100 µl of nucleofection buffer (25 mM HEPES, pH 7.4, 120 mM KCl, 2 mM MgCl2, 10 mM K2HPO4, 5 mM L-cysteine) containing 3 µg of MICL-specific siRNA (siMICL) or negative control siRNA (siCtrl). The samples were transferred into an electroporation cuvette, and transfections were performed using the program setting U-002. After nucleofection, cells were immediately transferred into prewarmed RPMI 1640 complete medium containing 0.3 mM dibutyryl cAMP, 10% FBS, 10 mM HEPES, 1 mM Na+ pyruvate and maintained at 37°C in a 5% CO2 humidified atmosphere. Two days after nucleofection, cells were harvested and resuspended in Mg2+-free HBSS containing 1.6 mM CaCl2 for analysis.
Flow cytometry analysis
To monitor the plasma membrane expression of MICL on neutrophils stimulated with different agonists, neutrophils were transferred into tubes cooled in an ice bath to terminate the stimulations, then centrifuged at 400 × g for 2 min at 4°C. The cell pellets were then resuspended in buffer with 50C1 (1 µg/ml) or the IgG2a isotype control antibody and incubated for 30 min on ice, washed, and centrifuged, and the resuspended cell pellets were incubated for a further 30 min on ice with FITC-labeled goat antimouse Fcγ-specific IgG (diluted 1:100 in HBSS/bovine serum albumin (HBSS/BSA) solution). Cells were then washed twice in HBSS/BSA and analyzed by flow cytometry using a FACSCanto II flow cytometer obtained from BD Biosciences (San Jose, CA, USA). To monitor the antibody-induced internalization of cell surface MICL, freshly isolated neutrophils were incubated with 50C1 antibody (1 µg/ml) or IgG2a isotype antibodies for the indicated times at 37°C and then washed, and the resuspended cell pellets were incubated for a further 30 min on ice with FITC-labeled goat antimouse Fcγ-specific IgG (diluted 1:100 in HBSS/BSA solution). In some experiments, neutrophils were treated with 10 µM colchicine or diluent (dimethyl sulfoxide (DMSO)) for 30 min at 37°C and centrifuged to wash away the drug prior to stimulation with 1 mg/ml MSU for 20 min at 37°C.
Trichloroacetic acid protein precipitation
Human neutrophils (20 × 106 cells/ml) were stimulated with MSU crystals (1 µg/ml) at 37°C and quickly centrifuged, then supernatants were precipitated with 15% trichloroacetic acid for 30 min at 4°C. Samples were then centrifuged at 16,000 × g for 5 min and washed three times with acetone. Pellets were dried at 95°C for 2 min and resuspended in modified Laemmli buffer.
Enzyme-linked immunosorbent assay
The assessment of the extracellular IL-8 was performed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (human IL-8 cytoset, no. CHC1303) from Invitrogen. All samples were measured in duplicate. Briefly, dibutyryl cAMP-differentiated PLB-985 cells were stimulated for 3 h at 37°C with 1 mg/ml MSU crystals in RPMI 1640 and centrifuged (16,000 × g for 5 min), then the supernatants were harvested and filtered. Extracellular IL-8 was quantified using commercially available ELISA kits (BD Biosciences). All samples were measured in triplicate. For the quantitation of extracellular IL-1β, neutrophils primed with TNF-α or incubated in buffer were stimulated for 8 h with MSU at 37°C, and IL-1β was quantified in the cell-free supernatant using a commercially available ELISA kit obtained from eBioscience (catalog no. 88-7010-22; San Diego, CA, USA). All samples were measured in duplicate. To determine the effect of colchicine on the MSU-induced production of IL-8, neutrophils were treated with 10 µM colchicine or diluent (DMSO) for 30 min at 37°C prior to adding MSU (1 mg/ml) or buffer to the neutrophil-colchicine mixture and incubating it for a further 3 h at 37°C. It is of note that the effect of colchicine on neutrophils is reversible (data not shown). For colchicine to be effective, it should not be removed from the neutrophil suspension for the duration of the experiment for assays longer than 20 min.
Electrophoresis and immunoblotting
For Western blot analysis, cell suspensions were transferred directly into the same volume of 2× boiling modified Laemmli sample buffer (1× buffer: 62.5 mM Tris·HCl (pH 6.8), 4% (wt/vol) sodium dodecyl sulfate (SDS), 5% (vol/vol) β-mercaptoethanol, 8.5% (vol/vol) glycerol, 2.5 mM orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 0.025% bromophenol blue) to terminate cell activation with MSU, then boiled for 7 min. Proteins were separated by SDS-PAGE on 10% acrylamide gels under nonreducing conditions and transferred to polyvinylidene fluoride (PVDF) membranes. Blocking agents and antibodies were diluted in a Tris-buffered saline Tween 20 (TBST) solution (25 mM Tris·HCl, pH 7.8, 190 mM NaCl, 0.15% vol/vol Tween 20). PVDF membranes were incubated in blocking solution (5% wt/vol dried milk in TBST) prior to immunoblotting with the anti-MICL antibody (HB3 antibody) or anti-flotillin-1 antibody. Gelatin solution (2% wt/vol) was used as a blocking solution prior to immunoblotting with the antiphosphotyrosine (4G10) antibody. Anti-MICL and anti-flotillin antibodies were diluted 1:1,000 and antiphosphotyrosine antibodies was diluted 1:2,000. Horseradish peroxidase-labeled sheep antimouse IgG was diluted 1:20,000 in TBST solution. Chemiluminescence reagents were used to detect antibodies with a maximal exposure time of 5 min. All the immunoblots presented were controlled for equal protein loading with an anti-p85 of PI3K antibody.
Neutrophils (1 × 107 cells/ml) were incubated for 30 min at 37°C with 1 μM Fura-2AM, washed once in HBSS, resuspended to a concentration of 5 × 106 cells/ml and transferred to a temperature-controlled (37°C) cuvette compartment of a spectrofluorometer (SLM 8000; SLM Instruments, Urbana, IL, USA). Cell-associated fluorescence was monitored at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. The internal calcium concentrations were calculated as described by Grynkiewicz et al. .
Statistical analyses were performed using a two-tailed Student's paired t-test on the raw data with GraphPad Prism 4 software (GraphPad Software, La Jolla, CA, USA). Significance was considered to be attained at a value of P < 0.05.
Monosodium urate crystals reduce myeloid inhibitory C-type lectin-like receptor expression in human neutrophils
data not shown), modulated MICL plasma membrane expression. Similarly, we detected no change in the surface expression of MICL in response to the particulate stimulus, nonopsonized zymosan (Figure 1B). MSU is thus the only stimulus tested that can directly induce the internalization of cell surface MICL. To determine whether the internalization of MICL observed with the above stimuli could be induced with the anti-MICL antibody (clone 50C1), neutrophils were incubated with 50C1 antibody, and internalization was assessed by flow cytometry with a fluorochrome-conjugated secondary antibody as described in the Methods section. We show that 50C1 can induce the internalization of cell surface MICL in human neutrophils (Figure 1C).
Myeloid inhibitory C-type lectin-like receptor negatively regulates the production of IL-8 by monosodium urate crystal-activated human neutrophils
To verify that the expression of MICL can be knocked down in PLB-985 cells, the cells were transfected with control siRNA or MICL siRNA, and the cell surface expression of MICL was determined by flow cytometry. MICL siRNA significantly diminishes the cell surface expression of MICL (P = 0.021) (Figure 3B). Moreover, the silencing of MICL expression has no effect on the differentiation of PLB-985 cells. The level of expression of Mac-1 and FLPR1 is the same in differentiated cells transfected with the control or MICL siRNA (data not shown).
Having demonstrated that MICL expression can be silenced by MICL-specific siRNA in neutrophil-like PLB-985 cells, we next examined how the diminution in MICL expression affects MSU-induced IL-8 production. The amount of IL-8 measured in the cell-free supernatant of MSU-activated, dibutyryl cAMP-differentiated PLB-985 silenced for MICL expression was significantly superior in comparison to cells transfected with control siRNA (Figure 3C). To confirm this observation in human neutrophils, the internalization of cell surface MICL was induced with the 50C1 antibody prior to stimulation with MSU, as shown in Figure 1C. Neutrophils incubated with 50C1 prior to stimulation with MSU produced significantly larger amounts of IL-8 in the cell-free supernatant (Figure 3D) than in those treated with an isotype antibody (IgG2a). Thus, using two different experimental strategies, we have produced evidence that a reduction in the cell surface expression of MICL potentiates MSU-induced IL-8 production in neutrophils.
Production of IL-1 by monosodium urate crystal-activated human neutrophils is not regulated by myeloid inhibitory C-type lectin-like receptor
Diminution of myeloid inhibitory C-type lectin-like receptor expression enhances monosodium urate crystal-induced signaling in human neutrophils
Together, the above findings are suggestive of a negative regulatory role for MICL because a decrease in its cell surface expression enhances two of the earliest signaling events in neutrophils upon stimulation with MSU, the tyrosine phosphorylation of intracellular proteins and an increase in intracellular levels of cytoplasmic free calcium.
Colchicine reduces the internalization of myeloid inhibitory C-type lectin-like receptor on human neutrophils
Colchicine inhibits the production of IL-8 by monosodium urate crystal-activated human neutrophils
Inhibitory receptors are essential for the maintenance of immune homeostasis by abrogating signaling pathways that lead to cellular activation . Even though inhibitory receptors may contribute to the pathogenesis of inflammatory diseases by virtue of their ability to modulate leukocyte activation, little is known about their roles in disease. This is the first report of the modulation of the expression of an inhibitory receptor by the etiological agent of gout. The activation of human neutrophils with MSU results in the loss of MICL expression. The functional significance of the diminution of MICL expression is an enhancement in the MSU-induced release of IL-8. This finding is of clinical relevance because IL-8 is a potent neutrophil chemoattractant. The loss of MICL expression may thus be a requisite for the recruitment and/or perpetuation of neutrophil-driven gout flares. Moreover, we also show that an inflammatory drug used to treat gout, colchicine, inhibits the effect of MSU on MICL expression.
Our observation that MSU downregulates MICL expression adds to a growing list of proinflammatory stimuli that diminish the cell surface expression of MICL in neutrophils. MSU is the first damage-associated molecular pattern demonstrated to modulate MICL expression. The biological significance of the negative modulation of MICL expression in neutrophils by proinflammatory stimuli other than MSU (mostly TLR agonists) remains largely unexplored, rendering the interpretation of these results challenging. One report has investigated the functional effect of the diminution of cell surface MICL on cytokine synthesis in monocyte-derived dendritic cells . In that report, Chen et al. demonstrated that a diminution in the expression of cell surface MICL inhibited or augmented cytokine production in a stimulus-dependent manner. In response to LPS, the production of TNF-α, IL-12p40 and IL-12p70 was suppressed upon MICL internalization. In contrast, the production of TNF-α, IL-12p40, IL-12p70, IL-6 and IL-10 was enhanced in response to CD40 ligand. Since inhibitory receptors can dampen cellular activation or, in some cases, activate the cells, it remains unclear whether MICL is an inhibitory receptor. We used a siRNA approach to resolve this issue. Our demonstration that the silencing of MICL expression significantly enhances the release of IL-8 by MSU-activated neutrophils strongly supports the notion that MICL acts as an inhibitory receptor in human neutrophils.
The modulation of IL-8 production by MICL in neutrophils is of relevance to gout. The massive recruitment of neutrophils to the inflamed joint is a pathological hallmark of gout, and this recruitment is reduced in mice deficient in chemokine (C-X-C motif) receptor 2 (CXCR2). It is thus reasonable to propose that the MSU-induced downregulation of MICL expression releases the inhibitory activity of this receptor in neutrophils permitting the activation of the neutrophil by this nonmicrobial agent. Interestingly, MICL does not regulate the MSU-induced production of IL-1β. The molecular mechanisms underlying the selective nature of MICL regulation of the production of cytokines by MSU-activated neutrophils remain to be identified. Whether MICL modulates the release of other chemotactic factors or cytokines by neutrophils remains to be determined. With regards to the ability of MICL to modulate additional MSU-induced neutrophil responses, we did not observe any modulation of MSU-induced degranulation or 5-lipoxygenase production in human neutrophils subsequent to the antibody-induced internalization of MICL (data not shown). These observations suggest that MICL modulates a subset of molecular pathways employed by MSU to activate human neutrophils.
At the molecular level, we show that MICL modulates early MSU-induced signaling events in neutrophils. The stimulation of neutrophils with MSU induced the loss of cell surface MICL leading to the enhancement of the MSU-induced increase in the concentration of intracellular calcium and tyrosine phosphorylation of intracellular substrates. These observations corroborate the observed enhancement in MSU-induced IL-8 production upon the downregulation of MICL expression. Both MSU-induced tyrosine phosphorylation and IL-8 production depend on the activation of Src kinases in neutrophils. The inhibition by MICL of MSU-induced early signaling events is consistent with the known mode of action of inhibitory receptors and is strongly suggestive that MICL may regulate several MSU-induced neutrophil effector functions driven by downstream signaling events.
Neutrophils pretreated with colchicine internalize significantly less cell surface MICL in the presence of MSU. This observation provides additional evidence for the ability of MICL to negatively regulate the MSU-induced activation of neutrophils. We propose that colchicine preserves the cell surface expression and consequently the inhibitory activity of MICL, shifting the balance between pro- and anti-inflammatory signals toward the latter. It thus follows that MSU may induce tyrosine phosphorylation of intracellular substrates and the mobilization of intracellular calcium in part by internalizing MICL, because these neutrophil responses are enhanced subsequent to the antibody-induced internalization of MICL and inhibited by colchicine. Regarding the MSU-induced secretion of IL-8, the ability of colchicine to inhibit this neutrophil response to MSU remains unexplored. We thus investigated the effect of colchicine on the production of this cytokine in response to MSU. Colchicine downregulates the MSU-induced release of IL-8 in human neutrophils (Figure 8). Together, these observations strongly suggest that MICL regulates very early signaling events involving Src kinases because MSU-induced tyrosine phosphorylation of intracellular substrates, as well as the increases in the concentrations of free cytosolic calcium and the production of IL-8, are all Src tyrosine kinase-dependent events. MICL is the first inhibitory pathway identified that could partly explain the antiphlogistic activity of colchicine. Since colchicine destabilizes microtubules by binding α and β monomers of tubulin, it is not unreasonable to suggest that MICL interacts directly or indirectly with the microtubule network.
A limitation of our experimental approach is that the internalization of MICL was induced prior to MSU stimulation to investigate the effect of MICL on MSU-induced responses. Although this approach is widely used to investigate inhibitory receptors whose ligands remain unidentified, it remains to be determined whether cells expressing a mutant form of MICL that is resistant to MSU-induced internalization and/or degradation respond more weakly to MSU.
As mentioned above, MSU are the first damage-associated molecular pattern shown to modulate MICL expression and consequentially its function. The regulation of MICL expression by damage-associated molecular patterns may differ from that of pathogen-associated molecular patterns and other proinflammatory stimuli. Indeed, MSU is the only stimulus that we studied that modulates MICL expression in resting neutrophils. Moreover, studies that previously published the diminution of cell surface MICL by stimuli other than damage-associated molecular patterns were performed on primed neutrophils. We show that in resting neutrophils, however, MICL expression is not modulated by proinflammatory stimuli that are not damage-associated molecular patterns. Together, these observations indicate that neutrophils have to receive two stimuli and/or signals prior to mobilizing cell-surface MICL when activated with non-damage-associated molecular pattern stimuli. In contrast, damage-associated molecular patterns seem to be able to override the necessity of a primary signal (neutrophil priming) and affect MICL expression directly.
Our findings strongly suggest that MICL may play a pathogenic role in gout by being one of the targets of MSU. We propose that MSU downregulate the inhibitory activity of MICL by diminishing its expression, resulting in the full activation of human neutrophils. In contrast, colchicine has the opposite effect on the cell surface expression of MICL. MICL is thus a potential therapeutic target for gout. Further characterization of the role of MICL in MSU-induced neutrophil activation and the molecular events driving MICL function is necessary to shed light on the pathogenic role of MICL in gout and its potential as a drug target. Since neutrophils are the principal players in the inflammatory flares typical of gout, it is not unreasonable to suggest that MICL may also play a similar pathogenic role in other chronic diseases characterized by recurrent, neutrophil-driven inflammatory episodes, such as rheumatoid arthritis. It is thus of interest to gain further insight into the potential role of MICL in the effector phase of these chronic inflammatory diseases.
C-type lectin-like domain
- dibutyryl cAMP:
N6:2'-O-dibutyryladenosine-3':5'-cyclic monophosphate sodium salt
immunoreceptor tyrosine-based inhibitory motifs
myeloid inhibitory C-type lectin-like receptor
monosodium urate crystals
The authors thank Dr Alexandre Brunet for expert technical assistance in flow cytometry analysis. We also thank Myriam Vaillancourt for her technical assistance in IL-8 ELISA analysis. This research was funded by operating funds awarded to MJGF by the Canadian Arthritis Network (CAN), the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Crohn's and Colitis Foundation of Canada (CCFC). LM is a recipient of the Canadian Arthritis Network post-doctoral award and VG received a Ph.D. scholarship from Fonds de recherche Québec-Santé and the CCFC. MJGF is a recipient of the Arthritis Society Investigator Award.
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