Published online before print May 29, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ, Department of Medicine, Faculty of Medicine, Laval University, Québec, Canada
1 Correspondence: Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ, Local T1-49, 2705, Boulevard Laurier, Sante-Foy, Québec, Canada, G1V 4G2. E-mail: paul.naccache{at}crchul.ulaval.ca
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: inflammation • gout • PI(3 • 4 • 5)P3 • phospholipase D • degranulation • tyrosine phosphorylation
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Neutrophils play a crucial role in host defense against injury and infection as well as in inflammatory responses by virtue of their ability to mount a series of effector responses. MSU crystals cause a massive infiltration of neutrophils in the joint fluid and synovial membrane, where they phagocytose crystals actively [2 ]. If animals are depleted of polymorphonuclear leukocytes, the inflammatory response to injections of MSU crystals is suppressed [3 ], and moreover, a number of agents, which suppress neutrophil functions, prevent and/or terminate acute gouty inflammation [4 ]. Direct MSU crystal interactions with neutrophils and inflammatory mediators that potentiate neutrophil activation are therefore crucial to the induction and perpetuation of gout attacks. MSU crystals induce secretion of a variety of cytokines (reviewed in ref. [5 ]), prostanoids [6 7 ], chemotactic factors {including IL-8, MCP-1 [8 ], and unidentified factors (crystal-induced chemotactic factor [9 ] and others [10 ])}, which drive the inflammatory process. These cytokines can amplify the inflammatory process through increased inflammatory cell recruitment by up-regulation of adhesion molecules and stimulation of the acute-phase response.
One of the earliest events observed upon stimulation of neutrophils with MSU crystals is an increase in the level of tyrosine phosphorylation of various proteins [11 ]. The protein tyrosine kinase Syk, which has been associated largely with the phagocytic response by FcRs [12 ] and with spreading mediated by integrins [13 ], has been identified previously in our laboratory as one of the major proteins tyrosine-phosphorylated and activated in human neutrophils upon stimulation with MSU crystals [14 ]. Syk is a 72-kDa protein kinase, which plays a central role in coupling immune recognition receptors to multiple downstream signaling pathways in hematopoietic cells. Our previous results showed that piceatannol, a specific Syk inhibitor, diminished the tyrosine phosphorylation patterns, the mobilization of intracellular calcium, the production of superoxide anions, and the phospholipase D (PLD) activity stimulated by MSU crystals [14 ].
Together with Syk, Src family kinases play crucial roles in multiple neutrophil intracellular signaling pathways. A hierarchical activation of Src family kinases and Syk has been proposed following Fc
RIIa engagement [15
].
The PI-3Ks phosphorylate PI, PI-4 phosphate [PI(4)P], and PI-4,5 biphosphate [PI(4,5)P2] to form PI(3)P, PI(3,4)P2, and PI-3,4,5 trisphosphate [PI(3,4,5)P3], respectively. These lipids serve as phospholipase substrates for the generation of soluble (inositol phosphate) and membrane-associated (diacylglycerol) second messengers; they also interact directly with intracellular proteins, affecting their location and/or activity, and finally, they can alter the local membrane topology by electrostatic interactions [16
]. Class Ia enzymes of the PI-3K family are heterodimeric proteins, each of which consists of a catalytic subunit of 110–120 kDa and an associated regulatory subunit called p85 proteins 
and ß. The tyrosine phosphorylation of p85 is indicative of its activation. Under resting conditions, p85 serves to stabilize and inactivate the p110 catalytic subunit [17
].
Syk is required for the activation of the PI-3K family in response to a variety of signals including engagement of the B cell antigen receptor [18
] and macrophage or neutrophil FcRs [19
]. Moreover, the p85 PI-3K subunit was the major Syk-binding protein identified in yeast two-hybrid screens using libraries from two different sources [20
].
Although activation of PI-3Ks has been well documented for other stimuli [21 22 ], little is known about its involvement in the responses of human neutrophils to MSU crystals Increased PI-3K activity has been reported in tyrosine-phosphorylated immunoprecipitates from plasma-opsonized MSU, crystal-stimulated human neutrophils [23 ]. However, the signal transduction pathways leading to the activation of PI-3Ks in response to MSU crystals and its functional significance remain to be investigated.
We show here that MSU crystals induce the activation of Class Ia PI-3Ks in human neutrophils and that this activation depends on the kinase activity of Syk. Our results also indicate that activated Syk and p85 are found in specialized microdomains insoluble in the nonionic detergent Nonidet P-40 (NP-40) upon MSU crystal stimulation. Moreover, we show that MSU crystals induce the formation of a Syk/p85 complex, which is dependent on Src family kinases. Our results also indicate that the stimulation of the activity of PLD and of the degranulation response induced by MSU crystals depends on the Syk-mediated activation of PI-3Ks. These events are likely to be relevant to the tissue damage that occurs in gouty arthritis.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Dextran T-500 and Ficoll-Paque were purchased from Wisent (Saint-Bruno, QC, Canada). 1-O-[3H]Alkyl-2-lyso-phosphatidylcholine was from Perkin Elmer (Woodbridge, ON, Canada). Wortmannin and piceatannol were purchased from Biomol (Plymouth Meeting, PA, USA). LY294002 (440204), 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2; 529573), and 4-amino-7-phenylpyrazol[3,4-d] pyrimidine (PP3; 529574) were purchased from Calbiochem (La Jolla, CA, USA). Protein A Sepharose beads (71-5280-04) were purchased from Amersham Biosciences (Baie d’Urfé, QC, Canada). NP-40 was from Calbiochem. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), aprotinin, and leupeptin were obtained from Roche Diagnostics (Indianapolis, IN, USA). Di-isopropylfuorophosphate (DFP) was from Serva Electrophoresis (Heidelberg, Germany). 4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP) and PMSF were purchased from Sigma Aldrich (Oakville, ON, Canada).
Triclinical MSU monohydrate crystals were synthesized and characterized as described previously [24 ].
Neutrophil purification
Venous blood was collected from healthy adult volunteers in isocitrate, anticoagulant solution. Neutrophils were separated as described previously [25
]. Briefly, whole blood was centrifuged at 180 g for 10 min, and the resulting platelet-rich plasma was discarded. Leukocytes were obtained following sedimentation in 2% Dextran T-500. Mononuclear cells were removed by centrifugation on Ficoll-Paque cushions, and contaminating erythrocytes in the neutrophil pellets were removed by a 20-s hypotonic lysis. Neutrophils were resuspended at 40 x 106 cells/ml in HBSS 1x, pH 7.4, containing 1.6 mM Ca2+ but no Mg2+.
Cell stimulation
When indicated, neutrophils (40x106 cells/ml) were preincubated at 37°C for 10 min with the specified concentrations of inhibitors (40 µM piceatannol; 10 µM PP2, PP3, or LY294002; 30 nM wortmannin) or an equal volume of DMSO (Me2SO). The Me2SO concentration never exceeded 0.1%. In cell-based assays, these concentrations of piceatannol, wortmannin, and LY294002 have been described previously as exhibiting a relatively high selectivity for Syk and PI-3Ks, respectively [16
26
]. Neutrophil suspensions were then stimulated at 37°C with 3 mg/ml MSU crystals for the indicated time periods.
Western blotting
For whole cell analysis, stimulations were stopped by transferring 100 µl of the cell suspension to an equal volume of boiling 2x Laemmli’s sample buffer (1x is 62.5 mM Tris-HCl, pH 6.8, 4% SDS, 5% ß-ME, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM pNPP, 12.5 µg/ml leupeptin, 12.5 µg/ml aprotinin, 0.00125% bromophenol blue) and boiled for 7 min. Samples were then loaded onto 7.5–20% gradient SDS-polyacrylamide gels. Separated proteins were transferred from the gels to Immobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). After incubations with the desired antibodies [mouse antiphosphotyrosine: 1:4000 dilution; mouse antiflotillin-1 and goat anti-LDH: 1:2000 dilution; rabbit antiphospho-p85, anti-p85, antiphospho-AKT (Thr308), antiphospho-AKT (Ser473), anti-AKT, and mouse anti-Syk: 1:1000 dilution] and the corresponding second antibody (1:20,000 dilution), the membranes were revealed using the ECL detection system.
Preparation of NP-40-insoluble fractions
Neutrophils were processed and stimulated with MSU crystals as described above. The reactions were stopped at the indicated times by a 10-s centrifugation at 6000 g. The supernatants were discarded, and the cells were lysed in 1 ml ice-cold hypotonic lysis buffer (20 mM Tris-HCl, 0.1% NP-40, 10 mM NaCl, 2 mM EDTA, 10 µg/ml aprotinin and leupeptin, 2 mM Na3VO4, 10 mM pNPP, 2 mM PMSF, 3 mM DFP, pH 7.2) as described previously [27
]. The samples were vortexed, kept on ice for 5 min, and then centrifuged at 16,000 g at 4°C for 5 min. An aliquot of the supernatants was transferred to the same volume of 2x Laemmli’s sample buffer and boiled for 7 min. The insoluble pellets were resuspended in 0.1% NP-40 lysis buffer and transferred to the same volume of 2x Laemmli’s sample buffer. When loading the SDS-polyacrylamide gels, adjustments were made for all samples to be equivalent with respect to cell number.
Immunoprecipitations under native conditions
Neutrophils were processed and stimulated with MSU crystals as described above. The stimulations were stopped by transfer to an ice bath followed by a 10-s spin in a microcentrifuge at 6000 g. The pellets were resuspended in cold lysis buffer (10 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA, 0.6% CHAPS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM sodium orthovanadate, 250 µg/ml soybean trypsin inhibitor, 3 mM DFP, 1 mM PMSF). After 5 min on ice, the cell lysates were centrifuged at 16,000 g for 5 min at 4°C. The supernatants were incubated at 4°C with gentle rotation for 3 h in the presence of prewashed protein-A Sepharose beads linked to an anti-p85 antibody (4 µg for 50 µl beads) or to an anti-Syk antibody (0.8 µg for 50 µl beads). For p85, the immunoprecipitations were preceded by 1 h of preclearing with protein-A Sepharose beads, which were then collected and washed three times with cold lysis buffer. Laemmli’s sample buffer (50 µl) was added to the beads, which were boiled for 7 min.
Immunoprecipitations under denaturing conditions
This protocol has been described already in our laboratory as being useful for preserving the stability of the tyrosine-phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification [28
29
]. Briefly, neutrophils were processed and stimulated with MSU crystals as described above. The total lysates and the soluble NP-40 fractions were transferred in the same volume of 2x denaturing lysis buffer (125 mM Tris-HCl, 6% SDS, 2% ß-ME, 5 mM Na3VO4, 25 µg/ml aprotinin and leupeptin, 0.0025% bromophenol blue, at pH 6.8) and boiled for 10 min. The insoluble NP-40 fractions were resuspended in NP-40 lysis buffer, transferred in the same volume of 2x denaturing lysis buffer, and boiled for 10 min. The samples were filtered through Sephadex G-10 columns to remove the denaturing agents. The denatured samples were incubated with the anti-Syk antibody (0.8 µg bound to 50 µl protein-A Sepharose beads) for 3 h at 4°C on a rotator platform. The beads were then collected and washed twice with ice-cold NP-40 buffer (62.5 mM Tris-base, 1% NP-40, 1% glycerol, 137 mM NaCl, 2 mM Na3VO4, 10 µg/ml aprotinin and leupeptin, at pH 8.0). The beads were boiled 10 min in 100 µl 2x Laemmli’s sample buffer.
Assay for Class Ia PI-3K activity
PI-3K activity was determined in vitro using a competitive PI-3K ELISA kit (K-1000) from Echelon Biosciences Inc. (Salt Lake City, UT, USA). This kit measures PI-3K activity as a conversion of PI(4,5)P2 into PI(3,4,5)P3. We used this kit in conjunction with p85 immunoprecipitates (see Immunoprecipitations under native conditions in Materials and Methods). Thus, we specifically determined the Class Ia activity of PI-3K. Briefly, PI-3K bound to the protein-A Sepharose beads was incubated for 1 h with 10 µM diC8PI(4,5)P2 substrate at room temperature in 50 µl buffer containing 4 mM MgCl2, 20 mM Tris-HCl (pH 7.4), 10 mM NaCl, and 25 µM ATP. The beads were removed by centrifugation, and the supernatant or known concentrations of PI(3,4,5)P3 were incubated for 1 h with 50 µl PI(3,4,5)P3-binding reagent and then transferred to a detection plate coated with PI(3,4,5)P3. Plate-bound binding reagent was quantified by using a secondary detection reagent, peroxidase, and peroxidase substrate with reaction product measured by absorbance at 450 nm.
PLD measurements—phosphatidylethanol (PEt) formation
Neutrophils were labeled with 1-O-[3H]alkyl-2-lyso-phosphatidylcholine (2 µCi/107 cells) for 90 min as described previously [30
]. The cells were then washed and resuspended at 10 x 106 cells/ml in HBSS. Cell suspensions (0.5 ml) were stimulated with 3 mg/ml MSU crystals for 15 min in the presence of 1% ethanol. Incubations were stopped by adding 1.8 ml chloroform/methanol/HCl (50:100:1) and unlabeled PEt as a standard. Lipids were extracted and dried under nitrogen. The lipid extracts were dissolved in 40 µl chloroform/methanol (2:1) and spotted on prewashed silica gel thin-layer chromatography plates. [3H]PEt was separated from the other lipids using the solvent mixture chloroform/methanol/acetic acid (65:15:2). Lipids were visualized by Coomassie brilliant blue staining (0.03% dye; 35% MeOH; 200 mM NaCl), and the different lipid classes were scraped off the plates. Radioactivity in PEt was monitored by liquid scintillation counting, and the results corrected for background radioactivity and quenching.
Degranulation
Neutrophils were processed and stimulated with MSU crystals as described above. The stimulations were stopped by a quick spin (15 s, 6000 g), and the supernatants were harvested, filtered on 0.22 µm nylon syringe filters, and kept on ice. The extent of lysozyme release was assessed by adding 100 µl supernatant to 900 µl of a 0.25 mg/ml Micrococcus lysodeikticus solution prepared in a 0.1 M PO4 buffer. The loss of absorbance was then read at 450 nm for a period of 2 min. To obtain a percentage value for MSU crystal-induced degranulation, the absorbance values were compared, and the maximal loss of absorbance was obtained by lysing the cells with 0.1% Triton X-100.
Human albumin levels in MSU crystal-stimulated neutrophil supernatants were determined by capture ELISA (Bethyl Laboratories Inc., Montgomery, TX, USA), according to the manufacturer’s recommendations. The amount of total albumin released was obtained by lysing the cells with 0.1% Triton X-100. All samples were measured in triplicate, and the results are expressed as the mean ± SEM percentage of total albumin released in at least five experiments.
Densitometric analysis
Densitometric analysis was performed using Image J software on the scanned Western blot results. The measurements of pixel intensity of each blot lane were plotted using GraphPad Prism 4 Software.
Statistical analysis
Statistical analyses were performed using the Student’s paired t-test (two-tailed) using GraphPad Prism 4 software on the nonprocessed data. Every inhibitory condition was compared with the control condition, and significance was considered to be attained when P was less than 0.05.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The tyrosine phosphorylation of the p85 regulatory subunit of Class Ia PI-3Ks is indicative of its activation [17 ]. As shown in Figure 1A (left panel), MSU crystals induce a transient tyrosine phosphorylation of p85 with a peak of intensity observed between 2 and 5 min of stimulation. As it was shown previously in our laboratory that Syk tyrosine kinase plays a key role in the responses of human neutrophils to stimulation with MSU crystals [14 ], we investigated the effect of piceatannol, a Syk-specific inhibitor [26 ], on the tyrosine phosphorylation of p85 induced by MSU crystals. The cells were incubated for 10 min at 37°C with 40 µM piceatannol prior to MSU crystal stimulation. These conditions were observed previously to be sufficient to significantly diminish the stimulation of tyrosine phosphorylation of Syk and its tyrosine kinase activity in neutrophils stimulated with MSU crystals [14 ]. As shown in Figure 1A (right panel), piceatannol significantly inhibited the stimulation of tyrosine phosphorylation of p85 induced by MSU crystals in human neutrophils. A quantitative evaluation of these results (by densitometry) is provided in the lower panel of Figure 1A .
 View larger version (18K): [in a new window] |
Figure 1. MSU crystal stimulation induces PI-3K activation in human neutrophils in a Syk-dependent manner. Neutrophils (40x106 cells/ml) were preincubated with 1 mM DFP for 10 min at room temperature. When indicated, piceatannol (40 µM) or an equal volume of its solvent (Me2SO) was added for 10 min at 37°C. The neutrophils were then stimulated with 3 mg/ml MSU crystals, and the stimulations were stopped at the indicated times. (A) Cells were lysed in nondenaturing lysis buffer containing 0.6% CHAPS. The 16,000 g supernatants were immunoprecipitated (IP) for p85 protein and revealed with an antiphosphotyrosine antibody (pY) or with an anti-p85 antibody (p85) as described in Materials and Methods. WB, Western blot. (B) The p85 immunoprecipitates were also used for performing PI-3K activity ELISA according to the manufacturer’s recommendations. Class Ia PI-3K activity was determined as a conversion of PI(4,5)P2 into PI(3,4,5)P3 (PIP3). The results shown are representative of at least three independent experiments.
|
The serine/threonine kinase AKT is a major target of PI-3Ks. Phosphoinositides activate phosphoinositide-dependent kinases 1 (PDK1) and 2 (PDK2), which phosphorylate AKT on Thr308 and Ser473, respectively [32 ]. We therefore examined the state of activation of AKT following the stimulation of neutrophils by MSU crystals. As shown in Figure 2A and 2B , MSU crystals induced a transient phosphorylation of AKT on Thr308 and Ser473. As observed for p85 (Fig. 1A) , piceatannol drastically diminished the MSU crystal-induced phosphorylation of AKT (Fig. 2A and 2B) . The inactive analog of piceatannol, transtilbene, did not have any effect on the phosphorylation of p85 or AKT (data not shown), confirming that the effect of piceatannol was a result of its inhibitory effect on Syk.
 View larger version (25K): [in a new window] |
Figure 2. MSU crystal stimulation induces AKT phosphorylation in human neutrophils in a Syk-dependent manner. Neutrophils (40x106 cells/ml) were preincubated with 1 mM DFP for 10 min at room temperature. When indicated, piceatannol (40 µM) or an equal volume of its solvent (Me2SO) was added for 10 min at 37°C. The neutrophils were then stimulated with 3 mg/ml MSU crystals, and the stimulations were stopped at the indicated times. The whole cell lysates were analyzed by Western blotting for Thr308 (A)- or Ser473 (B)-phosphorylated AKT (pAKT) and for AKT as a loading control. The results shown are representative of at least three independent experiments.
|
Tyrosine-phosphorylated p85 and Syk are located in NP-40-insoluble fractions in human neutrophils stimulated with MSU crystals
Specialized microdomains, insoluble in nonionic detergents, rich in cholesterol and sphingolipids, have been shown to concentrate signaling molecules such as tyrosine kinases and adaptor proteins and are believed to be involved in the activation of signaling cascades and in membrane trafficking in a variety of cells including neutrophils [27
33
34
]. Accordingly, tyrosine-phosphorylated substrates and enzyme activities were found to be increased and concentrated in NP-40-insoluble fractions upon stimulation of human neutrophils with MSU crystals [29
].
We therefore monitored the distribution of p85 between the NP-40-soluble and -insoluble fractions following stimulation of human neutrophils with MSU crystals. Neutrophils (stimulated or not with MSU crystals) were lysed in NP-40, and the NP-40-soluble and -insoluble fractions were prepared and immunoblotted for p85 as described in Materials and Methods. The results summarized in Figure 3A show that the amount of p85 in the NP-40-insoluble fractions more than doubled following stimulation of the cells with MSU crystals. Flotillin, a detergent-resistant membrane (DRM) marker, was found to be contained nearly exclusively within the NP-40-insoluble fraction.
 View larger version (22K): [in a new window] |
Figure 3. p85 translocates into a NP-40-insoluble fraction upon MSU crystal stimulation. Neutrophils (40x106 cells/ml) were preincubated with 1 mM DFP for 10 min at room temperature. Where indicated, neutrophils were incubated with piceatannol (40 µM) or with an equal volume of Me2SO for 10 min at 37°C. The cells were then stimulated with 3 mg/ml MSU crystals, and the stimulations were stopped on ice at the indicated times. The NP-40-insoluble fractions were prepared as described in Materials and Methods. (A) The total cell lysates, the soluble and insoluble fractions, were probed with an anti-p85 antibody (p85) or an antiflotillin antibody (flotillin). (B) The insoluble fractions were probed with an anti-p85 antibody (p85). When loading the SDS-polyacrylamide gels, adjustments were made for all samples to be equivalent with respect to cell number. The results shown are representative for at least three different experiments.
|
We next investigated the distribution of the phosphorylated p85 and Syk in the NP-40-soluble and -insoluble fractions following stimulation with MSU crystals. These experiments were carried out as described above except that immunoblotting was achieved using an antiphospho-p85 or an antiphosphotyrosine antibody following the immunoprecipitation under denaturing conditions of Syk in the various fractions (the experimental details are described in Materials and Methods). We observed that the phosphorylated form of p85 is found predominantly in the NP-40-insoluble fraction following stimulation with MSU crystals (Fig. 4A ). The distribution of phospho-p85 among these fractions therefore closely parallels that of p85. As shown in Figure 4B , the NP-40-insoluble fractions also contained nearly all the tyrosine-phosphorylated Syk.
 View larger version (56K): [in a new window] |
Figure 4. Tyrosine-phosphorylated p85 and Syk are located in the NP-40-insoluble fraction upon MSU crystal stimulation. Neutrophils (40x106 cells/ml) were preincubated with 1 mM DFP for 10 min at room temperature. The cells were then stimulated with 3 mg/ml MSU crystals, and the stimulations were stopped after 5 min. The NP-40-insoluble fractions were prepared as described in Materials and Methods and analyzed with an antiphospho-p85 (p-p85) or an anti-p85 antibody (A). For Syk, the samples were immunoprecipitated under denaturing conditions and probed with an antiphosphotyrosine or an anti-Syk antibody (B). When loading the SDS-polyacrylamide gels, adjustments were made for the samples to be equivalent with respect to cell number. The results shown are representative of three independent experiments.
|
MSU crystal stimulation induces an interaction between the p85 regulatory subunit of PI-3Ks and Syk in human neutrophils
CHAPS is a zwitterionic detergent used to solubilize proteins. Zwitterions are polar and usually have a high solubility in water and a poor solubility in most organic solvents. CHAPS is used as a nondenaturing solvent in the process of protein purification and is especially useful in purifying membrane proteins, which are often sparingly soluble or insoluble in aqueous solutions as a result of their natively hydrophobic, cellular environment [35
]. As shown in Figure 5
and contrary to what was observed using NP-40 (see Figs. 3
and 4
), Syk and p85 are nearly, completely soluble in CHAPS, even after stimulation with MSU crystals, thus making it possible to perform immunoprecipitations under native conditions and to study protein–protein interactions.
 View larger version (32K): [in a new window] |
Figure 5. p85 and Syk are entirely soluble in the zwitterionic detergent CHAPS. Neutrophils (40x106 cells/ml) were preincubated with 1 mM DFP for 10 min at room temperature. The cells were then stimulated with 3 mg/ml MSU crystals, and the stimulations were stopped on ice after 5 min. The CHAPS-soluble and -insoluble fractions were prepared as described in Materials and Methods (see Immunoprecipitations under native conditions) and analyzed with an anti-p85 or an anti-Syk antibody. When loading the SDS-polyacrylamide gels, adjustments were made for the samples to be equivalent with respect to cell number. The results shown are representative of at least five different experiments.
|
We next assessed whether an interaction between p85 and Syk could be detected following the stimulation of human neutrophils by MSU crystals. By a two-way CHAPS coimmunoprecipitation technique (details in Materials and Methods, Immunoprecipitations under native conditions), we show here that stimulation of human neutrophils with MSU crystals induces the time-dependent formation of a complex containing p85 and Syk (Fig. 6A ).
 View larger version (31K): [in a new window] |
Figure 6. MSU crystal stimulation induces an interaction between the Class Ia PI-3K regulatory subunit and Syk in human neutrophils. (A) Neutrophils (40x106 cells/ml) were preincubated with 1 mM DFP for 10 min at room temperature. The cells were then stimulated with 3 mg/ml MSU crystals, and the stimulations were stopped on ice at the indicated times. The CHAPS p85 and Syk immunoprecipitations were performed as described in Materials and Methods and probed with an antiphosphotyrosine, an anti-Syk, or an anti-p85 antibody. (B) Prior to stimulation, neutrophils were preincubated with piceatannol (40 µM) or with an equal volume of Me2SO and then treated as described above. (C) Prior to stimulation, neutrophils were preincubated with PP2 (10 µM) or with an equal concentration of PP3 and then treated as described above. The results shown are representative of five independent experiments.
|
Together with Syk, Src family kinases have crucial roles in multiple neutrophil intracellular signaling pathways. In many cases, they seem to operate together, and the general hypothesis is that activation of Src family kinases precedes and modulates the activation of Syk tyrosine kinase [36 ]. We therefore hypothesized that the MSU crystal-induced coimmunoprecipitation between p85 and Syk could depend on the Src-mediated activation of Syk. To confirm this hypothesis, we evaluated the potential effects of PP2, a Src kinase-specific inhibitor [37 ], on the interaction between p85 and Syk induced by MSU crystals. As shown in Figure 6C , PP2 abolished the tyrosine phosphorylation of Syk completely and nearly completely inhibited the coimmunoprecipitation of p85 and Syk. We did not observe any effect of PP2 on the MSU crystal-induced insolubilization of p85 (data not shown). Together with the inhibition of the MSU crystal-induced p85/Syk interaction by PP2, this result suggests that the localization of p85 to detergent-resistant domains is not sufficient for it to interact with Syk. The initial, Src-mediated tyrosine phosphorylation of Syk is, conversely, essential for the formation of the p85/Syk complex.
When compared with Me2SO, the inactive analog of PP2, PP3, affected neither the phosphorylation of Syk nor its association to p85 (data not shown). The same results were obtained when p85 was immunoprecipitated (data not shown).
Functional consequences of the MSU crystal-induced PI-3K activation in human neutrophils
It was described previously in our laboratory that MSU crystal stimulation induces the activity of PLD in human neutrophils [38
]. In view of the above results linking stimulation of human neutrophils to the activation of PI-3Ks, we investigated the role of the latter in the stimulation of the activity of PLD. Neutrophils were preincubated with the two unrelated PI-3K inhibitors, wortmannin and LY294002, which are cell-permeable, low molecular-weight compounds with in vitro IC50 of approximately 5 nM and 1 µM, respectively. The results of these studies, summarized in Figure 7
, show that both compounds inhibited the stimulation of the activity of PLD in a concentration-dependent manner (IC50 of 8 nM and 2 µM for wortmannin and LY294002, respectively). Maximal inhibition (80%) of the MSU crystal-induced PLD activity was obtained with 
20 nM wortmannin and 10 µM LY294002. No further inhibition of MSU crystal-induced PLD activity was observed by increasing the concentrations of wortmannin and LY294002 up to 1 µM and 100 µM, respectively.
 View larger version (15K): [in a new window] |
Figure 7. MSU crystal-induced PLD activity depends on PI-3Ks. Neutrophils were preincubated with wortmannin or LY294002 at the indicated concentrations. The cells were stimulated with 3 mg/ml MSU crystals for 15 min, and the samples were then processed for PLD activity as described in Materials and Methods. The radioactivity was monitored by liquid scintillation counting, and the results were corrected for background radioactivity and quenching. The results shown are the mean value of three independent experiments.
|
 View larger version (12K): [in a new window] |
Figure 8. MSU crystal-induced neutrophil degranulation depends on PI-3Ks. Neutrophils (40x106 cells/ml) were preincubated with wortmannin (30 nM), LY294002 (10 µM), or an equal volume of Me2SO as vehicle for 10 min and then stimulated with 3 mg/ml MSU crystals for 15 min at 37°C. The stimulations were stopped by a quick spin (15 s, 16,000 g), and the supernatants were harvested and analyzed, as described in Materials and Methods, for the amount of lysozyme released (A) or for the amount of albumin released (B). The results shown are the mean value of six independent experiments.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The major characteristics of gout include rapid onset, self-limitation, and phagocytosis of the MSU crystals by polymorphonuclear leukocytes within the synovial cavity, resulting in the release of inflammatory mediators such as leukotriene B4, platelet-activating factor, PGE2, IL-8, lysosomal proteases, superoxide, and a still-unidentified chemotactic factor [6 8 10 42 ].
Gout has become more common and more clinically complex in recent years, particularly in older subjects [43 ]. In addition, an association of hyperuricemia with premature cardiovascular disease and mortality has become more evident over time [44 ]. The understanding of the molecular mechanisms involved in generating the pathologic effects of MSU crystals is essential to the ongoing search for more effective therapies for gouty arthritis, as the lack of growth in available therapeutic options for chronic hyperuricemia has led to a limited number of support options [45 ].
Recent studies have shown that PI-3Ks play major roles in phagocytosis, superoxide anion production, and chemotaxis (reviewed in ref. [31 ]). We show here that MSU crystals induce the tyrosine phosphorylation of the p85 regulatory subunit of Class Ia PI-3Ks in a Syk tyrosine kinase-dependent manner, as this phosphorylation is diminished by piceatannol. We also show that in human neutrophils, MSU crystals increase PI-3K activity, and this effect is inhibited significantly by piceatannol. As the PI-3K activity was determined using p85 immunoprecipitates, these results identify Class Ia PI-3K as the target of stimulation by MSU crystals. The effects of MSU crystals on PI-3K were Syk-dependent, as piceatannol inhibited the activation of PI-3K as well as that of the PI-3K effector AKT (as monitored by the Thr308 and the Ser473 phosphorylations of AKT).
The translocation of signaling proteins into nonionic, detergent-resistant domains has already been described in human neutrophils stimulated, among others, by cross-linking of FcRIIa or FcRIIIb. For example, we have observed recently that the cross-linking of FcRIIIb, which mediates, at least in part, the responses of human neutrophils to MSU crystals [46
], induces a significant increase in the amount of the receptor in high-density DRMs [47
]. Moreover, tyrosine phosphorylation profiles and enzyme activities were found to be increased and concentrated in NP-40-insoluble fractions upon stimulation of human neutrophils [29
]. Accordingly, we found here an increase in the amount of p85 associated with the cytoskeleton and/or DRM-containing, NP-40-insoluble fraction derived from human neutrophils stimulated with MSU crystals. Furthermore, we observed that piceatannol did not diminish the MSU crystal-induced p85 translocation into these detergent-resistant structures, suggesting that neither the kinase activity of Syk nor the tyrosine phosphorylation of p85 is required for the insolubility of p85. Finally, we found that upon stimulation by MSU crystals, tyrosine-phosphorylated p85 and Syk are localized into a NP-40-insoluble fraction, whose functions appear to be related to signal transmission and membrane trafficking. These data provide additional evidence that detergent-resistant microdomains function as signaling platforms in human neutrophils. Furthermore, they underline the similarities between the signal transduction pathways activated following the engagement of FcRIIIb and by MSU crystals.
The insoluble status of p85 and Syk in NP-40 precluded any attempts at examining potential interactions between these two proteins by coimmunoprecipitations in this detergent. Conversely, we found Syk and p85 to be predominantly soluble in CHAPS even after stimulation by MSU crystals and were thus able to show a coimmunoprecipitation of these two proteins.
The formation of Syk- and p85-containing complexes has been described previously as necessary for the FcR-mediated phagocytosis of opsonized particles in COS-1 cells stably transfected with FcRIIa [48
]. The signaling pathway activated by MSU crystals appears to be similar, as MSU crystals induced the formation of a complex containing p85 and Syk, which was not affected by piceatannol but was abrogated completely by PP2, a Src family kinase inhibitor, suggesting that Src-mediated tyrosine phosphorylation of Syk is essential for it to interact with p85. Our results are in accordance with recent studies, which proposed that p85 interacted directly with Syk, and these interactions were mediated by the Src homology 2 domains of p85, binding to specific phosphotyrosines on Syk, thus providing a mechanism by which signals could be transmitted directly from activated, phosphorylated Syk to p85 [20
]. Conversely, neither the tyrosine kinase activity of Syk nor its autophosphorylation appear to be necessary for p85 to interact with Syk, as piceatannol affected neither the coimmunoprecipitation nor the insolubilization of p85 induced by MSU crystals. However, the tyrosine kinase activity of Syk and/or its autophosphorylation is essential for the subsequent MSU crystal-induced activation of PI-3Ks, as shown by the effects of piceatannol on the phosphorylation of p85 and the PI-3K major target AKT and on the PI-3K activity.
Evidence for the functionality of the signaling events described above was also obtained. It was described previously in our laboratory that MSU crystals stimulate the activity of PLD in human neutrophils [38 ] and that this activation is Syk-dependent [14 ]. We show here that the activation of PLD by MSU crystals is dependent on PI-3Ks, as wortmannin and LY294002 inhibited it significantly. This result suggests that the effects of piceatannol on MSU crystal-induced PLD activation observed previously are exerted indirectly via the inhibition of one or more members of the Class Ia PI-3K family.
Previous studies have identified a role for PLD in the regulation of neutrophil degranulation in response to different stimuli [39
49
]. In inflammatory diseases such as rheumatoid arthritis and gout, the neutrophil is thought to be largely responsible for the tissue damage caused by the excessive release of the cytolytic enzymes of its granules into the synovial fluid. Moreover, we have shown recently that MSU crystal-stimulated neutrophils release a potent activation factor within 15 min of stimulation [10
]. This still-unidentified activation factor is thought to amplify and prolong the gouty inflammation. We show here that MSU crystals induce a significant degranulation of human neutrophils as measured by the amounts of lysozyme and albumin (markers of the azurophil, specific, gelatinase, and secretory granules, respectively) released into the extracellular milieu. PI-3Ks seem to play an important role in the MSU crystal-induced neutrophil degranulation, as wortmannin and LY294002 diminished it significantly. Further studies of the MSU crystal-induced degranulation of human neutrophils are required, as the contents of the granules are various and exert contradictory effects. Although the cytolytic enzymes contained within the neutrophil granules could be largely responsible for the tissue damage occurring in patients suffering from gout, the opioid peptides contained within the same granules counteract the inflammatory pain in the early stages of inflammation, thus having a beneficial role [50
]. Moreover, the secretory vesicles represent a specialized, membrane-like, intracellular compartment of human neutrophils enriched in GPI-anchored proteins such as FcRIIIb and CD14, both of which have been shown to play a role in the recognition of MSU crystals [41
46
51
].
In conclusion, this is the first report of the activation of Class Ia PI-3Ks by MSU crystals in human neutrophils and of the dependence of this response on the tyrosine kinase Syk. Moreover, the results of this study also document the degranulation that accompanies the stimulation of human neutrophils with MSU crystals and show that the Syk-PI-3K axis is critical for this response. Further studies of the intracellular signal transduction pathways activated by MSU crystals are required to identify the complex of receptors with which MSU crystals interact (FcRIIIb, CD14, TLRs, and others), which still remains largely uncharacterized, and the elucidation of which could provide critical clues as to the cellular mechanisms involved in the inflammatory reaction characteristic of gout, and in so doing, indicate novel and more efficient, therapeutic targets for the treatment of gouty arthritis.
Received March 19, 2007; revised April 24, 2007; accepted April 30, 2007.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, interleukin-1ß, interleukin-8, and interleukin-1 receptor antagonist in monosodium urate crystal-induced rabbit arthritis Lab. Invest. 78,559-569[Medline] receptor signaling in macrophages and neutrophils Mol. Cell. Biol. 18,4209-4220RIIa Biochim. Biophys. Acta 1357,348-358[Medline], regulate formyl-methionyl-leucyl-phenylalanine-stimulated chemotaxis and superoxide production in differentiated neutrophil-like PLB-985 cells J. Immunol. 176,7621-7627
R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol J. Biol. Chem. 269,29697-29703 RIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase J. Immunol. 155,2085-2095[Abstract]RIIIB (CD16) and CD11b in response to inflammatory microcrystals FASEB J. 12,209-220 receptor phagocytosis and endocytosis J. Leukoc. Biol. 80,1553-1562 receptor activation of neutrophils and provides specificity between high-valency immune complexes and fMLP signaling pathways J. Leukoc. Biol. 61,522-528[Abstract]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
