FASEB J. Avanti Polar Lipids
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(The FASEB Journal. 1998;12:209-220.)
© 1998 FASEB

Crystal-induced neutrophil activation VI. Involvement of Fc{gamma}RIIIB (CD16) and CD11b in response to inflammatory microcrystals

Frédéric Barabéa, Caroline Gilberta, Nathalie Liaoa, Sylvain G. Bourgoinb and Paul H. Naccachea,1

a Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL
b Departments of Medicine and Physiology, Faculty of Medicine, Laval University, Ste-Foy, Québec, G1V 4G2, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Fc{gamma}RIIIB (CD16) in response to inflammatory microcrystals. FASEB J. 12, 209–220 (1998)


Key Words: tyrosine phosphorylation • inflammation • gout • signal transduction • opsonin receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE ROLE OF THE DEPOSITION of monosodium urate (MSU) crystals in synovial fluid and the synovium is well established in the etiology of acute gouty arthritis and the development of the articular symptoms of pain and tenderness. These clinical manifestations of crystal deposition are the result of an important intra-articular inflammatory reaction in which polymorphonuclear neutrophils play a critical role in the phagocytosis of MSU crystals (1–4). Another crystal-induced articular disease, articular chondrocalcinosis (pseudogout), which is caused by calcium pyrophosphate dihydrate (CPPD) crystals, also produces joint inflammation (5–7).

In the past 20 years, the physiologic consequences of the stimulation of neutrophils by MSU and/or CPPD crystals have been intensively investigated. Various studies have shown that neutrophil–crystal interactions lead to the production and secretion of proinflammatory products such as lysosomal enzymes (8), oxygen-derived free radicals (9, 10), eicosanoids (11, 12), interleukin 1 (IL-1) (13), and IL-8 (14, 15). Also, activation of phospholipases A2 (16) and D (17), intracellular calcium mobilization (12, 18, 19), inositol 1,4,5 triphosphate formation (20), and increased levels of protein tyrosine phosphorylation (21–23) have been demonstrated in neutrophils in response to stimulation by microcrystals. Although neutrophils are the predominant cell type involved in gout, other cells of the articular milieu such as monocytes and fibroblast-like synoviocytes also respond to MSU crystals and release IL-1 (24), IL-6 (25), IL-8 (15, 26), and tumor necrosis factor {alpha} (27), all of which may contribute in varying degrees to the inflammatory response.

Despite the advances in our understanding of neutrophil responses to pathogenic crystals, much remains to be clarified about the signal transduction pathways involved, particularly the initial determinants of their interaction with neutrophils. Several lines of evidence indicate that very early synovial fluids are poor in proteins and that MSU crystals during the initiation phases of the inflammatory response have adsorbed little proteins; these include predominantly immunoglobulins (and no lipoproteins) (28). Neutrophils, platelets, and monocytes repeatedly have been shown to respond to naked crystals (see references in paragraph above). The interaction of naked MSU crystals with platelets has been shown to be mediated, at least in part, by their ability to interact with membrane integrins (GPIIb/IIIa) (29).

The present studies were initiated to further identify the pathway (or pathways) involved in the activation of human neutrophils by inflammatory microcrystals—in particular, to characterize the surface antigens they react with during initiation of the inflammatory reaction. The focus of these investigations was to examine the role of Fc{gamma} receptors, both Fc{gamma}RIIA (CD32) and Fc{gamma}RIIIB (CD16), and of ß2-integrins (CD11b/CD18) in the responses of human neutrophils to inflammatory microcrystals. The results obtained provide evidence that the activation of human neutrophils by MSU crystals (as monitored by stimulation of tyrosine phosphorylation, mobilization of calcium, the production of superoxide anions, and stimulation of the activity of PLD) is mediated primarily by Fc{gamma}RIIIB, and secondarily by CD11b.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
VIFcRIII anti-Fc{gamma}RIII antibodies were purchased from Accurate Antibodies, Inc. (Westbury, N.Y.); the 3G8 anti-Fc{gamma}RIII antibodies were purified from ascites fluid of mouse inoculated with the hybridoma 3G8, generously provided by Dr. J. Unkeless (Mount Sinai School of Medicine, New York, N.Y.). The anti-Cbl and the anti-Syk antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.) and the antiphosphotyrosine antibody (UBI 05–321, clone 4G10) from Upstate Biotechnology, Inc. (Lake Placid, N.Y.). Anti-Fc{gamma}RII (IV.3, hybridoma HB 217), anti-CD11b (OKM1, hybridoma CRL 8026), anti-CD11a (hybridoma TS1/22.1.1.13), and anti-CD18 (TS1/18.1.2.11 hybridoma HB 203) were purified from ascites fluid of mice inoculated with the hybridomas, which were obtained from the American Type Culture Collection (ATCC) (Rockville, Md.). The anti-CD11b antibody VIM12 was generously provided by Dr. W. Knapp (Institute of Immunology, University of Vienna, Vienna, Austria), and the anti-CD11b (MEM 170) and anti-CD16 (MEM 154) antibodies were kindly provided by Dr. V. Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic). GAM-FITC was obtained from Jackson Immune Research (West Grove, Pa.).

Reagents
Di-isopropylfluorophosphate and fMet-Leu-Phe were purchased from Sigma Chemical Co. (St. Louis, Mo.). Triclinic MSU and CPPD crystals were kindly provided by Drs. R. de Médicis and A. Lussier (University of Sherbrooke, Sherbrooke, Québec, Canada) and prepared as previously described (21). The crystals used in this study were characterized by X-ray diffraction (Geigerflex D/max, Rigaku, Mass.) and examined under phase and polarization microscopy and by scanning electron microscopy. Several distinct lots of crystals (sizes between 10 and 20 µM, specific areas between 0.7 and 2.4 m2/g) were used with identical results (data not shown). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was supplied by the Genetics Institute (Cambridge, Mass.). Dextran T-500, Ficoll paque, and Sephadex G-10 were purchased from Pharmacia Biotech (Dorval, Québec, Canada); fura-2/AM was from Molecular Probes (Junction City, Oreg.). Cytochrome c was obtained from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.).

Neutrophil purification
Venous blood was collected in isocitrate anticoagulant solution from healthy adult volunteers after receiving their signature on a consent form approved by the Centre de Recherche du CHUL human experimentation ethics committee. Neutrophils were purified as previously described (21) and resuspended in Hanks' balanced salt solution (HBSS) containing 0.8 mM calcium and no magnesium (pH 7.4).

Cell culture
HL-60 cells were obtained from ATCC. They were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, glutamine (2 mM), penicillin (100 µg/ml), and streptomycin (100 units/ml). Differentiation toward granulocytic phenotype was started by the addition of 1.25%. The cells were used between passages 4 and 12.

Tyrosine phosphorylation
Neutrophil suspensions (40x106 cells/ml) were preincubated at 4°C for 15 min with the indicated concentrations of antibodies or an equal volume of HBSS. Neutrophils were stimulated at 15 x 106 cells/ml at 37°C with 3 mg/ml of crystals for 5 min. In some experiments, cells were stimulated with 10-7 M fMet-Leu-Phe for 1 min or with GM-CSF (3 nM) for 10 min. After stimulation, 100 µl of the cells was added to an equal volume of boiling 2X Laemmli sample buffer (1X is 62.5 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitro-phenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol blue) and boiled for 7 min. Samples were then subjected to 7.5–20% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon PVDF membranes (Millipore Corporation, Bedford, Mass.). Immunoblotting was performed using the 4G10 antiphosphotyrosine antibody at a final dilution of 1/4000 and revealed by the ECL detection system, as previously described (30).

Immunoprecipitation
Neutrophils [pretreated with diisopropylfluoro-phosphate (1 mM) for 10 min at room temperature] were preincubated as described above and stimulated at 40 x 106 cells/ml at 37°C with 3 mg/ml of MSU for 5 min. Aliquots (500 µl) of the cells were lysed in an equal volume of boiling lysis buffer (1X is 62.5 mM Tris-HCl, pH 6.8, 3% SDS, 1.5% ß-mercaptoethanol, 8.5% glycerol, 2.5 mM orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.025% bromophenol blue) and boiled for 7 min. Immunoprecipitation was performed as previously described (31). Briefly, the lysates were filtered through Sephadex G-10 columns in order to remove the denaturing agents. The filtered lysates were precleared with protein A Sepharose at 4°C for 30 min in the presence of 1% NP-40, 0.05% BSA, 2 mM orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The samples were then immunoprecipitated with 1.5 µg of anti-Cbl or anti-syk for 90 min at 4°C. Fifty microliters of protein A-Sepharose-conjugated beads (from a 30% slurry) were then added and the samples were incubated for 1 h at 4°C. The agarose beads were collected and washed four times with lysis buffer containing 1% NP-40 and no SDS or ß-mercaptoethanol. Sample buffer (40 µl, 2X) was added to the beads, which were boiled for 7 min. The samples were then electrophoresed as described above. The membranes were first blotted with the anti-phosphotyrosine antibodies and then incubated for 30 min at 56°C in stripping buffer (2% SDS, 100 mM ß-mercaptoethanol, 62.5 mM Tris-base, pH 6.7). The membranes were immunoblotted with anti-Cbl (final dilution 1/1000) or anti-syk (final dilution 1/500) as previously described (31).

Calcium mobilization
The cells (107 cells/ml) were incubated at 37°C with 1 µM fura-2/AM. Neutrophils were washed twice in HBSS to remove the extracellular probe, resuspended at 5 x 106 cells/ml, and transferred to the thermostatted (37°C) cuvette compartment of a spectrofluorimeter (SLM 8000, Aminco, Urbana, Ill.). The fluorescence of the cells 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 Tsien et al. (32).

Phospholipase D measurements
Neutrophils were prelabeled with [3H]lyso-PAF (2 µCi/107 cells) for 90 min. The cells were then washed and resuspended at 8 x 106 cells/ml. Half a milliliter of the cell suspensions was used for each sample. The cells were preincubated at 37°C for 1 min with 2.5 and 5 µg/ml of the antibodies indicated or an equal volume of HBSS. Ethanol (final concentration 1.0% vol/vol) was added immediately before addition of MSU crystals (3 mg/ml). The reactions were allowed to proceed for 5 min and were stopped by adding 1.8 ml of cold chloroform/methanol/HCl (50:100:1, vol/vol/vol) to 0.5 ml of the cell suspensions. The lipids were extracted according to the Bligh and Dyer procedure (33). Five micrograms of phosphatidylethanol (PEt) standard was added to each sample. Total lipid extracts were dried under nitrogen and resuspended in 50 µml of chloroform/methanol (2:1, vol/vol). The lipids were spotted on prewashed silica gel 60 TLC plates and PEt was separated from the other lipids by using the solvent mixture chloroform/methanol/acetic acid (65:15:2, vol/vol/vol). To visualize the different lipids, the plates were immersed in Coomassie brilliant blue solution (0.03% dye, 35% methanol and 200 mM NaCl). The different lipid classes were scraped off the plates in 1-cm lanes. Radioactivity was monitored by liquid scintillation counting and the results were corrected for background radioactivity and quenching.

Superoxide anion measurements
Superoxide production was measured by using the reduction of cytochrome c as previously described (34). Briefly, neutrophils (15x106 cells/ml) were incubated for 15 min at 4°C with 10 µg/ml of the indicated antibodies or an equal volume of HBSS. The cells were resuspended at 1 x 106 cells/ml with 130 µM cytochrome c and stimulated for 5 min at 37°C with the crystals (1.5 mg/ml). The reactions were stopped by transferring the tubes to an ice bucket for 15 min and then centrifuged for 10 min at 1500 g. The optical density of the supernatants was read at 540 and 550 nm, and the amount of superoxide produced was calculated from the difference between these two readings by using an extinction coefficient of 21.1.

Flow cytometry
Cell suspensions (107 cells/ml) were preincubated at 4°C for 30 min with 10 µg/ml of the indicated antibody (or an equal amount of isotype-matched control antibody). The cells were then washed twice in HBSS. GAM-FITC (100 µl of a 1/100 solution) was added for 30 min at 4°C in the dark, followed by two more washes. The fluorescence of the cells was then evaluated by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of crystal-induced tyrosine phosphorylation by anti-Fc{gamma}RIII antibodies
Neutrophils were incubated in the presence or absence of increasing concentrations (0 to 25 µg/ml) of IgM anti-Fc{gamma}RIII antibodies (VIFcRIII) or control IgM antibodies at 4°C for 15 min, transferred to 37°C, and stimulated with MSU crystals for 5 min. The levels of tyrosine phosphorylation in the cell samples were then determined by immunoblotting with anti-phosphotyrosine antibodies. The results of these experiments are illustrated in Fig. 1. As previously observed (21, 22, 35, 36), the addition of MSU crystals to human neutrophils increased the levels of tyrosine phosphorylation in a characteristic manner, with a predominance of bands in the 60–70 kDa region in addition to the bands at 120 and 140–150 kDa. The antibodies alone did not induce any response because they were not cross-linked. However, preincubation of the cells with VIFcRIII led to a concentration-dependent reduction of the tyrosine phosphorylation response to the crystals, with a decrease in intensity of all bands ( Fig. 1A). The control IgM antibodies had no effect ( Fig. 1B), thereby providing evidence for the specificity of the effects of the anti-Fc{gamma}RIII antibodies. The tyrosine phosphorylation response to MSU crystals were also inhibited by another anti-CD16 antibody (MEM 154) (data not shown). The inhibitory effects of VIFcRIII were maintained for at least 10 min of stimulation with MSU crystals, as equivalent levels of inhibition were observed when cells were processed for immunoblotting after 5 or 10 min of stimulation with the crystals ( Fig. 2).



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  		Figure 1. Concentration dependence of the effects of VIFcRIII (anti-CD16) on the tyrosine phosphorylation response induced by MSU microcrystals in human neutrophils. The cells were incubated with the indicated concentrations of VIFcRIII (A) or a control IgM (B), as described in Materials and Methods. The cells were stimulated with MSU crystals (3 mg/ml) for 5 min at 37°C. The cells were then processed and blotted with anti-phosphotyrosine antibodies. The data are from one experiment representative of at least four others.



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  		Figure 2. Maintenance of the inhibitory effects of VIFcRIII on the tyrosine phosphorylation induced by MSU crystals. The cells were incubated with 25 µg/ml for 15 min at 4°C as described in Materials and Methods. They were then stimulated with MSU crystals (3 mg/ml) for 5 or 10 min and processed as described in Materials and Methods for blotting with anti-phosphotyrosine antibodies. The data are from one experiment representative of three others.

The inhibitory effects of VIFcRIII extended to the effects of CPPD microcrystals. As shown in Fig. 3, CPPD crystals induced a pattern of tyrosine phosphorylation closely resembling that of MSU-stimulated crystals (21). Preincubation with VIFcRIII (25 µg/ml) reduced intensity of staining of most, if not all, bands whose phosphorylation was increased upon stimulation by CPPD crystals.



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  		Figure 3. Effect of VIFcRIII on the tyrosine phosphorylation response induced by CPPD crystals in human neutrophils. The incubation conditions were as in Fig. 1 except that CPPD crystals (3 mg/ml) were used, the antibody concentration was 25µg/ml, and incubation time with the crystals was 15 min. The data are from one experiment representative of at least three others.

The inhibitory effects of VIFcRIII were not reproduced by 3G8, which also recognize Fc{gamma}RIII, but at a different epitope (data not shown). 3G8 at concentrations up to 25 µg/ml had no effect on MSU crystal responses. The integrity of 3G8 was verified by testing its ability to label neutrophils for flow cytometry analyses (data not shown).

The generality of the inhibitory effects of the anti-Fc{gamma}RIII antibodies (VIFcRIII) was tested by examining their potential effects on the tyrosine phosphorylation responses to soluble agonists. Two classes of agonists were used: chemotactic factors, which interact with G-protein-coupled receptors, and the growth factor GM-CSF, the receptors of which belong to the hematopoietin cytokine receptor superfamily. As shown in Fig. 4, neutrophils responded to fMet-Leu-Phe and GM-CSF with characteristic patterns of tyrosine phosphorylation (35). The latter differ qualitatively (different ratios of intensity of the tyrosine phosphorylation of the various bands) (21) and pharmacologically (the response to the crystals is inhibited by colchicine; that to other neutrophil agonists are not) (22) from that induced by inflammatory microcrystals. VIFcRIII, at concentrations that maximally inhibited response to the microcrystals (25 µg/ml), had no effect on the increases in tyrosine phosphorylation induced by either of the two soluble agonists.



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  		Figure 4. Lack of effect of VIFcRIII on the tyrosine phosphorylation responses to GM-CSF and fMet-Leu-Phe. Neutrophils were preincubated with VIFcRIII (25 µg/ml) as described in Materials and Methods, stimulated with fMet-Leu-Phe (107 M, 1 min) or GM-CSF (3 nM, 10 min), and processed for immunoblotting with anti-phosphotyrosine antibodies. The data are from a single experiment representative of three others.

The second class of low-affinity Fc{gamma} receptors on neutrophils, Fc{gamma}RIIA or CD32, does not appear to play a direct role in mediating the effects of MSU crystals. As shown in Fig. 5, antibody IV.3, which blocks (among others) the adhesion of neutrophils to IgG-coated surfaces (37), had no effect on the stimulation of tyrosine phosphorylation induced by MSU crystals. Concentrations of antibody IV.3 (anti-CD32) as high as 50 µg/ml were tested and found not to affect the tyrosine phosphorylation responses to MSU crystals (data not shown). The activity of these antibodies was verified in parallel by demonstrating that their cross-linking induced an increase in the level of tyrosine phosphorylation in neutrophils (data not shown).



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  		Figure 5. Lack of effect of antibody IV.3 (anti-Fc{gamma}RIIA) on the tyrosine phosphorylation response induced by MSU crystals in human neutrophils. Neutrophils were treated with antibody IV.3 (25 µg/ml) and then stimulated with MSU crystals (3 mg/ml, 5 min at 37°C). The antiphosphotyrosine blot shown here is representative of at least three others.

The effects of anti-Fc{gamma}RIII antibodies on MSU-stimulated tyrosine phosphorylation of specific substrates were examined next ( Fig. 6). In these experiments, the cells were preincubated or not with VIFcRIII and stimulated for 5 min with MSU crystals (3 mg/ml). Cell lysates were immunoprecipitated with anti-syk or anti-Cbl antibodies. The precipitates were immunoblotted sequentially with anti-phosphotyrosine and then anti-syk or anti-Cbl antibodies. As shown in Fig. 6A, a basal level of tyrosine phosphorylation of syk was observed, which increased significantly upon stimulation by MSU crystals. The anti-Fc{gamma}RIII antibodies alone had no effect on the basal level of phosphorylation of syk, but inhibited completely the response to MSU crystals. Figure 6B demonstrates that equal immunoprecipitation and loading of syk had taken place in all lanes. Figure 6C illustrates that MSU crystals increased tyrosine phosphorylation of the proto-oncogene Cbl and that this response, as that of syk, was abrogated in the presence of anti-Fc{gamma}RIIIB antibodies. Figure 6D provides evidence for equal loading of Cbl.



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  		Figure 6. Inhibition by VIFcRIII of the stimulation of the tyrosine phosphorylation of syk and Cbl induced by MSU crystals in human neutrophils. The cells were preincubated with VIFcRIII as described in Materials and Methods before being stimulated with MSU crystals (3 mg/ml, 5 min at 37°C). The cells were then lysed and treated for immunoprecipitation with anti-syk (A, B) or anti-Cbl (C, D) antibodies as described in Materials in Methods. After blotting with anti-phosphotyrosine antibodies (A, C), the membranes were stripped and reblotted with anti-syk (B) or anti-Cbl (D) antibodies. The data are from a single experiment representative of at least four others.

Inhibition of crystal-induced tyrosine phosphorylation by anti-CD11b antibodies
CD16 on neutrophils is a GPI-linked isoform of Fc{gamma}RIII that has no transmembrane or cytoplasmic domains. Signal transduction through Fc{gamma}RIIIB has been postulated to depend on its interaction with CD11b (38–40). The communication between CD16 and CD11b is thought to be mediated by lectin-like intramembrane interactions (41, 42). Antibody VIM12 is an anti-CD11b antibody that recognizes an epitope near the polysaccharide binding domain of CD11b (43). As shown in Fig. 7, VIM12 inhibited in a concentration-dependent manner (1–25 µg/ml) the stimulation of tyrosine phosphorylation induced by MSU crystals in neutrophils. An isotype-matched control antibody had no effect ( Fig. 7B). On the other hand, MEM 170, another anti-CD11b antibody, also inhibited the tyrosine phosphorylation response to MSU crystals (data not shown).



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  		Figure 7. Concentration dependence of the effects of VIM12 (anti-CD11b) on the tyrosine phosphorylation response induced by MSU microcrystals in human neutrophils. The cells were incubated with the indicated concentrations of VIM12 (A) or a control, isotype-matched IgG (B) as described in Materials and Methods. The cells were stimulated with MSU crystals (3 mg/ml) for 5 min at 37°C. The cells were then processed and blotted with anti-phosphotyrosine antibodies. The data are from one experiment representative of at least four others.

The effects of VIM12 were not reproduced by OKM-1 (an anti-CD11b antibody that recognizes a epitope distinct from that interacting with VIM12) or by TS1/18.1.2.11 (an anti-CD18 antibody), which were without effect on the tyrosine phosphorylation responses to MSU crystals ( Fig. 8). Preliminary experiments indicated that TS1/22.1.1.13 (an anti-CD11a antibody) was similarly without effect on responses to MSU crystals (data not shown).



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  		Figure 8. Lack of effect of OKM1 (anti-CD11b) and TS1/18.1.2.11 (anti-CD18) on the tyrosine phosphorylation response of human neutrophils to MSU crystals. The cells were preincubated with 25 µg/ml of either antibody, as described in Materials and Methods. They were then stimulated with MSU crystals (3 mg/ml) for 5 min at 37°C and processed for blotting with anti-phosphotyrosine antibodies. The data are representative of at least three independent experiments.

Functional responses
The effects of the anti-Fc{gamma}RIII antibodies on responses to the crystals other than tyrosine phosphorylation were investigated next. In these experiments, the antibody concentrations were adjusted to give a final ratio of about 0.7 µg antibody/106cells, conditions found to provide an optimal signal-to-noise ratio (data not shown). MSU and CPPD crystals have previously been shown to induce a mobilization of calcium (12, 20). To accommodate the requirements of maintaining ionic balance in the cells and the fluorescent probe loading protocol, antibodies were added to the cells 1 min before the addition of MSU crystals. As shown in Fig. 9 and reported previously (12), MSU crystals (1.5 mg/ml) induced a rapid mobilization of calcium in human neutrophils. The addition of VIFcRIII (5 µg/ml) reduced to a large degree the increase in the level of free cytoplasmic calcium induced by microcrystals. Higher concentrations of the antibodies interfered with the calcium assay and could not be used. In addition, the kinetics of the antibody-resistant portion of the mobilization of calcium was significantly slower than that observed in control cells. A control IgM had no effect on the calcium response to microcrystals. VIM12 (1–5 µg/ml) also inhibited the mobilization of calcium induced by MSU crystals (data not shown).



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  		Figure 9. Inhibition by VIFcRIII of the mobilization of calcium induced by MSU crystals in human neutrophils. The cells were loaded with fura-2 as described in Materials and Methods. The antibodies (VIFcRIII or IgM control) were added at the first arrow (marked Ab) and the crystals (1.5 mg/ml) at the second arrow (marked MSU). The data are from a single experiment representative of at least four other independent determinations.

Preincubation with anti-Fc{gamma}RIII antibodies was also found to inhibit the activation of PLD by MSU microcrystals ( Fig. 10). In these experiments, PLD activity was monitored by following the formation, in the presence of ethanol, of PEt. The amount of PEt formed in response to MSU crystals decreased as a function of the concentration of VIFcRIII used. The control IgM had no effect on the response of the cells. A 39 ± 5% (P<0.01) and 57 ± 6% (P<0.01) inhibition of the MSU-stimulated formation of PEt was observed at 2.5 and 5.0 µg/ml VIFcRIII, respectively. At 25 µg/ml, VIFcRIII completely inhibited the responses to the crystals, but the control IgM (at the same concentration) started exhibiting nonspecific effects and inhibited the formation of PEt by about 30% (results not shown).



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  		Figure 10. Inhibition by VIFcRIII of the stimulation of the activity of PLD induced by MSU crystals in human neutrophils. The cells were labeled and processed for the determination of PLD activity as described in Materials and Methods. The antibodies (VIFcRIII or IgM control) were added 1 min before the addition of MSU crystals (3 mg/ml). The data represent the means ±SEM of three independent experiments.

A partial inhibition of superoxide production in response to MSU crystals was also observed. The cells (15x106 cells/ml) were preincubated with 10 µg/ml VIFcRIII or VIM12 for 15 min at 4°C. VIFcRIII-treated neutrophils produced only 66 ± 9% of the amount of superoxide of control cells (P<0.01). A control IgM had no significant effect. VIM12 also inhibited the superoxide response induced by MSU crystals, the treated cells producing only 65 ± 8% (P<0.01) of the amount of superoxide of control cells. An isotype-matched control IgG had no significant effect.

Tyrosine phosphorylation responses of HL-60 cells to MSU crystals
The data presented above suggest that the responses of human neutrophils to MSU crystals are dependent on interactions with CD16, and possibly also CD11b. A corollary of this hypothesis is that other cells that express these opsonin receptors should also respond to the crystals, and conversely, cells that do not express them should not.

HL-60 cells are a promyelocytic leukemia cell line that can be induced to exhibit a neutrophil-like phenotype upon differentiation by a variety of agents, including Me2SO4. Undifferentiated HL-60 cells express neither CD16 nor CD11b/CD18 (or only at very low levels) and do not exhibit a significant response (increase in tyrosine phosphorylation) to MSU crystals (data not shown). The addition of Me2SO4 to HL-60 cells increases the surface expression of both CD11b/CD18 and CD16 ( Fig. 11). Concomitant with the expression of these receptors, HL-60 cells acquire the ability to respond to MSU crystals. As shown in Fig. 11, the addition of MSU crystals to Me2SO4-differentiated HL-60 cells results in the stimulation of a pattern of tyrosine phosphorylation that closely resembles that observed in human neutrophils.



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  		Figure 11. Tyrosine phosphorylation responses of HL-60 cells to the addition of MSU crystals. HL-60 cells were induced to differentiate into granulocyte-like cells by the addition of 1.25% Me2SO4. On the day indicated, the cells were stimulated with MSU crystals (3 mg/ml) and processed for blotting with anti-phosphotyrosine antibodies or for flow cytometry analysis of the expression of CD16 (using antibody 3G8) or CD11b (using antibody OKM1). MF: Mean fluorescence; %: % of labeled cells; ND: not determined. The data are from a single experiment representative of at least three others.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data obtained in the course of the present investigation provide evidence linking Fc{gamma}RIIIB (CD16) and CD11b to the mediation of the activation of human neutrophils by inflammatory microcrystals. To the best of our knowledge, these data represent the first identification of surface antigens that MSU and CPPD crystals interact with on human neutrophils. Several lines of evidence support the view that MSU and CPPD crystals interact primarily with CD16 on the surface of human neutrophils and secondarily with CD11b. These (opportunistic) interactions could play an important part in explaining the phlogistic potential of inflammatory microcrystals, as they appear to be critical to the subsequent activation of neutrophils.

These conclusions are based on the inhibitory effects of anti-Fc{gamma}RIII and anti-CD11b antibodies. The anti-Fc{gamma}RIII antibodies (VIFcRIII) inhibited tyrosine phosphorylation (including that of Cbl and syk), mobilization of calcium, activation of PLD, and the production of superoxide induced by MSU crystals. These four parameters are of particular functional relevance to the phlogistic activities of MSU and CPPD microcrystals. The pattern of tyrosine phosphorylation to MSU and CPPD crystals is qualitatively distinct (21) and pharmacologically specific (exclusive sensitivity to colchicine) (22) to these agonists. Calcium is a well-established second messenger; inflammatory microcrystals are particularly potent activators of PLD (17), an enzymatic pathway capable of producing several products with signaling potential (phosphatidic and lysophosphatidic acid, diglyceride); and the activation of NADPH oxydase is believed to be involved in many manifestations of inflammatory disorders. The ability of anti-Fc{gamma}RIII antibodies to inhibit these various responses therefore profoundly affects activation of the cells by inflammatory microcrystals.

Neutrophils constitutively express two classes of Fc{gamma} receptors: Fc{gamma}RIIA and Fc{gamma}RIIIB. The data presented above link Fc{gamma}RIIIB to the responses of human neutrophils to MSU and CPPD crystals. Fc{gamma}RIIIB is a phosphatidylinositol-glycan-linked Fc{gamma} receptor that lacks transmembrane and cytoplasmic domains (44). Is is presently unclear how Fc{gamma}RIIIB transmits signals to the inside of the cells. However, studies of transfected cells and of neutrophils with antibodies have provided evidence for a close physical association between Fc{gamma}RIIIB and CD11b/CD18 (39, 40, 45, 46) as well as a role for Fc{gamma}RIIA (47–49). Cross-linking Fc{gamma}RIIIB with VIFcRIII stimulated a pattern of tyrosine phosphorylation in human neutrophils resembling that induced by MSU crystals in that it included predominantly bands in the 60–70 and 120 kDa regions (results not shown). Previous studies have described degranulation, calcium, and (in some studies) oxydative responses to the cross-linking of Fc{gamma}RIIIB in neutrophils (48, 50–52). We have also observed that the tyrosine phosphorylation response to cross-linking of Fc{gamma}RIII is—similar to that induced by microcrystals and contrary to the responses to chemotactic factors (22)—inhibited by the microtubule-disrupting agent colchicine (data not shown).

Evidence for a mediatory role of CD11b was also obtained. VIM12 is an anti-CD11b antibody that reacts with a region of this surface antigen close to the polysaccharide binding region and is apparently involved in intramembrane, lectin-type interaction with CD16 (43). The lack of effect of OKM1, another CD11b antibody with a different epitope specificity, suggests that CD11b acts by mediating the effect of the engagement of CD16 by MSU crystals and not by interacting directly with MSU crystals. The relative lack of tyrosine phosphorylation response of HL-60 cells exposed to Me2SO4 for short periods of time (1–3 days), which express nearly uniformly CD11b (but not CD16) to the crystals, is consistent with this hypothesis. It is only when a significant percentage of HL-60 cells express CD16 (and maintain their CD11b complement) that they exhibit significant responses to MSU. These data indicate that the involvement of CD11b is secondary to the interaction of the crystals with CD16. The stimulated interactions between CD16 and CD11b/CD18 (39, 40, 45, 46) may provide a mechanistic explanation for these data.

The data presented above provide no evidence for a (direct) role of Fc{gamma}RII in mediating human neutrophil responses to MSU crystals. Antibody IV.3, which is known to inhibit the adhesion of neutrophils to IgG-coated plates (37) and binding of immune complexes (53), had no effect on any of the parameters of neutrophil activation by MSU crystals tested. Although no evidence supporting a role of CD32 has been obtained in the course of the present study, the possibilities that these interactions may take place cannot be excluded. More studies with additional anti-Fc{gamma}RII antibodies with distinct epitope specificities and with different cell types (e.g., CD16-, CD32+) are required.

A partial sensitivity of some neutrophil responses to inflammatory microcrystals (calcium mobilization, superoxide production) had previously been noted (18–20). The data described above indicate that these interactions are only secondary to the initial activation of CD16/CD11b. The magnitude of the inhibition by antiCD16/CD11b antibodies far exceeds that observed with pertussis toxin (35), whereas the responses to fMLP (an agonist whose receptors belong to the 7-transmembrane spanning family) are not sensitive to these antibodies. The calcium responses to the crystals are to a large extent sensitive to tyrosine kinase inhibitors (54; unpublished observations), whereas those same responses elicited by fMLP are completely insensitive to the same inhibitors (unpublished observations). In addition, we have preliminary data, not described here, showing that the expression of CD16 at the surface of human neutrophils decreases during the time required for pertussis toxin to penetrate the cells. Thus, it is possible that the partial sensitivity to pertussis toxin described previously was uncovered only because of a decrease in the expression of the primary "receptors" the crystals interact with.

The ability of MSU crystals to stimulate the tyrosine phosphorylation of syk had not been reported previously. This tyrosine kinase has been closely related to the mechanisms of activation of phagocytic cells upon engagement of Fc{gamma} receptors (55, 56). The observation that MSU crystals increase the tyrosine phosphorylation level of syk is therefore consistent with the hypothesis that Fc{gamma} receptors are involved in the interaction of inflammatory microcrystals with neutrophils. The ability of anti-Fc{gamma}RIII antibodies to inhibit the MSU-stimulated tyrosine phosphorylation of syk further supports this conclusion.

Cbl is a proto-oncogene (57, 58) that has been shown to be tyrosine phosphorylated in response to a variety of agonists (59–65), particularly in response to ligation of Fc{gamma} receptors (66, 67). Even though the precise role of Cbl is unknown, it has been shown to associate in a stimulus-dependent manner with various elements of the tyrosine phosphorylation signaling pathway (61–63, 65, 68–73), thereby suggesting that it is intimately involved in these responses (58). We have previously observed that Cbl is tyrosine phosphorylated in human neutrophils exposed to phagocytic agonists (data not shown). The data presented in Fig. 6confirm that tyrosine phosphorylation levels of Cbl are increased upon stimulation by MSU crystals. The ability of the anti-Fc{gamma}RIII antibodies to abrogate this response therefore provides additional evidence for a mediatory role of CD16 in the responses elicited by MSU crystals.

The lack of effect of VIFcRIII on the responses to chemotactic factors (fMet-Leu-Phe, IL-8) or to GM-CSF illustrates two distinct points. First, it demonstrates that the effects of the antibody are not generally deleterious to the cells, since they remain capable of responding normally to several types of agonists. Second, the specificity demonstrated of the effects of anti-Fc{gamma}RIII antibodies support the hypothesis that the interaction of inflammatory microcrystals with CD16 is an integral part of their phlogistic potential. MSU and CPPD microcrystals interact in an opportunistic manner with CD16. This leads to a potentially unregulated activation of this pathway and may play a critical part in the acute inflammatory reactions associated with these agonists. These data elicit the hypothesis that similar events may be involved in other clinical settings in which inflammatory reactions are associated with the presence of immune complexes (e.g., rheumatoid arthritis, systemic scleroderma, autoimmune diseases).


   ACKNOWLEDGMENTS
 
Supported in part by grants and fellowships from the Medical Research Council of Canada and from the Arthritis Society of Canada. The authors wish to thank Ms. Claire Léveillée for insightful discussions, Mr. Maurice Dufour, Ms. Danielle Harbour, and Mr. Sylvain Levasseur for their expert help with the flow cytometry analyses, PLD measurements, hybridoma cultures, and antibody purification, respectively.


   FOOTNOTES
 
1 Correspondence: CHUL, Room T1–49, 2705 Boulevard Laurier, Ste-Foy, Québec, G1V 4G2, Canada. E-mail: paul.naccache{at}crchul.ulaval.ca

2 Abbreviations: ATCC, American Type Culture Collection; MSU, monosodium urate; CPPD, calcium pyrophosphate dihydrate; PLD, phospholipase D; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; PEt, phosphatidylethanol; HBSS, Hanks' balanced salt solution; SDS, sodium dodecyl sulfate.

Received for publication July 15, 1997. Accepted for publication October 20, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gordon, T. P., Terkeltaub, R., and Ginsberg, M. H. (1988) Gout: crystal-induced inflammation. In Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, I. M., and Snyderman R., eds) 775–783, Raven Press, New York
  2. Terkeltaub, R. A. (1995) Gout: questions that still need to be answered. Ann. Rheum. Dis. 54, 79–81[Free Full Text]
  3. Seegmiller, J. E., Howell, R. R., and Malawista, S. E. (1962) The inflammatory reaction to sodium urate. J. Am. Med. Assoc. 180, 125–131
  4. Faires, J. S., and McCarty, D. J. (1962) Acute arthritis in man and dog after synovial injection of sodium urate crystals. Lancet 2, 682–685
  5. de Medicis, R., Lussier, A., Kahan, A., and Tetrault, L. (1983) Phagocytosis of sodium urate and calcium pyrophosphate crystals by leukocytes of gouty, pseudogouty and health donors. Eur. J. Rheum. Inflam. 6, 143–148[Medline]
  6. Roch-Arveiller, M., Legros, R., Chanaud, B., Muntaner, O., Strzalko, S., Thuret, A., Willoughby, D. A., and Giroud, J. P. (1990) Inflammatory reactions induced by various pyrophosphate crystals. Biomed. Pharmacother. 44, 467–474[Medline]
  7. Schumacher, H. R., Fishbein, P., Phelps, P., Tse, R., and Krauser, R. (1975) Comparison of sodium urate and calcium pyrophosphate crystal phagocytosis by polymorphonuclear leukocytes. Arthritis Rheum. 18, 783–792[Medline]
  8. Ginsberg, H., Kozin, F., Chow, D., May, J., and Skosey, J. L. (1977) Adsorption of polymorphonuclear leukocyte lysosomal enzymes to urate crystals. Arthritis Rheum. 20, 1538–1542[Medline]
  9. Simchowitz, L., Atkinson, J. P., and Spilberg, I. (1982) Stimulation of the respiratory burst in human neutrophils by crystals phagocytosis. Arthritis Rheum. 25, 181–188[Medline]
  10. Abramson, S., Hoffstein, S. T., and Weissmann, G. (1982) Superoxide anion generation by human neutrophils exposed to monosodium urate. Arthritis Rheum. 25, 174–180[Medline]
  11. Serhan, C. N., Lindberg, U., Weissmann, G., and Samuelsson, B. (1984) Formation of leukotrienes and hydroxy acids by human leukocytes exposed to monosodium urate. Prostaglandins 27, 563–581[Medline]
  12. Poubelle, P. E., de Médicis, R., and Naccache, P. H. (1987) Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-coupling sequence of human neutrophils. Biochem. Biophys. Res. Commun. 149, 649–657[Medline]
  13. Roberge, C. J., DeMédicis, R., Dayer, J. M., Rola-Pleszczynski, M., Naccache, P. H., and Poubelle, P. E. (1994) Crystal-induced neutrophil activation. IV. Differential production of biologically active IL-1 and IL-1 receptor antagonist. J. Immunol. 152, 5485–5494[Abstract]
  14. Miller, E. J., and Brelsford, W. G. (1993) Interleukin-8—the major neutrophil chemotaxin in a case of pseudogout. J. Rheumatol. 20, 1250–1252[Medline]
  15. Hachicha, M., Naccache, P. H., and McColl, S. R. (1995) Inflammatory microcrystals differentially regulate the secretion of macrophage inflammatory protein 1 and interleukin 8 by human neutrophils: a possible mechanism of neutrophil recruitment to sites of inflammation in synovitis. J. Exp. Med. 182, 2019–2025[Abstract/Free Full Text]
  16. Bomalaski, J. S., Baker, D. G., Brophy, L. M., and Clark, M. A. (1990) Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein. J. Immunol. 145, 3391–3397[Abstract]
  17. Naccache, P. H., Bourgoin, S., Plante, E., Roberge, C. J., Demedicis, R., Lussier, A., and Poubelle, P. E. (1993) Crystal-induced neutrophil activation. II. evidence for the activation of a phosphatidylcholine-specific phospholipase-D. Arthritis Rheum. 36, 117–125[Medline]
  18. Terkeltaubm, R. A., Sklar, L. A., and Mueller, H. (1990) Neutrophil activation by inflammatory microcrystals of monosodium urate monohydrate utilizes pertussis toxin-insensitive and -sensitive pathways. J. Immunol. 144, 2719–2724[Abstract]
  19. Naccache, P. H., Grimard, M., Roberge, C. J., Gilbert, C., Lussier, A., de Médicis, R., and Poubelle, P. E. (1991) Crystal-induced neutrophil activation. I. Initiation and modulation of calcium mobilization and superoxide production by microcrystals. Arthritis Rheum. 33, 333–342
  20. Onello, E., Traynor-Kaplan, A., Sklar, L., and Terkeltaub, R. (1991) Mechanism of neutrophil activation by an unopsonized inflammatory particulate. Monosodium urate crystals induce pertussis toxin-insensitive hydrolysis of phosphatidylinositol 4,5-bisphosphate. J. Immunol. 146, 4289–4294[Abstract]
  21. Gaudry, M., Roberge, C. J., de Médicis, R., Lussier, A., Poubelle, P. E., and Naccache, P. H. (1993) Crystal-induced neutrophil activation. III. Inflammatory microcrystals induce a distinct pattern of tyrosine phosphorylation in human neutrophils. J. Clin. Invest. 91, 1649–1655
  22. Roberge, C. J., Gaudry, M., De Médicis, R., Lussier, A., Poubelle, P. E., and Naccache, P. H. (1993) Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine. J. Clin. Invest. 92, 1722–1729
  23. Burt, H. M., Jackson, J. K., Dryden, P., and Salari, H. (1993) Crystal-induced protein tyrosine phosphorylation in neutrophils and the effect of a tyrosine kinase inhibitor on neutrophil responses. Mol. Pharmacol. 43, 30–36[Abstract]
  24. DiGiovine, F. S., Malawista, S. E., Nuki, G., and Duff, G. W. (1987) Interleukin 1 (IL1) as a mediator of crystal arthritis: stimulation of T cell and synovial fibroblast mitogenesis by urate crystal-induced IL1. J. Immunol. 10, 3213–3218
  25. Guerne, P. A., Terkeltaub, R., Zuraw, B., and Lotz, M. (1989) Inflammatory microcrystals stimulate interleukin-6 production and secretion by human monocytes and synoviocytes. Arthritis Rheum. 32, 1443–1152[Medline]
  26. Terkeltaub, R., Zachariae, C., Santoro, D., Martin, J., Peveri, P., and Matsushima, K. (1991) monocyte-derived neutrophil chemotactic factor/interleukin-8 is a potential mediator of crystal-induced inflammation. Arthritis Rheum. 34, 894–903[Medline]
  27. Di Giovine, F. S., Malawista, S. E., Thornton, E., and Duff, G. W. (1991) Urate crystals stimulate production of tumor necrosis factor alpha from human blood monocytes and synovial cells. J. Clin. Invest. 87, 1375–1381
  28. Ortizbravo, E., Sieck, M. S., and Schumacher, H. R. (1993) Changes in the proteins coating monosodium urate crystals during active and subsiding inflammation—immunogold studies of synovial fluid from patients with gout and of fluid obtained using the rat subcutaneous air pouch model. Arthritis Rheum. 36, 1274–1285[Medline]
  29. Jaques, B. C., and Ginsberg, M. H. (1982) The role of cell surface proteins in platelet stimulation by monosodium urate crystals. Arthritis Rheum. 25, 508–520[Medline]
  30. Al-Shami, A., Bourgoin, S. G., and Naccache, P. H. (1996) Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. I. Tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters. Blood 89, 1035–1044[Abstract/Free Full Text]
  31. Al-Shami, A., Gilbert, C., Barabé, F., Gaudry, M., and Naccache, P. H. (1997) Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. J. Immunol. Methods 202, 183–191[Medline]
  32. Grynkievicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450[Abstract/Free Full Text]
  33. Bligh, E. A., and Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917
  34. Naccache, P. H., Gilbert, C., Caon, A. C., Gaudry, M., Huang, C. K., Bonak, V. A., Umezawa, K., and McColl, S. R. (1990) Selective inhibition of human neutrophil functional responsiveness by erbstatin, an inhibitor of tyrosine protein kinase. Blood 76, 2098–2104[Abstract/Free Full Text]
  35. Rollet, E., Caon, A. C., Roberge, C. J., Liao, N. W., Malawista, S. E., McColl, S. R., and Naccache, P. H. (1994) Tyrosine phosphorylation in activated human neutrophils—comparison of the effects of different classes of agonists and identification of the signaling pathways involved. J. Immunol. 153, 353–363[Abstract]
  36. Roberge, C. J., Gaudry, M., Gilbert, C., Malawista, S. E., De Médicis, R., Lussier, A., Poubelle, P. E., and Naccache, P. H. (1996) Paradoxical effects of colchicine on the activation of human neutrophils by chemotactic factors and inflammatory microcrystals. J. Leukocyte Biol. 59, 864–871[Abstract]
  37. Kusunoki, T., Tsuruta, S., Higashi, H., Hosoi, S., Hata, D., Sugie, K., Mayumi, M., and Mikawa, H. (1994) Involvement of CD11b/CD18 in enhanced neutrophil adhesion by Fc gamma receptor stimulation. J. Leukocyte Biol. 55, 735–742[Abstract]
  38. Zhou, M.-j., and Brown, E. J. (1994) CR3 (Mac-a, alphaMbeta2, CD11b/CD18) and FcgammaRIII cooperate in generation of a neutrophil respiratory burst: requirement for FcgammaRII and tyrosine phosphorylation. J. Cell Biol. 125, 1407–1416[Abstract/Free Full Text]
  39. Zhou, M. J., Todd, R. F., Vandewinkel, J. G. J., and Petty, H. R. (1993) Cocapping of the leukoadhesin molecules complement receptor type-3 and lymphocyte function-associated antigen-1 with fc-gamma receptor-III on human neutrophils—possible role of lectin-like interactions. J. Immunol. 150, 3030–3041[Abstract]
  40. Krauss, J. C., Poo, H. Y., Xue, W., Mayobond, L., Todd, R. F., and Petty, H. R. (1994) Reconstitution of antibody-dependent phagocytosis in fibroblasts expressing Fc gamma receptor IIIB and the complement receptor type 3. J. Immunol. 153, 1769–1777[Abstract]
  41. Sehgal, G., Zhang, K., Todd, R. F., Boxer, L. A., and Petty, H. R. (1993) Lectin-like inhibition of immune complex receptor-mediated stimulation of neutrophils. Effects on cytosolic calcium release and superoxide production. J. Immunol. 150, 4571–4579[Abstract]
  42. Galon, J., Gauchat, J. F., Mazieres, N., Spagnoli, R., Storkus, W., Lotze, M., Bonnefoy, J. Y., Fridman, W. H., and Sautes, C. (1996) Soluble Fc gamma receptor type III (fc gamma RIII, CD16) triggers cell activation through interaction with complement receptors. J. Immunol. 157, 1184–1192[Abstract]
  43. Stockl, J., Majdic, O., Pickl, W. F., Rosenkranz, A., Prager, E., Gschwantler, E., and Knapp, W. (1995) Granulocyte activation via a binding site near the C-terminal region of complement receptor type 3 alpha-chain (CD11b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol-anchored fc gamma RIIIB (CD16) molecules. J. Immunol. 154, 5452–5463[Abstract]
  44. Hulett, M. D., and Hogarth, M. P. (1994) Molecular basis of the Fc receptor function. Adv. Immunol. 57, 1–19[Medline]
  45. Petty H. R., and Todd R. F. (1993) Receptor-receptor interactions of complement receptor type-3 in neutrophil membranes. J. Leukocyte Biol. 54, 492–494[Abstract]
  46. Poo, H., Krauss, J. C., Mayobond, L., Todd, R. F., and Petty, H. R. (1995) Interaction of fc gamma receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies. J. Mol. Biol. 247, 597–603[Medline]
  47. Edberg, J. C., and Kimberly, R. P. (1994) Modulation of Fcgamma and complement receptor function by the glycosyl-phosphatidylinositol-anchored form of FcgammaRIII. J. Immunol. 152, 5826–5835[Abstract]
  48. Salmon, J. E., Millard, S. S., Brogle, N. L., and Kimberly, R. P. (1995) Fc gamma receptor IIIb enhances Fc gamma receptor IIa function in an oxidant-dependent and allele-sensitive manner. J. Clin. Invest. 95, 2877–2885
  49. Unkeless, J. C., Shen, Z., Lin, C. W., and DeBeus, E. (1995) Function of human Fc gamma RIIA and Fc gamma RIIIB. Semin. Immunol. 7, 37–44[Medline]
  50. Mackenzie, S. J., and Kerr, M. A. (1995) IgM monoclonal antibodies recognizing Fc alpha R but not Fc gamma RIII trigger a respiratory burst in neutrophils although both trigger an increase in intracellular calcium levels and degranulation. Biochem. J. 306, 519–523
  51. Kobayashi, K., Takahashi, K., and Nagasawa, S. (1995) The role of tyrosine phosphorylation and Ca2+ accumulation in Fc gamma-receptor-mediated phagocytosis of human neutrophils. J. Biochem. Tokyo 117, 1156–1161[Abstract/Free Full Text]
  52. Hoffmeyer, F., Witte, K., Gebhardt, U., and Schmidt, R. E. (1995) The low affinity Fc gamma RIIa and Fc gamma RIIIb on polymorphonuclear neutrophils are differentially regulated by CD45 phosphatase. J. Immunol. 155, 4016–4023[Abstract]
  53. Strohmeier, G. R., Brunkhorst, B. A., Seetoo, K. F., Meshulam, T., Bernardo, J., and Simons, E. R. (1995) Role of the Fc gamma R subclasses Fc gamma RII and Fc gamma RIII in the activation of human neutrophils by low and high valency immune complexes. J. Leukocyte Biol. 58, 415–422[Abstract]
  54. Burt, H. M., Jackson, J. K., and Salari, H. (1994) Inhibition of crystal-induced neutrophil activation by a protein tyrosine kinase inhibitor. J. Leukocyte Biol. 55, 112–119[Abstract]
  55. Indik, Z. K., Park, J. G., Pan, X. Q., and Schreiber, A. D. (1995) Induction of phagocytosis by a protein tyrosine kinase. Blood 85, 1175–1180[Abstract/Free Full Text]
  56. Indik, Z. K., Park, J. G., Hunter, S., and Schreiber, A. D. (1995) The molecular dissection of Fc gamma receptor mediated phagocytosis. Blood 86, 4389–4399[Abstract/Free Full Text]
  57. Langdon, W. Y., Hartley, J. W., Klinken, S. P., Ruscetti, S. K., and Morse, H. C. (1989) v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc. Natl. Acad. Sci. USA 86, 1168–1172[Abstract/Free Full Text]
  58. Langdon, W. Y. (1995) The cbl oncogene: a novel substrate of protein tyrosine kinases. Aust. N.Z. J. Med. 25, 859–864[Medline]
  59. Tanaka, S., Neff, L., Baron, R., and Levy, J. B. (1995) Tyrosine phosphorylation and translocation of the c-cbl protein after activation of tyrosine kinase signaling pathways. J. Biol. Chem. 270, 14347–14351[Abstract/Free Full Text]
  60. Galisteo, M. L., Dikic, I., Batzer, A. G., Langdon, W. Y., and Schlessinger, J. (1995) Tyrosine phosphorylation of the c-cbl proto-oncogene protein product and association with epidermal growth factor (EGF) receptor upon EGF stimulation. J. Biol. Chem. 270, 20242–20245[Abstract/Free Full Text]
  61. Meisner, H., Conway, B. R., Hartley, D., and Czech, M. P. (1995) Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol. Cell. Biol. 15, 3571–3578[Abstract/Free Full Text]
  62. Fukazawa, T., Reedquist, K. A., Trub, T., Soltoff, S., Panchamoorthy, G., Druker, B., Cantley, L., Shoelson, S. E., and Band, H. (1995) The SH3 domain-binding T cell tyrosyl phosphoprotein p120—Demonstration of its identity with the c-cbl protooncogene product and in vivo complexes with fyn, grb2, and phosphatidylinositol 3-kinase. J. Biol. Chem. 270, 19141–19150[Abstract/Free Full Text]
  63. Reedquist, K. A., Fukazawa, T., Panchmoorthy, G., Langdon, W. Y., Shoelson, S. E., Druker, B. J., and Band, H. (1996) Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G. J. Biol. Chem. 271, 8435–8442[Abstract/Free Full Text]
  64. Sawasdikosol, S., Ravichandran, K. S., Lee, K. K., Chang, J.-H., and Burakoff, S. J. (1996) Crk interacts with tyrosine-phosphorylated p116 upon T cell activation. J. Biol. Chem. 270, 2893–2896[Abstract/Free Full Text]
  65. Cory, G. O. C., Lovering, R. C., Hinshelwood, S., MacCarthy-Morrogh, L., Levinsky, R. J., and Kinnon, C. (1995) The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase. J. Exp. Med. 182, 611–615[Abstract/Free Full Text]
  66. Matsuo, T., Hazeki, K., Hazeki, O., Katada, T., and Ui, M. (1996) Specific association of phosphatidylinositol 3-kinase with the protooncogene product cbl in Fc gamma receptor signaling. FEBS Lett. 382, 11–14[Medline]
  67. Marcilla, A., Rivero-Lezcano, O. M., Agarwal, A., and Robbins, K. C. (1996) Identification of the major tyrosine kinase substrate in signaling complexes formed after engagement of Fcgamma receptors. J. Biol. Chem. 270, 9115–9120[Abstract/Free Full Text]
  68. Ribon, V., Hubbell, S., Herrera, R., and Saltiel, A. R. (1996) The product of the cbl oncogene forms stable complexes in vivo with endogenous crk in a tyrosine phosphorylation-dependent manner. Mol. Cell. Biol. 16, 45–52[Abstract/Free Full Text]
  69. Fukazawa, T., Miyake, S., Band, V., and Band, H. (1996) Tyrosine phosphorylation of cbl upon epidermal growth factor (EGF) stimulation and its association with EGF receptor and downstream signaling proteins. J. Biol. Chem. 271, 14554–14559[Abstract/Free Full Text]
  70. Fournel, M., Davidson, D., Weil, R., and Veillette, A. (1996) Association of tyrosine protein kinase zap-70 with the protooncogene product p120*-cbl in T lymphocytes. J. Exp. Med. 183, 301–306[Abstract/Free Full Text]
  71. Tezuka, T., Umemori, H., Fusaki, N., Yagi, T., Takata, M., Kurosaki, T., and Yamamoto, T. (1996) Physical and functional association of the cbl protooncogene product with an src-family protein tyrosine kinase, p53/56(lyn), in the B cell antigen receptor-mediated signaling. J. Exp. Med. 183, 675–680[Abstract/Free Full Text]
  72. Reedquist, K. A., Fukazawa, T., Panchamoorthy, G., Langdon, W. Y., Shoelson, S. E., Druker, B. J., and Band, H. (1996) Stimulation through the T cell receptor induces cbl association with crk proteins and the guanine nucleotide exchange protein c3G. J. Biol. Chem. 271, 8435–8442
  73. Uddin, S., Gardziola, C., Dangat, A., Yi, T. L., and Platanias, L. C. (1996) Interaction of the c-cbl proto-oncogene product with the tyk-2 protein tyrosine kinase. Biochem. Biophys. Res. Commun. 225, 833–838[Medline]



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[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Scott, H. Ma, S. Viriyakosol, R. Terkeltaub, and R. Liu-Bryan
Engagement of CD14 Mediates the Inflammatory Potential of Monosodium Urate Crystals
J. Immunol., November 1, 2006; 177(9): 6370 - 6378.
[Abstract] [Full Text] [PDF]

Home page
 ANN INTERN MEDHome page
H. K. Choi, D. B. Mount, and A. M. Reginato
Pathogenesis of Gout
Ann Intern Med, October 4, 2005; 143(7): 499 - 516.
[Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
N. Dalbeth and D. O. Haskard
Mechanisms of inflammation in gout
Rheumatology, September 1, 2005; 44(9): 1090 - 1096.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Liu-Bryan, K. Pritzker, G. S. Firestein, and R. Terkeltaub
TLR2 Signaling in Chondrocytes Drives Calcium Pyrophosphate Dihydrate and Monosodium Urate Crystal-Induced Nitric Oxide Generation
J. Immunol., April 15, 2005; 174(8): 5016 - 5023.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. Ryckman, C. Gilbert, R. de Medicis, A. Lussier, K. Vandal, and P. A. Tessier
Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils
J. Leukoc. Biol., August 1, 2004; 76(2): 433 - 440.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. Ratthe and D. Girard
Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15R{alpha} chain
J. Leukoc. Biol., July 1, 2004; 76(1): 162 - 168.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Barabe, E. Rollet-Labelle, C. Gilbert, M. J. G. Fernandes, S. N. Naccache, and P. H. Naccache
Early Events in the Activation of Fc{gamma}RIIA in Human Neutrophils: Stimulated Insolubilization, Translocation to Detergent-Resistant Domains, and Degradation of Fc{gamma}RIIA
J. Immunol., April 15, 2002; 168(8): 4042 - 4049.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
R. Liu, K. Aupperle, and R. Terkeltaub
Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells
J. Leukoc. Biol., December 1, 2001; 70(6): 961 - 968.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
P. Desaulniers, M. Fernandes, C. Gilbert, S. G. Bourgoin, and P. H. Naccache
Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals
J. Leukoc. Biol., October 1, 2001; 70(4): 659 - 668.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Gilbert, F. Barabe, E. Rollet-Labelle, S. G. Bourgoin, S. R. McColl, B. B. Damaj, and P. H. Naccache
Evidence for a Role for SAM68 in the Responses of Human Neutrophils to Ligation of CD32 and to Monosodium Urate Crystals
J. Immunol., April 1, 2001; 166(7): 4664 - 4671.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. N. Serhan, I. M. Fierro, N. Chiang, and M. Pouliot
Cutting Edge: Nociceptin Stimulates Neutrophil Chemotaxis and Recruitment: Inhibition by Aspirin-Triggered-15-Epi-Lipoxin A4
J. Immunol., March 15, 2001; 166(6): 3650 - 3654.
[Abstract] [Full Text] [PDF]

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