ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 2, February 2014, pp 470–478 DOI 10.1002/art.38245 © 2014, American College of Rheumatology
The p38-Mediated Rapid Down-Regulation of Cell Surface gp130 Expression Impairs Interleukin-6 Signaling in the Synovial Fluid of Juvenile Idiopathic Arthritis Patients Nora Honke,1 Kim Ohl,1 Anastasia Wiener,1 Jeff Bierwagen,1 Joachim Peitz,2 Stefano Di Fiore,3 Rainer Fischer,3 Norbert Wagner,1 Stefan Wu ¨ller,1 and Klaus Tenbrock1 Objective. Interleukin-6 (IL-6) signaling plays an important proinflammatory role, but this role is restricted by regulatory mechanisms that, for example, reduce the cell surface availability of the signaltransducing chain of the IL-6 receptor, gp130. The aim of this study was to determine whether the inflammatory environment in arthritic joints has an impact on monocytic gp130 surface expression and the extent to which regulatory processes in the synovial fluid (SF) can be reproduced in an in vitro model. Methods. Flow cytometry and live cell imaging were used to measure the cell surface expression and internalization of gp130. STAT-3 phosphorylation was monitored by flow cytometry and Western blotting. Results. In patients with juvenile idiopathic arthritis (JIA), levels of cell surface gp130 expression in SF monocytes were reduced compared to those in peripheral blood (PB) monocytes. These reduced levels were reproduced when PB monocytes from healthy donors were stimulated with SF, and this reduction was dependent on p38 MAPK. The induction of p38 by IL-1 in PB monocytes interfered with IL-6 signaling due to the reduced cell surface expression of gp130. Conclusion. These results suggest that p38mediated proinflammatory stimuli induce the downregulation of gp130 on monocytes and thus restrict
gp130-mediated signal transduction. This regulatory mechanism could be of relevance to processes in the inflamed joints of patients with JIA. Juvenile idiopathic arthritis (JIA) constitutes a group of childhood inflammatory arthritic disorders. It is the most common pediatric rheumatic disease, defined by onset before the age of 16 years and persistence of symptoms for at least 6 weeks. Patients have chronic recurrent inflammation of the joints that targets the synovial membrane, cartilage, and bone. The resulting joint effusion, swelling, pain, restricted range of motion, and systemic effects on growth can severely reduce the quality of life (1). Oligoarthritis is the most common form of arthritis in childhood; it predominately affects girls of preschool age and is sometimes associated with the occurrence of antinuclear antibodies and uveitis (2). Oligoarthritis can be treated by intraarticular injection of corticosteroids. During this procedure synovial fluid (SF) and SF cells can be obtained. The cause of JIA is unknown, but multiple factors contribute to its pathogenesis. In addition to genetic and environmental elements, cytokines play a pivotal role in the course of each subtype of the disease. Increased levels of proinflammatory cytokines, such as tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), and IL-6, in the SF of patients with JIA induce several processes that promote joint destruction (3,4). IL-6 has both proinflammatory and antiinflammatory properties, and it is strongly expressed in the serum and SF of patients with oligoarticular JIA (5). IL-6 shares the gp130 subunit with related cytokines, and this subunit functions as a signal transduction component when IL-6 binds to its specific ␣ receptor, IL-6 receptor (IL-6R). The signaling complex comprises 2 gp130 subunits and 2 IL-6R subunits, which together
1
Nora Honke, MSc, Kim Ohl, PhD, Anastasia Wiener, MSc, Jeff Bierwagen, Norbert Wagner, MD, Stefan Wu ¨ller, MD, Klaus Tenbrock, MD: RWTH Aachen University, Aachen, Germany; 2 Joachim Peitz, MD: University of Cologne, Cologne, Germany; 3 Stefano Di Fiore, MSc, Rainer Fischer, PhD: Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany. Address correspondence to Klaus Tenbrock, MD, Department of Pediatrics, Medical Faculty, RWTH Aachen University, Pauwelsstrasse 30, Aachen NRW 52062, Germany. E-mail:
[email protected]. Submitted for publication April 26, 2013; accepted in revised form October 17, 2013. 470
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ulate the activity and differentiation of T cells (13). They are also precursors of macrophages and osteoclasts (14), both of which promote joint destruction and bone resorption (15,16). In this report we provide evidence that the expression of the IL-6 receptor complex on human monocytes is differentially regulated by an inflammatory environment and that cytokine cross-talk plays an important role in the SF of patients with JIA. Our results show that IL-1 regulates IL-6 signaling via p38 by restricting the availability of gp130 on the cell surface.
activate JAKs that are constitutively associated with gp130. This process triggers 3 different pathways, predominantly involving signaling proteins from the STAT family (for review, see ref. 6). Phosphorylated STAT-3 activates several target genes with different biologic roles, depending on the cell type, duration, and strength of the signal. Dysregulated IL-6 signaling is associated with severe autoimmune diseases, such as inflammatory bowel disease and rheumatoid arthritis (RA). Therefore, different regulatory mechanisms exist to ensure that gp130-mediated signaling is tightly controlled. Suppressor of cytokine signaling (SOCS) proteins are expressed in response to STAT signaling and act as classic feedback inhibitors by inactivating JAKs (7). More recently, it has been shown that proinflammatory cytokines, such as IL-1, TNF␣, and lipopolysaccharide (LPS), rapidly inhibit IL-6 signaling through a SOCS-3–independent mechanism that instead involves p38 MAPK (8,9). In hepatocytes, p38-dependent activation of the downstream MAPK leads to the phosphorylation of gp130 on serine residue 782, which is located close to the dileucine internalization motif, promoting the internalization and degradation of the receptor (10). In inflamed joints, where many different cytokines act together and the biologic outcome depends critically on the ability of a specific cell type to integrate the diverse signals, cytokine cross-talk can be particularly important. Human monocytes express large amounts of gp130 on the surface, and nearly all CD14⫹ cells are also gp130⫹ (ref. 11 and Honke et al: unpublished observations). Monocytes are abundant in the SF of patients with JIA and RA and play an important role in pathogenesis (12). Monocytes are potent producers of proinflammatory cytokines, which allows them to mod-
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MATERIALS AND METHODS Materials. Recombinant human IL-1 and IL-6 were purchased from PeproTech and Miltenyi Biotech, respectively. The p38 inhibitor SB202190 was obtained from Calbiochem, and actinomycin D was obtained from Sigma-Aldrich. RPMI 1640 and protease inhibitors were obtained from Invitrogen. Ficoll and Percoll were purchased from GE Healthcare. Patients. All patients (n ⫽ 18) were diagnosed as having oligoarticular JIA and were receiving nonsteroidal antiinflammatory drugs before therapeutic aspiration of SF and instillation of corticosteroids. Cells were pelleted by centrifugation and supernatants were stored at ⫺20°C. Ethical approval for all experiments was obtained from the local ethics committee. Cell isolation. Human mononuclear cells from patients with JIA were isolated onto a Ficoll gradient as paired samples from peripheral blood (PB) and SF. Cells from healthy donors (which were, in part, from buffy coats provided by the local blood bank, Transfusionsmedizin, Universitätsklinikum Aachen, Germany) were isolated from PB by the same procedure. Monocytes were isolated by centrifugation using 2 different Percoll gradients as previously described (17). Flow cytometry. Samples were prepared from PB and SF by washing in phosphate buffered saline (PBS) and fixing in 1% paraformaldehyde before lysing the erythrocytes. After washing, cells were incubated with antibodies against gp130
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Figure 1. Reduced expression of gp130 and interleukin-6 receptor (IL-6R) on peripheral blood (PB) monocytes and synovial fluid (SF) monocytes. Cells were prepared directly from PB or SF and stained for CD14, gp130, and IL-6R. Gating was set on CD14. A, Representative histograms of gp130 and IL-6R expression within the monocyte population. PB monocytes from 1 patient were compared to SF monocytes from the same patient. B, Mean fluorescence intensity (MFI) of gp130 on CD14⫹ cells from individual patients (n ⫽ 18). C, MFI of IL-6R on CD14⫹ cells from individual patients (n ⫽ 15). In B and C, symbols represent individual patients; bars show the mean ⫾ SEM. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by Wilcoxon’s 2-tailed matched pairs test.
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Figure 2. Soluble factors in synovial fluid (SF) cause the downregulation of gp130. A, SF mononuclear cells were isolated by Ficoll gradient centrifugation and either immediately prepared for fluorescence-activated cell sorting analysis or cultured at 37°C for different periods of time in RPMI supplemented with 10% fetal calf serum prior to flow cytometry analysis. The cells were stained with antibodies against gp130, interleukin-6 receptor (IL-6R), and CD14. Gating was set on CD14, and mean fluorescence intensity (MFI) values for gp130 (n ⫽ 5 patients) or IL-6R (n ⫽ 4 patients) were determined. Bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by one-way analysis of variance with Tukey’s multiple comparison test. B, Peripheral blood (PB) mononuclear cells were isolated by Ficoll gradient centrifugation and stimulated with 10% SF or human serum (HS) (as a control) for 20 minutes. Cells were stained for gp130 and CD14 and analyzed by flow cytometry. CD14⫺ cells were excluded by gating, and the mean value in untreated controls was set to 1. Symbols represent paired samples (n ⫽ 25) treated with human serum and with SF; bars show the mean ⫾ SEM. ⴱⴱⴱ ⫽ P ⬍ 0.001 by Wilcoxon’s 2-tailed matched pairs test.
(B-R3 or AM64; Abcam and BD Biosciences, respectively), IL-6R (47.7G7.1F2 or 17506; eBioscience and R&D Systems, respectively), and CD14 (MEM-18; Immunotools) for 20 minutes. Phosphorylated STAT-3 was stained by permeabilizing the cells in ice-cold methanol for at least 10 minutes prior to
washing and incubation with anti–pSTAT-3 (4/P-STAT3; BD Biosciences) and anti-CD14 antibodies. After isolation of mononuclear cells by densitygradient centrifugation, cells (5 ⫻ 105) were incubated with antibodies against gp130 and CD14 for 20 minutes on ice. For intracellular staining, the cells were fixed in 1.5% paraformaldehyde for 10 minutes and treated as described above. Phosphorylated gp130 was examined by the use of an anti– phosphorylated gp130 antibody (Ser782; Bioss). Matched isotype control antibodies (from Immunotools and Bioss) were used to control staining specificity. Flow cytometry was carried out using a FACSCanto II (BD Biosciences). Confocal microscopy. We seeded 1,500 peripheral blood mononuclear cells (PBMCs) per well into 96-well plates coated with poly-L-lysine and allowed them to settle overnight. Nonadherent lymphocytes were removed by washing with PBS, and the cells were incubated with antibodies against gp130 (BR-3) and CD14 (MEM-18) for 30 minutes on ice. Cells were washed with PBS (to remove nonbound antibodies) and RPMI 1640 without phenol red, and supplemented with 10% fetal calf serum (FCS) with or without the addition of 20 ng/ml of IL-1 prior to microscopy. Images were acquired automatically with an Opera high-content screening system (PerkinElmer) (10 positions per well using a 40⫻ air objective). The images were evaluated under blinded conditions, and cells were assessed based on the following scoring system: 0 ⫽ no internalization, 0.5 ⫽ early internalization (appearance of some rafts), 1 ⫽ medium internalization (appearance of many rafts), and 1.5 ⫽ late internalization (appearance of many rafts and intracellular staining). Immunoblot analysis. For the analysis of whole cell extracts, 4 ⫻ 106 cells were lysed, separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. Y705-phosphorylated STAT-3 was detected by probing the membrane with ␣–pSTAT-3 (Cell Signaling Technology) and the appropriate horseradish peroxidase–coupled secondary antibody (DakoCytomation). A chemiluminescence kit (Millipore) was used to visualize the protein bands. The blot was stripped and reprobed with STAT-3 (124H6; Cell Signaling Technology) to detect total STAT-3 levels. RNA isolation, complementary DNA (cDNA) synthesis, and quantitative real-time polymerase chain reaction (PCR). Total RNA was extracted from 4 ⫻ 106 cells using an RNeasy Mini Kit (Qiagen) and transcribed to cDNA using a First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions. Standard real-time PCR was carried out on a TaqMan 7900 (Applied Biosystems) using the DNA intercalating dye SYBR Green. The expression of SOCS-3 was normalized to the expression of 3 different housekeeping genes: -actin, RPL13A, and GAPDH. We used the following primer sequences: for -actin, GAC-TAC-CTCATG-AAG-ATC-CTC-ACC (forward) and TCT-CCT-TAATGT-CAC-GCA-CGA-TT (reverse); for RPL13A, 5⬘-AGGTAT-GCT-GCC-CCA-CAA-AAC-3⬘ (forward) and 5⬘-TGTAGG-CTT-CAG-ACG-CAC-GAC-3⬘ (reverse); for GAPDH, GAA-GGT-GAA-GGT-CGG-AGT-CAA-C (forward) and CAG-AGT-TAA-AAG-CAG-CCC-TGG-T (reverse); and for SOCS-3, 5⬘-CAC-CTG-GAC-TCC-TAT-GAG-AAA-GTCA-3⬘ (forward) and 5⬘-GGG-GCA-TCG-TAC-TGG-TCCAGG-AA-3⬘ (reverse).
DOWN-REGULATION OF CELL SURFACE gp130 EXPRESSION BY INFLAMMATION
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Figure 3. Internalization of gp130 is dependent on p38. A, DMSO or SB202190 (5 M) was added to peripheral blood mononuclear cells (PBMCs) 30 minutes prior to stimulation with 10% synovial fluid (SF) for 20 minutes. The mean fluorescence intensity (MFI) of gp130 within the monocyte population was analyzed by flow cytometry, with mean values set to 1. Values after DMSO and SB202190 treatment of PBMCs from 11 individuals are shown. ⴱ ⫽ P ⬍ 0.05 by paired 2-tailed t-test. B, Representative live cell images in the presence or absence of interleukin-1 (IL-1) are shown, revealing staining with antibodies against gp130 and CD14. Boxed areas in the left panels of the merge images are shown at higher magnification in the right panels. FITC ⫽ fluorescein isothiocyanate; APC ⫽ allophycocyanin. C, Cells in the images were assigned an internalization score as described in Materials and Methods. Bars show the mean ⫾ SEM (n ⫽ 231 for untreated cells and n ⫽ 61 or n ⫽ 46 for treated cells). ⴱⴱⴱ ⫽ P ⬍ 0.001 by one-way analysis of variance with Dunn’s multiple comparison test. D, PBMCs (n ⫽ 6) were stimulated and analyzed as described in A, but the concentration of SB202190 was 1 M, and 20 ng/ml IL-1 was added instead of SF. Symbols represent samples; bars show the mean ⫾ SEM. ⴱⴱ ⫽ P ⬍ 0.01 by paired 2-tailed t-test.
RESULTS Synovial fluid down-regulates the IL-6 receptor complex on monocytes. We used flow cytometry to investigate the cell surface expression of the IL-6 receptor complex on PB and SF monocytes, and found that both components of the complex (gp130 and IL-6R) were expressed at significantly lower levels in SF monocytes as compared to PB monocytes (Figure 1). It has previously been shown that proinflammatory cytokines restrict IL-6 signaling by modulating receptor availability. We, in turn, sought to determine whether soluble factors present in the SF were responsible for the down-regulation of gp130 and IL-6R. SF mononuclear
cells (SFMCs) were isolated by density-gradient centrifugation, incubated in RPMI supplemented with 10% FCS, and rested for up to 60 minutes. We measured the expression of gp130 and IL-6R over time and found that the expression of gp130 on the monocyte cell surface increased significantly within 1 hour of culture, whereas IL-6R levels remained constant (Figure 2A). To determine whether SF could subsequently down-regulate the expression of gp130 on the cell surface, we incubated PBMCs isolated from healthy donors with 10% SF in RPMI. As expected, this resulted in rapid (within 20 minutes) and significant downregulation of gp130 in monocytes (Figure 2B). These
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Figure 4. Interleukin-1 (IL-1) inhibits IL-6–induced STAT-3 phosphorylation via a p38-dependent mechanism. A, Monocytes were isolated by Ficoll and Percoll gradient centrifugation and stimulated with IL-6 (20 ng/ml) for 10 minutes. For costimulation, IL-1 (20 ng/ml) was added 20 minutes prior to stimulation with IL-6. Cell lysates were prepared and analyzed by Western blotting, using an antibody against Y705-phosphorylated STAT-3. After detection, the blot was stripped and reprobed with an antibody against STAT-3. B, Peripheral blood (PB) mononuclear cells were stimulated as described for A. DMSO or SB202190 (1 M) was added 30 minutes before the addition of IL-1. Mean fluorescence intensity (MFI) values for pSTAT-3 within the monocyte population were determined by fluorescence-activated cell sorting analysis. Representative histograms are shown. Bars show the mean ⫾ SEM (n ⫽ 5). ⴱⴱⴱ ⫽ P ⬍ 0.001 by one-way analysis of variance with Bonferroni adjustment for multiple comparisons.
experiments showed that soluble factors in the SF induce the down-regulation of cell surface gp130 expression on monocytes.
The down-regulation of gp130 is mediated by p38. Previous studies have revealed that IL-6 signaling in different cell types is regulated by cross-talk involving stress-induced p38 MAPK (9,18). For example, Radtke et al have shown that p38 mediates the internalization and degradation of gp130 in hepatocytes following stimulation with IL-1 or TNF␣ (10). We therefore investigated whether inhibition of p38 would prevent the down-regulation of gp130 in PBMCs stimulated with SF. PBMCs were preincubated with the p38-specific inhibitor SB202190 or the carrier DMSO prior to the addition of SF. As shown in Figure 3A, the cell surface expression of gp130 on monocytes was rescued by preincubation with the p38 inhibitor. Next we assessed whether IL-1 itself can regulate IL-6 signaling in monocytes by down-regulating gp130 surface expression, as has been seen in hepatocytes. Live cell imaging was used to monitor receptor molecules on the cell surface after labeling with an antibody, and their uptake was examined by confocal microscopy. Images were acquired immediately and after incubation for 10 minutes. Consistent with the findings of previous studies (19), the normal rate of constitutive internalization (indicated by the increasing internalization score in the absence of IL-1 after 10 minutes [Figure 3C]) was significantly and rapidly enhanced in the presence of IL-1 (Figures 3B and C). To determine the role of p38 in this process, gp130 surface expression was analyzed by flow cytometry in the presence of SB202190. As when SF was added to PBMCs, the loss of gp130 from the cell surface was efficiently prevented by inhibiting p38 (Figure 3D). These data suggest that p38 plays a role in the downregulation of gp130 by SF and IL-1. The preincubation of PBMCs with a p38-specific inhibitor prevented the loss of gp130 (induced by IL-1 or SF) from the monocytic cell surface. However, preincubation of PBMCs with anakinra, an IL-1 receptor antagonist, did not result in reconstitution of cell surface gp130 expression after stimulation with SF (data not shown), which supports the notion that multiple factors, rather than a single cytokine, are responsible for gp130 downregulation by SF. STAT-3 phosphorylation induced by IL-6 is inhibited by IL-1 via a SOCS-3–independent, but p38dependent, mechanism. The impact of reduced cell surface gp130 expression on downstream signaling was tested by measuring IL-6–induced STAT-3 activation. Prestimulation of monocytes with IL-1 resulted in a significant reduction in IL-6–induced STAT-3 phosphorylation (Figure 4A), consistent with results of pre-
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Figure 5. Interleukin-1 (IL-1) inhibits IL-6–induced STAT-3 phosphorylation via a SOCS-3–independent mechanism. A, Peripheral blood mononuclear cells (PBMCs) were stimulated with IL-6 (20 ng/ml) for 10 minutes. For costimulation, IL-1 (20 ng/ml) was added 20 minutes prior to IL-6. DMSO or actinomycin D (5 g/ml) was added 30 minutes before the addition of IL-1. Mean fluorescence intensity (MFI) values for pSTAT-3 within the monocyte population were determined by fluorescence-activated cell sorting analysis. Representative histograms are shown. Bars show the mean ⫾ SEM (n ⫽ 4). ⴱⴱⴱ ⫽ P ⬍ 0.001 by one-way analysis of variance with Bonferroni adjustment for multiple comparisons. B, PBMCs were stimulated with IL-6 (20 ng/ml) for 10 minutes, or for 1 hour as a positive control. For costimulation, IL-1 (20 ng/ml) was added 20 minutes prior to IL-6. RNA was isolated and used to prepare cDNA for quantitative real-time polymerase chain reaction. SOCS-3 expression was adjusted to the expression of the housekeeping genes RPL13A, -actin, and GAPDH. Bars show the mean ⫾ SEM fold SOCS-3 expression (n ⫽ 4) compared with that in untreated cells (set at 1). NS ⫽ not significant.
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proinflammatory cytokines such as TNF␣ and IL-1 can enhance IL-6–induced feedback via SOCS-3 by stabilizing SOCS-3 mRNA (20,21). Therefore we investigated whether IL-1 could reduce IL-6–induced STAT-3 activation independent of de novo protein synthesis and, thus, SOCS-3 expression. Figure 5A shows that the inhibitory effect of IL-1 was not influenced by treating
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vious studies on macrophages (8). We therefore used SB202190 to determine whether the inhibitory activity of IL-1 was p38 dependent. Although IL-6–induced STAT-3 phosphorylation was reduced to ⬃70% by IL-1 in the absence of SB202190, pretreatment with this inhibitor efficiently blocked the inhibitory effect of IL-1 (Figure 4B). It has previously been shown that
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Figure 6. Internalization of gp130 is induced by p38-mediated serine 782 phosphorylation of gp130. A, Peripheral blood mononuclear cells (PBMCs) were incubated with SB202190 (1 M) or the carrier DMSO for 30 minutes prior to interleukin-1 (IL-1) stimulation (20 ng/ml for 20 minutes). Gating was set on CD14 cells, and the percentage of cells positive for phosphorylated gp130 (serine 782) was determined by flow cytometry. Bars show the mean ⫾ SEM (n ⫽ 8). ⴱ ⫽ P ⬍ 0.05 by Wilcoxon’s 2-tailed matched pairs test. B, PBMCs (n ⫽ 5 donors) were isolated by Ficoll gradient centrifugation and stimulated with 10% synovial fluid (SF) for 20 minutes or left untreated (n ⫽ 13 experiments altogether). The percentage of phosphorylated gp130⫹ cells within the CD14⫹ population was analyzed by fluorescence-activated cell sorting. ⴱ ⫽ P ⬍ 0.05 by Wilcoxon’s matched pairs test. C, Cells were stimulated and analyzed as described in A, but the concentration of SB202190 was 5 M, and 10% SF was added instead of IL-1. The value in untreated controls was set at 1. Symbols represent individual samples (n ⫽ 15); bars show the mean ⫾ SEM. ⴱⴱ ⫽ P ⬍ 0.01 by Wilcoxon’s 2-tailed matched pairs test.
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the cells with actinomycin D. We also used real-time PCR to confirm that SOCS-3 mRNA was not significantly induced by IL-1 or IL-6 alone or in combination at the concentrations and stimulation times used in our experiments (Figure 5B). We conclude from these experiments that the down-regulation of gp130 expression by IL-1 on monocytes impairs IL-6 signaling. Inhibiting SOCS-3 synthesis did not influence these effects, indicating the presence of a SOCS-3–independent mechanism. However, the inhibition of IL-6–induced STAT-3 phosphorylation by IL-1 was prevented by blocking of p38 activity. Stimulation with IL-1 or SF promotes serine 782 phosphorylation of gp130. In hepatocytes, the internalization of gp130 in response to p38-inducing stimuli was found to require phosphorylation of serine residue 782 in the cytoplasmic tail, which is proximal to the di-leucine internalization motif (10). In primary monocytes, stimulation with IL-1 promoted the phosphorylation of gp130 serine 782 (Figure 6A), indicating that this residue may act as a stress sensor not only in hepatocytes, but also in monocytes. Preincubation with the p38 inhibitor SB202190 efficiently prevented phosphorylation of gp130 in response to IL-1 (Figure 6A). Based on these results, we investigated whether stimulation of PBMCs with SF could induce serine phosphorylation of gp130. Figure 6B shows that the incubation of PBMCs with SF resulted in an increased percentage of monocytes with phosphorylated gp130. Again, this action could be blocked by inhibition of p38 (Figure 6C), suggesting that phosphorylation of serine 782 requires activity of p38 and most likely is a critical factor in a stress-induced cascade that leads to diminished receptor availability on the monocytic cell surface.
DISCUSSION We have shown that p38-inducing stimuli rapidly reduce cell surface expression of the IL-6 receptor complex on human monocytes in an inflammatory environment. This action thus prevents an excessive IL-6– mediated response during inflammation. IL-6 is involved in the pathogenesis of JIA, as demonstrated by the successful clinical application of tocilizumab, an antibody against IL-6R (22,23). The previous finding that experimental arthritis cannot be induced in IL-6–deficient mice (24,25) further supports the notion that IL-6 has a pathogenic role. Interestingly, the administration of IL-6 alone is not sufficient to
restore the disease; restoration of the disease can be accomplished by administering a fusion protein comprising soluble IL-6R (sIL-6R) and IL-6 (25). This alternative form of IL-6 signaling through its soluble receptor is known as IL-6 trans-signaling and has been highlighted as a key pathway at sites of inflammation (25–28). The use of sIL-6 for signal transduction not only makes cells respond to IL-6 (even if they express gp130 only and do not express IL-6R), but also amplifies signaling in cells (like monocytes) that express both receptor components (29). This highlights the importance of strictly regulated gp130 cell surface expression. High levels of IL-6 and its soluble receptor are found in the SF of patients with oligoarticular JIA and other subtypes of JIA, and it has been suggested that IL-6–sIL-6R complexes contribute to osteoclastogenesis in arthritic joints (30,31). Normally, the activities of bone-forming osteoblasts and bone-resorbing osteoclasts are finely balanced, but this may be disrupted during inflammation (32). The destruction of articular cartilage and bone is a hallmark of JIA and RA (33). Osteoclasts develop from hematopoietic cells of the monocyte/macrophage lineage (34). It has been shown that IL-6 cooperates with macrophage colonystimulating factor (M-CSF) to induce the differentiation of CD14⫹ cells into osteoclasts. This process may not be relevant in normal bone physiology, but instead may be relevant during pathologic events featuring increased cytokine signaling (35). IL-6 has also been shown to up-regulate the expression of M-CSF receptors on monocytes (36), which may amplify the induction of osteoclastogenesis by IL-6. Stress-induced p38 MAPK has been highlighted as a key player in previous studies investigating the cross-talk regulation of IL-6 signaling (8,10,18,37). Furthermore, p38 plays a crucial role in the induction and maintenance of chronic inflammation and is strongly activated in inflammatory arthritis (38). In the present study, inhibition of p38 restored gp130 cell surface expression after incubation with SF (Figure 3A). However, complete recovery to the expression levels observed in untreated cells was not achieved, indicating that other factors may facilitate the down-regulation of gp130. One likely candidate is IL-6 itself, since endocytosis of the receptor complex has been observed after ligand binding (39). Furthermore, the IL-6 receptor complex on the surface of T cells is down-regulated by SF from RA patients, and this effect is blocked in the presence of an anti–IL-6R antibody (40). We have shown that the impact of SF on monocytic gp130 expression can be reproduced in the pres-
DOWN-REGULATION OF CELL SURFACE gp130 EXPRESSION BY INFLAMMATION
ence of IL-1 (Figures 3B and C), which is a potent inducer of p38 (41). The down-regulation of gp130 by IL-1 is less potent than the response of gp130 to SF, which is probably a reflection of the difference between the effects of a single cytokine and the cytokine milieu or other inflammatory mediators contained in SF. The inhibition of p38, in the case of IL-1–treated cells, restored gp130 expression to a level close to that in untreated cells (Figure 3D), which again suggests that other SF components contribute to the down-regulation of gp130. The restricted availability of gp130 on the cell surface after stimulation with IL-1 reduces the response to IL-6, as shown by the limited activation of STAT-3 when the cells were prestimulated with IL-1 (Figure 4A). Consistent with previously reported findings (8–10), these effects were independent of the feedback inhibitor SOCS-3 (Figure 4B). Inhibition of p38 was once again shown to reverse the effect of IL-1, which in our opinion reflects the reconstitution of gp130 cell surface expression. Considering the proinflammatory effects of IL-6 (e.g., on osteoclast formation), the immediate downregulation of the IL-6 receptor complex by SF could represent a mechanism to counterbalance an overwhelming proinflammatory reaction. Conversely, IL-6 demonstrates antiinflammatory activities in myeloid cells, primarily in macrophages (42). In such a scenario, proinflammatory cytokines such as IL-1 could intensify inflammation by down-regulating gp130. It remains to be determined whether and to what extent the loss of gp130 on the cell surface is translated into the induction of gene expression and how exactly this contributes to the pathogenesis of oligoarticular JIA.
ACKNOWLEDGMENTS We thank the donors and patients who participated in this study.
AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Tenbrock had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Honke, Wu ¨ller, Tenbrock. Acquisition of data. Honke, Wiener, Bierwagen, Peitz, Di Fiore. Analysis and interpretation of data. Honke, Ohl, Di Fiore, Fischer, Wagner, Wu ¨ller, Tenbrock.
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