Blockade of Soluble IL-6 Receptor Governs IL-6 Activit

The Journal of Immunology

Soluble IL-6 Receptor Governs IL-6 Activity in Experimental Arthritis: Blockade of Arthritis Severity by Soluble Glycoprotein 130

Mari A. Nowell,* Peter J. Richards,* Sankichi Horiuchi,† Naoki Yamamoto,† Stefan Rose-John,‡ Nicholas Topley,§ Anwen S. Williams,¶ and Simon A. Jones1*

-Studies in IL-6-deficient (IL-6؊/؊) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mecha nism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6؊/؊ mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. His- topathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasyno- vial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis. The Journal of Immunology, 2003, 171: 3202–3209.

lthough elevated IL-6 concentrations have been docu- restricted to hepatocytes, megakaryocytes, and leukocytes, which mented in the serum and synovium of arthritic patients infer that sIL-6R-mediated signaling represents the prominent (1–5), the molecular mechanism by which it contributes mechanism by which IL-6 elicits its effects in vivo. Analysis of A 2 to the pathogenesis of rheumatoid arthritis (RA) is the subject of synovial fluids from arthritic patients highlights that increases in much debate (6, 7). A pivotal role for IL-6 is, however, suggested synovial sIL-6R levels correlate with the degree of leukocyte in- by several experimental models of arthritis, in which induction of filtration and correspond with the more advanced stages of RA (5, disease in IL-6-deficient (IL-6Ϫ/Ϫ) mice results in no clinical signs 23). Synovial sIL-6R concentrations might therefore originate or significantly reduced arthritis (8–12). Indeed, many of the sys- from infiltrating leukocytes and may ultimately control IL-6 sig- temic symptoms associated with RA, such as high fever, autoan- naling within an inflamed arthritic joint. This is supported by in tibody production, and regulation of the acute-phase response, vitro studies, which have implicated the involvement of sIL-6R in have been attributed to IL-6, while Abs directed against IL-6 or its osteoclast formation, synovial fibroblast proliferation, and carti- cognate receptor (IL-6R) have proven to be clinically beneficial (5, lage degradation (23–26). 13–19). However, the manner by which localized IL-6 responses Using a murine model of monoarthritic Ag-induced arthritis, we are regulated within an inflamed joint and the consequence of its have examined the significance of sIL-6R signaling within the sy- action remain far from clear. novium and established that blockade of sIL-6R activity by its It is increasingly apparent that many of the activities assigned to natural antagonist soluble gp130 (sgp130) inhibits arthritis pro- IL-6 are mediated via soluble IL-6R (sIL-6R) (20, 21). This sol- gression. Thus, use of the inhibitory properties of sgp130 may uble receptor forms a stimulatory complex with IL-6, which trig- represent a novel therapeutic strategy for the treatment of RA. gers cellular events through the ubiquitously expressed signal- transducing subunit for all IL-6-related cytokines, gp130 (22). Materials and Methods Soluble IL-6R-mediated signaling thus enables activation of cell Synovial fluid types that would not normally respond to IL-6 itself (20, 21). In- Ethical approval was obtained from Bro-Taf Health Authority (Cardiff, terestingly, the cellular distribution of the cognate IL-6R is largely Wales, U.K.) before commencing the study. All patients were diagnosed as having RA for at least 3 mo. Such patients used nonsteroidal anti- inflammatory drugs, corticosteroids, and disease-modifying antirheumatic *Cardiff School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom; drugs (with the exception of TNF inhibitors) either alone or in combina- †Department of Microbiology, Tokyo Medical and Dental University, Tokyo, Japan; tion. These regimes are in line with conventional rheumatology clinical ‡Institu¨t fu¨r Biochemie, Christian-Albrechts-Universita¨t zu Kiel, Kiel, Germany; and practice. Synovial fluids were collected from RA (n ϭ 25) and osteoar- § ¶ Institute of Nephrology and Department of Rheumatology, University of Wales thritis (OA; n ϭ 15) patients during routine joint aspiration. Fluids were College of Medcine, Cardiff, Wales, United Kingdom rendered cell free by centrifugation and supernatants stored at Ϫ80°C. Received for publication April 28, 2003. Accepted for publication July 10, 2003. Before ELISA determination, synovial fluids were diluted 50:50 (v/v) in The costs of publication of this article were defrayed in part by the payment of page PBS containing 1% BSA. charges. This article must therefore be hereby marked advertisement in accordance Animals with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Experiments were undertaken in 7- to 8-wk-old male inbred C57BL/6J Address correspondence and reprint requests to Dr. Simon A. Jones, Cardiff School ϩ/ϩ Ϫ/Ϫ of Biosciences, Molecular Cell Biology Research Group, Cardiff University, Biomed- wild-type (IL-6 ) and IL-6 mice (27). Procedures were performed in ical Sciences Building, Museum Avenue (P.O. Box 911), Cardiff CF10 3US, Wales, accordance with Home Office-approved project license PPL-30/1820. U.K. E-mail address: [email protected] Induction of murine Ag-induced arthritis (AIA) 2 Abbreviations used in this paper: RA, rheumatoid arthritis; AIA, Ag-induced arthritis; DAB, diaminobenzidine; DS, differential mRNA splicing; HSF, human synovial fibro- Briefly, male mice were s.c. immunized with 1 mg/ml of methylated BSA blast; i.a., intra-articular; mBSA, methylated BSA; OA, osteoarthritis; PC, proteolytic (mBSA) emulsified with an equal volume of CFA and injected i.p. with (shedding) cleavage; RAPS, RA antigenic peptide-bearing soluble form; s, soluble. 100 ␮l heat-inactivated Bordetella pertussis toxin (all reagents were from

Sigma-Aldrich, St. Louis, MO). The immune response was boosted 1 wk ␮g) was reverse transcribed using random hexanucleotide primers and Su- later. Twenty-one days after the initial immunization, AIA was induced by perscript II Reverse Transcriptase (Life Technologies Invitrogen). ␤-Actin intra-articular (i.a.) administration of 10 mg/ml mBSA in the right hind oligonucleotide primers were used as a normalization control. RT-PCR for limb. To modulate IL-6 signaling in vivo, AIA was induced by i.a. admin- CCL2 and ␤-actin was performed, as described (32). istration with mBSA in conjunction with 50 ng/ml IL-6, 50 ng/ml HYPER- FACS IL-6, or 500 ng/ml sgp130 (R&D Systems, Minneapolis, MN). In all cases, the total volume administered i.a. was 10 ␮l. HYPER-IL-6 was prepared, Analysis of gp130 and IL-6R on HSF was performed, as previously de- as previously described (28). scribed (27). Assessment of anti-mBSA titers Statistical methods ϩ/ϩ Ϫ/Ϫ Anti-mBSA titers were monitored in sera from IL-6 and IL-6 mice Statistical analysis was performed using one-way unpaired ANOVA. Val- immunized with 1 mg/ml mBSA. Briefly, microtiter plates were coated ues of p Ͻ 0.05 were considered statistically significant. with 5 ␮g/ml mBSA and blocked with 5% (w/v) milk extract in PBS containing 0.05% Tween 20. Sera were diluted as indicated in PBS containing milk extract and Tween 20. Bound Abs were detected using HRP- Results conjugated goat anti-mouse IgG as a secondary Ab. Absorbance changes Detection of sIL-6R isoforms in synovial fluids of arthritic were monitored at 450 nm, and values were presented as the mean Ϯ SD patients from four mice per condition. Examination of human synovial fluids obtained from 40 indepen- Histological assessment of arthritis dent donors (25 RA; 15 OA) showed that synovial IL-6 levels were Arthritis severity was judged by comparing the difference between the AIA significantly elevated ( p Ͻ 0.05) in RA patients (51.17 Ϯ 9.46 (right hind limb) and control (left hind limb) joints. Animals were in- ng/ml) as compared with those diagnosed with OA (6.39 Ϯ 3.35 spected daily for arthritis development by measuring the knee joint diam- eter using a digital micrometer. Inflammatory parameters were assessed in ng/ml). Synovial concentrations of sIL-6R were also significantly six animals (per condition) sacrificed at days 3 and 21 post-i.a. over two more elevated in individuals with RA (28.54 Ϯ 2.39 ng/ml) than separate experiments. Joints were fixed in neutral buffered formal saline OA (14.78 Ϯ 2.11 ng/ml), and these levels directly correlated (r ϭ and decalcified with formic acid at 4°C before embedding in paraffin. Mid- 0.57) with those of IL-6 ( p Ͻ 0.001; Fig. 1). Moreover, when ␮ sagittal serial sections (of 7 m thickness) were stained with hematoxylin/ appreciating the significance of sIL-6R during disease, it is note- safranin-O. Two independent observers blinded to the experimental groups scored sections. Synovial hyperplasia (pannus formation), cellular exudate, worthy to consider that differential mRNA splicing (DS) and pro- and cartilage depletion/bone erosion were scored from 0 (normal) to 3 teolytic (shedding) cleavage (PC) of the cognate receptor both lead (severe). Synovial infiltrate was scored from 0 to 5. All parameters were to the generation of sIL-6R (termed DS-sIL-6R and PC-sIL-6R) subsequently summed to give an arthritis index (mean Ϯ SEM). (20). These isoforms can be distinguished using Abs against the Immunohistochemistry Localization of Ags in paraffin sections was detected using an HRP-DAB (diaminobenzidine) Cell & Tissue Staining kit from R&D Systems. To eliminate endogenous peroxidase activity, sections were treated with a peroxidase-blocking reagent (R&D Systems). Sections were subsequently treated with 10% normal donkey serum and an avidin/biotin blocking reagent (R&D Systems) to reduce nonspecific staining before labeling with either 2 ␮g/ml goat anti-CCL2, anti-CCR2, or isotype control Abs (Auto- gen Bioclear, Calne, U.K.). All reagents were diluted in PBS containing 1% BSA. Ab labeling was detected using a high sensitivity streptavidin- HRP conjugate and DAB chromogen (R&D Systems). Intensity of staining was measured in sections of synovium taken from three to four separate animals. Images were digitally captured using a Neotech ImageGrabber PCI, and the DAB staining intensity was analyzed using Aequitas IA soft- ware (Dynamic Data Links, Cambridge, U.K.). Three randomly chosen areas of synovium were measured per section. Human synovial fibroblasts (HSF) HSF were isolated from synovial tissue obtained after synovectomy from RA patients at the time of total knee joint replacement. HSF were isolated and maintained, as previously described (29). Briefly, HSF were digested for1hat37°C with 1 mg/ml collagenase (Sigma-Aldrich). Cells were cultured in DMEM medium containing 100 U/ml penicillin/streptomycin; 1 ␮M hy- drocortisone, 1ϫ insulin-transferrin-selenium-X, and 20% heat-inactivated FCS (from Life Technologies Invitrogen, Paisley, U.K.). All stimulations were performed on growth-arrested HSF, and cells were exclusively used between the third and seventh passage. Cells were stimulated with 20 ng/ml IL-6 and 0–100 ng/ml PC-sIL-6R (R&D Systems), or DS-sIL-6R, or 0–100 ng/ml HY- PER-IL-6, as indicated in the figure legends. DS-sIL-6R was prepared, as previously described (30, 31). In control experiments, 2 ␮g/ml of a blocking anti-gp130 Ab was added (mAb-228; R&D Systems). ELISA Matched Ab pairs and protein standards for use in IL-6, sIL-6R, CCL5, CXCL8, CXCL9, and CXCL10 ELISA were obtained from R&D Systems. CCL2, CCL3, CCL4, and CCL11 were quantified using matched Ab pairs FIGURE 1. Soluble isoforms of IL-6R are elevated in RA. Concentra- from BD PharMingen (San Diego, CA) and Amersham Pharmacia Biotech tions of sIL-6R in synovial fluid from RA (F) and OA (E) patients. Each (Piscataway, NJ). Assays were performed in accordance with the manu- data point represents an individual patient (25 RA patients and 15 OA facturer’s instructions. Detection of DS-sIL-6R was conducted, as previ- Ͻ ously described (31). patients). The horizontal bars designate the mean for each condition (p 0.001). A Pearson correlation coefficient value of 0.03 was obtained for RT-PCR sIL-6R and IL-6 and is displayed as an inset. DS-sIL-6R was detectable in RNA was extracted from HSF with 500 ␮l TRI-reagent (Sigma-Aldrich) approximately one-third of the above RA patients and in none of the OA and processed according to the manufacturer’s protocol. Total RNA (0.5 patient synovial fluid. 3204 SOLUBLE IL-6R GOVERNS IL-6 ACTIVITY

FIGURE 2. Ag-induced arthritis in IL-6Ϫ/Ϫ and IL-6ϩ/ϩ mice. AIA was induced in preimmunized IL- 6ϩ/ϩ and IL-6Ϫ/Ϫ mice by i.a. administration of mBSA in PBS. a, Joint swelling after i.a. injection. Data are mean Ϯ SEM of a single measurement of swelling from all mice remaining in the experiment before sacrifice for histology. Data from IL-6Ϫ/Ϫ mice injected (i.a.) with HYPER-IL-6 alone, without prior immunization with mBSA are also included (open boxes). b, Histopathological analysis of hematoxylin/ safranin-O-stained sections from experimental mice sacrificed on day 3. Six mice were sacrificed in each group over two separate experiments. Two blinded independent observers scored each slide. Synovial hyperplasia, synovial infiltrate, cellular exudate, and joint erosion values were totaled to give an arthritis /p Ͻ 0.001. Hematoxylin ,ءءء ;p Ͻ 0.01 ,ءء .index Safranin-O-stained midsagittal sections from IL-6ϩ/ϩ and IL-6Ϫ/Ϫ mice sacrificed on day 3 of AIA are shown in cÐe. Representative micrographs are shown from mBSA-challenged IL-6ϩ/ϩ mice (c); IL-6Ϫ/Ϫ mice (d); and IL-6Ϫ/Ϫ mice administered with HY- PER-IL-6 (e). Femural and tibial heads are on the left and right, respectively. The synovium is at the top of each micrograph, and the meniscus is in the center. Scale bar represents 100 ␮m.

novel COOH-terminal sequence (GSRRRGSCGL) of DS-sIL-6R, via sIL-6R (28). Administration of IL-6 only marginally increased which is introduced through the splicing process (30, 31). Using joint swelling over the first 3 days of arthritis induction, while HY- such an approach, we observed that expression of DS-sIL-6R was PER-IL-6 restored the profile of joint swelling to that encountered in confined to patients with RA (0.07 Ϯ 0.03 ng/ml) and was not IL-6ϩ/ϩ mice (Fig. 2a). In agreement with previous studies, anti- detected in any of the synovial fluids obtained from individuals mBSA titers were lower in IL-6Ϫ/Ϫ than IL-6ϩ/ϩ mice (9, 10); how- with OA (Fig. 1). These increases in DS-sIL-6R did not reflect ever, i.a. administration of HYPER-IL-6 did not affect the Ab titer systemic changes in sIL-6R levels (data not shown; see also Ref. detected in IL-6Ϫ/Ϫ mice, emphasizing that joint swelling in response 31), and implies that DS-sIL-6R production occurs locally within to HYPER-IL-6 was not Ab dependent (Fig. 3). Finally, to ensure that the inflamed synovium during active disease. Reconstitution of sIL-6R signaling in IL-6Ϫ/Ϫ mice restores experimental arthritis The monoarthritic AIA model has previously been used to show that IL-6 has an effect on both the induction and effector phase of arthritis (9). To examine the role of sIL-6R signaling within an arthritic joint, we have monitored AIA in wild-type (IL-6ϩ/ϩ) and IL-6Ϫ/Ϫ mice. As shown in Fig. 2a, IL-6ϩ/ϩ mice displayed sub- stantial joint swelling upon induction of arthritis with mBSA, which continued for ϳ14 days post-i.a. administration. Con- versely, the degree of joint swelling observed in IL-6Ϫ/Ϫ mice was markedly reduced, and by day 3 showed no significant difference from the contralateral nonarthritic limb (Fig. 2a). To confirm that the swelling response was the product of local IL-6 activity, sig- Ϫ/Ϫ FIGURE 3. Anti-mBSA titer determinations. Anti-mBSA titers were naling was reconstituted within the joint space of IL-6 mice by measured using ELISA in sera from immunized IL-6ϩ/ϩ (boxes) and IL- administration of mBSA in combination with 50 ng/ml IL-6 or 6Ϫ/Ϫ (circles) mice. Wild-type mice challenged with 500 ng/ml sgp130 HYPER-IL-6 (i.a.). HYPER-IL-6 is a unimolecular sIL-6R-IL-6 (open boxes) and IL-6-deficient mice challenged with 50 ng/ml HYPER- fusion protein, which prevents the natural dissociation-association IL-6 (E) are also shown. Absorbance changes were monitored at 450 nm. of the ligand receptor complex and ensures signaling only occurs Values represent the mean Ϯ SD from four mice per condition. The Journal of Immunology 3205

Table I. Joint inﬂammation and cartilage damage on day 3 of AIAa

Histopathological Parameter

Species Treatment Hyperplasia Synovial inﬁltrate Synovial exudate Erosions Arthritis Index

IL-6ϩ/ϩ PBS only 1.50 Ϯ 0.23 2.58 Ϯ 0.23 2.50 Ϯ 0.19 0.42 Ϯ 0.15 7.00 Ϯ 0.46 ءءءϮ 0.43 4.07 ءءϮ 0.00 0.00 ءءءϮ 0.19 1.24 ءIL-6Ϫ/Ϫ PBS only 1.07 Ϯ 0.19† 1.78 Ϯ 0.18 IL-6Ϫ/Ϫ HYPER-IL-6 1.50 Ϯ 0.25† 2.12 Ϯ 0.18† 2.25 Ϯ 0.21† 0.25 Ϯ 0.11† 6.13 Ϯ 0.43† ءءϮ 0.00† 3.4 Ϯ 1.07 0.00 ءءءIL-6ϩ/ϩ sgp130 0.90 Ϯ 0.41† 1.70 Ϯ 0.42† 0.80 Ϯ 0.33

a Synovial hyperplasia, cellular infiltrate, and exudate into the synovial cavity were observed in hematoxylin/safranin-O-stained sections. Cartilage erosion was assessed as loss of red staining on femoral and tibial heads in the stained sections. Histology was assessed by two blinded independent observers and scored from 0 to 3 (hyperplasia, exudate, and erosions) or 0 to 5 (infiltrate). A final arthritis index is given as a sum of all observations. Comparisons were made against control IL-6ϩ/ϩ mice values by one-way unpaired .p Ͻ 0.001 ءءء ;p Ͻ 0.01 ءء ;p Ͻ 0.05 ء ;ANOVA. †, NS the joint swelling associated with HYPER-IL-6 treatment was not defined, whereas the intensity of staining was dramatically increased simply inherent to this chimeric protein, HYPER-IL-6 was adminis- in IL-6Ϫ/Ϫ mice receiving HYPER-IL-6. Expression of CCL2 colo- tered i.a. to IL-6Ϫ/Ϫ mice that had not received prior challenge from calized with staining for CCR2 and supported a role for sIL-6R in mBSA, and were therefore not prone to Ag-induced arthritis. Under regulating mononuclear leukocyte infiltration (Fig. 4). these conditions, no joint swellings were encountered over the course The sIL-6R isoforms regulate CCL2 expression in synovial of the experiment, which infers that restoration of arthritis severity by fibroblasts in vitro HYPER-IL-6 is a product of mBSA-mediated Ag-induced arthritis (Fig. 2a). The immunolocalization of CCL2 within joint section emphasized To establish the severity of disease in IL-6Ϫ/Ϫ and IL-6ϩ/ϩ that synovial-lining cells may be major producers of this inflam- mice, histopathological analysis of joint sections was performed on matory chemokine. To address this issue, and to substantiate the samples collected on day 3 postarthritis induction. Arthritis sever- involvement of sIL-6R in leukocyte recruitment, we tested the ity was graded as an index of synovial hyperplasia, synovial inability of the sIL-6R isoforms to regulate chemokine expression in filtrate, cellular exudate, and joint erosion. Fig. 2b shows that ar- human synovial fibroblasts (HSF). FACS analysis of HSF using thritis was significantly more advanced in IL-6ϩ/ϩ mice than that Abs against the cognate IL-6R and the signal-transducing element observed in IL-6Ϫ/Ϫ mice. Indeed, all of the histological parame- gp130 confirmed that these cells express gp130, but show no detect- ters assessed were markedly reduced in IL-6Ϫ/Ϫ mice (Table I). able levels of IL-6R (Fig. 5a). Consequently, HSF only respond to However, reconstitution of sIL-6R-mediated signaling in IL-6Ϫ/Ϫ IL-6 in the presence of sIL-6R. As shown in Fig. 5b, stimulation of mice with HYPER-IL-6 restored the arthritic index to that encoun- HSF with IL-6 in combination with either PC- or DS-sIL-6R signif- tered in IL-6ϩ/ϩ mice (Fig. 2b and Table I). Representative mi- icantly increased the expression of CCL2 in both a time- and dose- crographs from each condition as seen on day 3 of arthritis are dependent manner. Similar data were also obtained using HYPER- presented in Fig. 2, cÐe. Administration (i.a.) of HYPER-IL-6 to IL-6 (Fig. 5b), while sIL-6R-mediated CCL2 production was mBSA-challenged IL-6Ϫ/Ϫ mice also significantly ( p Ͻ 0.05) in- specifically inhibited (70 Ϯ 2%; n ϭ 3 independent experiments) by creased the degree of joint erosion seen on day 21 in IL-6Ϫ/Ϫ mice; inclusion of a blocking anti-gp130 Ab at the time of stimulation. however, restoration of arthritis severity was not comparable to the Stimulation of HSF with IL-6 and it soluble receptor isoforms, extent observed in IL-6ϩ/ϩ mice (Table II). Consequently, sIL- however, failed to induce expression of the chemokines CCL3, 6R-mediated signaling may primarily influence early inflammatory CCL4, CCL5, CCL11, CXCL8, CXCL9, and CXCL10, while the processes; however, it is unclear whether a single administration of cellular expression of ICAM-1 and VCAM-1 remained unaltered. HYPER-IL-6 is sufficient to elicit events 21 days later. Blockade of experimental arthritis in IL-6ϩ/ϩ mice by sgp130 HYPER-IL-6 controls mononuclear cell infiltration in vivo To confirm a role for sIL-6R in the progression of AIA, IL-6ϩ/ϩ mice through induction of CCL2 were coadministered on the day of arthritis induction with mBSA and Based on the histological observations presented in Table I, we next 500 ng/ml sgp130 (i.a.). This naturally occurring antagonist selec- tested whether HYPER-IL-6 orchestrates mononuclear leukocyte in- tively inhibits the cellular events elicited by the sIL-6R/IL-6 complex filtration during experimental AIA. Immunohistochemistry using Abs and does not affect IL-6 signaling via its cognate IL-6R (33). Histo- against the CC-chemokine CCL2 and its receptor CCR2 was per- logical analysis of day 3 joint sections showed that all parameters of formed on sequential joint sections prepared from IL-6Ϫ/Ϫ mice, in disease severity were suppressed in response to sgp130, and no sig- which arthritis had been induced for 3 days. As shown in Fig. 4, nificant difference was observed between the histological scores at- CCL2 staining within the synovial tissue of IL-6Ϫ/Ϫ mice was poorly tributed to IL-6Ϫ/Ϫ mice and those assigned to IL-6ϩ/ϩ mice exposed

Table II. Joint inﬂammation and cartilage damage on day 21 of AIAa

Histopathological Parameter

Species Treatment Hyperplasia Synovial inﬁltrate Synovial exudate Erosions Arthritis Index

IL-6ϩ/ϩ PBS only 2.00 Ϯ 0.30 2.70 Ϯ 0.26 1.20 Ϯ 0.20 1.60 Ϯ 0.27 7.50 Ϯ 0.78 ءءءϮ 0.17 1.35 ءءءϮ 0.00 0.00 ءءءϮ 0.07 0.07 ءءءϮ 0.18 0.86 ءءءIL-6Ϫ/Ϫ PBS only 0.43 Ϯ 0.14 ءءءϮ 0.29 2.14 ءءءϮ 0.13 0.25 ءءϮ 0.11 0.17 ءءϮ 0.14 1.33 ءIL-6Ϫ/Ϫ HYPER-IL-6 0.67 Ϯ 0.18 ءϮ 0.30† 0.80 Ϯ 0.29† 3.80 Ϯ 1.34 0.70 ءϮ 0.45 1.50 ءIL-6ϩ/ϩ sgp130 0.80 Ϯ 0.36

a Synovial hyperplasia, cellular inﬁltrate, and exudate into the synovial cavity were observed in hematoxylin/safranin-O-stained sections. Scoring criteria were as described .p Ͻ 0.001 ءءء ;p Ͻ 0.01 ءء ;p Ͻ 0.05 ء ;in Table I. Comparisons were made against control IL-6ϩ/ϩ mice values by one-way unpaired ANOVA. †, NS 3206 SOLUBLE IL-6R GOVERNS IL-6 ACTIVITY

FIGURE 4. Immunohistochemical analysis of CCL2 and CCR2. Serial joint sections were prepared from AIA (sacriﬁced on day 3)-challenged IL-6Ϫ/Ϫ (a and d) and IL-6Ϫ/Ϫ mice receiving HY- PER-IL-6 (b and e). Sections were probed with anti- CCL2 (a and b) or anti-CCR2 (d and e) Abs. Scale bar represents 25 ␮m. Computer analysis of CCL2 (c) and CCR2 (f) immunostaining was performed in sections of synovium taken from four separate mice (mean Ϯ SEM of three microscopic ﬁelds of .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء .(view

to sgp130 (Fig. 6, Table I). Indeed, treatment of IL-6ϩ/ϩ mice with changes in anti-mBSA levels because sgp130 did not affect anti- sgp130 signiﬁcantly reduced the intensity of CCL2 staining to levels mBSA titers in IL-6ϩ/ϩ mice (Fig. 3). comparable with that seen in IL-6Ϫ/Ϫ mice (Fig. 6, e and f ). This To establish whether blockade of the cellular processes seen early suppression of arthritis severity in response to sgp130 was not due to in AIA development was also evident in the latter stages of the model,

FIGURE 5. The sIL-6R isoforms stimulate CCL2 expression in vitro. a, Expression of IL-6R subunits (IL-6R and gp130) on HSF was analyzed by flow cy- tometry, and the data were acquired from 5000 gated events. b, HSF were growth arrested for 48 h and stimulated as indicated with 20 ng/ml IL-6 and increasing doses of the sIL-6R isoforms over a period of 24 h. Time-dependent release of CCL2 in response to IL-6 and 50 ng/ml PC-sIL-6R or DS-sIL-6R. CCL2 levels were quantified using ELISA. Data represent the mean Ϯ SEM from five independent donors. Inset, RT-PCR analysis performed using CCL2 and ␤-actin- specific oligonucleotide primers. Data are shown for HSF stimulated with IL-6 in combination with PC- sIL-6R (PC) and DS-sIL-6R (DS). The time- and dose-dependent release of CCL2 in response to HY- PER-IL-6 is also shown. The Journal of Immunology 3207

FIGURE 6. Soluble gp130 ameliorates AIA. Mid- sagittal sections from IL-6ϩ/ϩ mice sacrificed on day 3 of AIA. Representative hematoxylin/safranin-O- stained sections are shown for IL-6ϩ/ϩ mice (a) and IL-6ϩ/ϩ mice administered with 500 ng/ml sgp130 (b). Scale bar represents 100 ␮m. c, Histopathological analysis of hematoxylin/safranin-O-stained sections from experimental mice sacrificed on day 3. Two blinded independent observers scored each slide. Sy- novial hyperplasia, synovial infiltrate, cellular exudate, and joint erosion figures were totaled to give an arthritis index. Six mice were analyzed in each group over two separate experiments. Immunohistochemical analysis of CCL2 in joint sections derived from IL- 6ϩ/ϩ mice 3 days after onset of AIA. dÐf, Represen- tative joint sections from IL-6ϩ/ϩ mice (d) and IL- 6ϩ/ϩ mice treated with sgp130 (e) are presented. A filled arrow indicates synovial infiltrate within the joint cavity. An open arrow indicates the subintimal synovium. Scale bar represents 10 ␮m. Intensity of CCL2 staining (f) was measured in sections of synovium taken from three mice (mean Ϯ SEM of three .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .(microscopic fields of view

arthritis was induced in IL-6Ϫ/Ϫ and IL-6ϩ/ϩ mice, and evidence of cartilage damage, and this was paralleled by data obtained from cartilage/bone erosion and synovial hyperplasia was monitored 21 sgp130-treated IL-6ϩ/ϩ mice. Indeed, sgp130 significantly reduced days later (Fig. 7). IL-6Ϫ/Ϫ mice displayed a highly significant re- synovial hyperplasia, synovial infiltrate/exudate, and the degree of duction (vs IL-6ϩ/ϩ mice) in all parameters of joint inflammation and joint destruction observed on day 21 (Table II).

FIGURE 7. Soluble gp130 reduces joint destruction. Hematoxylin/Safranin-O-stained midsagittal sections from IL-6ϩ/ϩ mice sacrificed on day 21 of AIA. Representative histology of IL-6ϩ/ϩ mice (a) and IL- 6ϩ/ϩ mice administered with 500 ng/ml sgp130 (b). A star indicates synovial hyperplasia with evidence of articular cartilage destruction. Scale bar represents 100 ␮m. c, Histopathological analysis of hematoxylin/ safranin-O-stained sections from IL-6Ϫ/Ϫ mice sacrificed on day 21. Two blinded independent observers scored each slide. Synovial hyperplasia, synovial infiltrate, cellular exudate, and joint erosion figures were totaled to give an arthritis index. Six mice were analyzed in .p Ͻ 0.05 ,ء .each group over two separate experiments 3208 SOLUBLE IL-6R GOVERNS IL-6 ACTIVITY

Discussion arthritis in mice possessing a mutated gp130 subunit has been re- Although high IL-6 concentrations have been documented in the lated to the inability of the T cell population to undergo apoptosis- serum and synovial fluids of patients with various arthritides, cell induced cell death (50). Consequently, sIL-6R signaling may not types resident to the joint (chondrocytes, synoviocytes, fibroblasts, only regulate leukocyte infiltration (as shown in this study), but and endothelial cells) lack expression of a cognate IL-6R (5). Con- might also exacerbate disease through maintaining leukocytes sequently, these cells remain unresponsive to IL-6 itself. IL-6 sig- within the inflamed joint. naling through its soluble receptor may therefore represent the The inability of IL-6 to solely restore experimental arthritis in Ϫ/Ϫ prominent mechanism by which IL-6 elicits its effect during ar- IL-6 mice is intriguing. This infers that sIL-6R must be gen- thritis. Analysis of synovial fluids from arthritic patients has high- erated within the synovium, but that the source of its production is lighted that increases in sIL-6R correlate with the extent of joint unlikely to originate for cell types resident to the joint. Activation destruction, and correspond with more advanced stages of RA (23, of leukocytes is known to liberate sIL-6R (27, 35–37), and leuko- 34). In this respect, in vitro studies have implicated an involvement cyte recruitment may be the rate-limiting event in regulating IL- 6-mediated responses. In this respect, i.a. administration of IL-6 for sIL-6R in synovial fibrosis, bone resorption, and other inflam- Ϫ/Ϫ matory processes (23–26). Using an experimental model of arthri- had no effect on the degree of leukocyte influx seen in IL-6 tis, we now conclude that sIL-6R governs the IL-6 signal within an mice (data not shown). Consequently, IL-6 may not restore the inflamed joint and contributes to an exacerbation of the disease arthritic severity because synovial sIL-6R concentrations within process. Specifically, i.a. reconstitution of sIL-6R-mediated sig- the joint are not sufficiently elevated to ensure optimal IL-6 sig- Ϫ Ϫ naling. For example, formation of the sIL-6R/IL-6 complex is naling within IL-6 / mice led to a thickening of the synovial known to prolong the circulating t of IL-6 (51). In this respect, lining, cellular hyperplasia, and increased leukocyte infiltration 1/2 specific blockade of sIL-6R-mediated signaling with the antagonist and joint destruction. sgp130 in IL-6ϩ/ϩ mice was shown to suppress arthritis severity, The absence of a cognate IL-6R on the major structural cells of while the unimolecular nature of HYPER-IL-6 ensures that recon- the joint infers that these are unlikely to contribute to synovial stituted signaling does not occur via the cognate IL-6R (21). Over- sIL-6R concentrations. Because expression of IL-6R is largely re- all, these data emphasize that sIL-6R controls IL-6 responses stricted to hepatocytes and leukocytes (20), increases in synovial within the inflamed joint, which in humans uses two distinct sIL-6R levels might originate from infiltrating leukocytes. In this sIL-6R isoforms (DS- and PC-sIL-6R). At present, the importance respect, leukocytes release sIL-6R following challenge with in- of each form is unclear; however, release of these isoforms in vivo flammatory mediators (27, 35–37), while the degree of leukocyte appears to depend on the individual’s age, disease condition, and infiltration into an arthritic joint correlates with synovial concen- stage of inflammation (20, 27, 31). During acute inflammation, trations of sIL-6R (5). Consequently, sIL-6R might act as a para- initial increases in sIL-6R correlate with neutrophil infiltration and crine mediator localized to its site of generation, and in this ca- are released via proteolytic cleavage, whereas increases in DS- pacity influences leukocyte recruitment during inflammation (21). sIL-6R coincide with the influx of mononuclear leukocytes (27). Several studies have highlighted a role for sIL-6R in the regulation We now report that detection of DS-sIL-6R is exclusively confined of chemokine and adhesion molecule expression (27, 36–41), to synovial fluids from RA patients, which did not reflect changes while sIL-6R-mediated signaling appears to control transition be- in systemic sIL-6R levels. This implies that DS-sIL-6R is secreted tween the early neutrophilic stage of acute inflammation and the within the inflamed synovium during active disease. In this re- more sustained mononuclear cell influx (27). Such a scenario may spect, DS-sIL-6R has been found to be released from monocytic be relevant to arthritis, in which neutrophils play an essential role cells and T lymphocytes (30, 31), and as such may represent a in the initiation and progression of RA, and chronic maintenance clinical marker of disease activity. Whether this means that DS- of mononuclear leukocytes within an arthritic joint exacerbates the sIL-6R activity is associated with mononuclear leukocyte re- ϩ/ϩ Ϫ/Ϫ disease (42, 43). Through analysis of AIA in IL-6 and IL-6 sponses remains to be determined. mice, we observed that IL-6 deficiency is associated with reduced The observation that sIL-6R-mediated signaling contributes to the synovial infiltration and is accompanied by a defect in both CCL2 degree of arthritis severity emphasizes that specific therapeutic tar- expression and the recruitment of leukocytes bearing the CCL2 geting of this soluble receptor may represent a novel strategy for the receptor CCR2. Leukocytes expressing CCR2 (monocytes and ac- treatment of chronic inflammatory conditions, such as RA. In this tivated T cells (44)) play a central role in the persistence of chronic respect, sgp130 has been shown to block experimental colitis (48), inflammation in RA, and CCL2 may invariably represent an im- while neutralizing Abs against either IL-6 or its cognate receptor have portant factor in the progression of arthritis. Examination of our proven to be effective in alleviating RA progression (15–19). In this pool of synovial fluids indicates that CCL2 levels are significantly present study, specific inhibition of sIL-6R-mediated signaling by more elevated in RA (3.61 Ϯ 0.92 ng/ml) than OA (1.66 Ϯ 0.41 sgp130 effectively suppressed the histopathological hallmarks of ar- ng/ml) patients, while blockade of CCL2 activity prevents onset of thritis in IL-6ϩ/ϩ mice. Significantly, several isoforms of sgp130 have autoimmune arthritis in MRL-1pr mice (45). Significantly, devel- been described (21). However, one form known as gp130-RAPS (RA opment of this arthritis model has been associated with elevated antigenic peptide-bearing soluble form) has recently been identified as sIL-6R levels (46). an RA autoantigen, and serum Abs against gp130-RAPS not only Although it is evident from our studies that sIL-6R-mediated correlate with indices of disease activity, but also neutralize gp130- signaling regulates the influx of mononuclear leukocytes, IL-6 may RAPS activity (52). Although the significance of these findings needs also contribute to their maintenance at sites of inflammation. IL-6 to be clarified, sgp130 may symbolize a valuable addition to the cur- has previously been reported to rescue T cells from apoptosis rent arsenal of therapeutics that have proven so effective in the man- through induction of Bcl-2 and Bcl-xL, while blockade of sIL-6R agement of chronic conditions such as RA. signaling in experimental colitis prevents disease onset by sup- pressing expression of these antiapoptotic regulators (47, 48). Such Acknowledgments processes may be relevant to arthritis, because T cells extracted We thank the Rheumatology staff at the University Hospital of Wales from RA tissue remain resistant to apoptosis and show increased (Cardiff, Wales, U.K.) and Royal Glamorgan Hospital (Llantrisant, Wales,

Bcl-xL expression (43, 49). Indeed, development of autoimmune U.K.) for collection of clinical samples. The Journal of Immunology 3209

References IL-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity 14:705. 1. Houssiau, F. A., J. Devogelaer, J. Van Damme, C. Nagant de Deuxchaisnes, and 28. Fischer, M., J. Goldschmitt, C. Peschel, J. P. G. Brakenhoff, K.-J. Kallen, J. Van Snick. 1988. Interleukin-6 in synovial fluid and serum of patients with A. Wollmer, J. Gro¨tzinger, and S. Rose-John. 1997. A bioactive designer cyto- rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum. 31:784. kine for the hematopoietic progenitor cell expansion. Nat. Biotechnol. 15:142. 2. Guerne, P. A., B. L. Zuraw, J. H. Vaughan, D. A. Carson, and M. Lotz. 1989. 29. Dayer, I. M., S. M. Krane, R. Graham, D. R. Russell, and D. R. Robinson. 1976. Synovium as a source of interleukin 6 in vitro. J. Clin. Invest. 83:585. Production of collagenase and prostaglandins by isolated adherent rheumatoid 3. Manicourt, D. H., R. Triki, K. Fukuda, J. P. Devogelaer, synovial cells. Proc. Natl. Acad. Sci. USA 73:945. C. Nagant de Deuxchaisnes, and E. J. M. A. Thonar. 1993. Levels of circulating tumor necrosis factor ␣ and interleukin-6 in patients with rheumatoid arthritis. 30. Horiuchi, S., Y. Koyanagi, Y. Zhou, H. Miyamoto, Y. Tanak, M. Waki, Arthritis Rheum. 36:490. A. Matsumoto, M. Yamamoto, and N. Yamamoto. 1994. Soluble interleukin-6 4. Arvidson, N. G., B. Gudbjornsson, L. Elfman, A. C. Ryden, T. H. To¨tterman, and receptors released from T cell or granulocyte/macrophage cell lines and human R. Ha¨llgren. 1994. Circadian rhythms of serum interleukin-6 in rheumatoid ar- peripheral blood mononuclear cells are generated through an alternative splicing thritis. Rheum. Dis. 53:521. mechanism. Eur. J. Immunol. 24:1945. 5. Desgeorges, A., C. Gabay, P. Silacci, D. Novick, P. Roux-Lombard, G. Grau, 31. Horiuchi, S., W. Ampofo, Y. Koyanagi, A. Yamashita, M. Waki, A. Matsumoto, J. M. Dayer, T. Vischer, and P. A. Guerne. 1997. Concentrations and origins of and M. Yamamoto. 1998. High-level production of alternatively spliced soluble soluble interleukin 6 receptor-␣ in serum and synovial fluid. J. Rheumatol. 24:1510. interleukin-6 receptor in serum of patients with adult T-cell leukemia/HTLV-1- 6. Van de Loo, F. A., S. Kuiper, F. H. van Enckevort, O. J. Antz, and associated myelopathy. Immunology 95:360. W. B. van der Berg. 1997. Interleukin-6 reduces cartilage destruction during exper- 32. Li, F. K., A. Davenport, R. Robson, P. Loetscher, R. Rothlein, J. D. Williams, and imental arthritis: a study in interleukin-6-deficient mice. Am. J. Pathol. 151:177. N. Topley. 1998. Leukocyte migration across human peritoneal mesothelial cells 7. Ji, H., A. Pettit, K. Ohmura, A. Ortiz-Lopez, V. Duchatelle, C. Degott, is dependent on directed chemokine secretion and ICAM-1 expression. Kidney E. Gravallese, D. Mathis, and C. Benoist. 2002. Critical roles for interleukin-1 Int. 54:2170. and tumor necrosis factor-␣ in antibody-induced arthritis. J. Exp. Med. 196:77. 33. Jostock, T., J. Mu¨llberg, S. O¨ zbek, R. Atreya, G. Blinn, N. Voltz, M. Fischer, 8. Alonzi, T., E. Fattori, D. Lazzaro, P. Costa, L. Probert, G. Kollias, M. Neurath, and S. Rose-John. 2001. Soluble gp130 is the natural inhibitor of soluble F. De Benedetti, V. Poli, and G. Ciliberto. 1998. Interleukin 6 is required for the interleukin-6 receptor transsignaling responses. Eur. J. Biochem. 268:160. development of collagen-induced arthritis. J. Exp. Med. 187:461. 34. Robak, T., A. Gladalska, H. Stepien, and E. Robak. 1998. Serum levels of in- 9. Oshima, S., Y. Saeki, T. Mima, M. Sasai, K. Nishioka, S. Nomura, M. Kopf, terleukin-6 type cytokines and soluble interleukin-6 receptor in patients with Y. Katada, T. Tanaka, M. Suemura, and T. Kishimoto. 1998. Interleukin 6 plays rheumatoid arthritis. Med. Inflamm. 7:347. a key role in the development of antigen-induced arthritis. Proc. Natl. Acad. Sci. 35. Jones, S. A., D. Novick, S. Horiuchi, N. Yamamoto, A. J. Szalai, and USA 95:8222. G. M. Fuller. 1999. C-reactive protein: a physiological activator of IL-6 receptor 10. Boe, A., M. Baiocchi, M. Carbonatto, R. Papoian, and O. Serlupi-Crescenzi. shedding. J. Exp. Med. 189:599. 1999. Interleukin 6 knock-out mice are resistant to antigen-induced experimental 36. Marin, V., F. A. Montero-Julian, S. Gres, V. Boulay, P. Bongrand, C. Farnarier, arthritis. Cytokine 11:1057. and G. Kaplanski. 2001. The IL-6-soluble IL-6R␣ autocrine loop of endothelial 11. Sasai, M., Y. Saeki, S. Oshima, K. Nishioka, T. Mima, T. Tanaka, Y. Katada, activation as an intermediate between acute and chronic inflammation: an exper- K. Yoshizaki, M. Suemura, and T. Kishimoto. 1999. Delayed onset and reduced imental model involving thrombin. J. Immunol. 167:3445. severity of collagen-induced arthritis in interleukin-6-deficient mice. Arthritis 37. Modur, V., Y. Li, G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre. 1997. Rheum. 42:1635. Retrograde inflammatory signaling from neutrophils to endothelial cells by sol- 12. De Hooge, A. S. K., F. A. J. van de Loo, O. J. Arntz, and W. B. van den Berg. uble IL-6 receptor ␣. J. Clin. Invest. 100:2752. 2000. Involvement of IL-6, apart from its role in immunity, in mediating a 38. Romano, M., M. Sironi, C. Toniatti, N. Polentarutti, P. Fruscella, P. Ghezzi, chronic response during experimental arthritis. Am. J. Pathol. 157:2081. R. Faggioni, W. Luini, V. van Hinsbergh, S. Sozzani, et al. 1997. Role of IL-6 13. Madhok, R., A. Crilly, J. Watson, and H. A. Capell. 1993. Serum interleukin-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. levels in rheumatoid arthritis: correlations with clinical and laboratory indexes of Immunity 6:315. disease activity. Ann. Rheum. Dis. 52:232. 39. Klouche, M., S. Rose-John, W. Schmiedt, and S. Bhakdi. 2000. Enzymatically 14. Keul, R., P. C. Heinrich, G. Mu¨ller-Newen, K. Muller, and P. Woo. 1998. A degraded, nonoxidized LDL induces human vascular smooth muscle cell activa- possible role for soluble IL-6 receptor in the pathogenesis of systemic onset tion, foam cell transformation and proliferation. Circulation 101:1799. juvenile chronic arthritis. Cytokine 10:729. 40. Matsumiya, T., T. Imaizumi, K. Fujimoto, X. Cui, T. Shibata, W. Tamo, 15. Wendling, D., A. Racadote, and J. Wijdenes. 1993. Treatment of severe rheumatoid M. Kumagai, K. Tanji, H. Yoshida, H. Kimura, and K. Datoh. 2001. Soluble arthritis by an anti-interleukin 6 monoclonal antibody. J. Rheumatol. 20:259. interleukin-6 receptor ␣ inhibits the cytokine-induced fractalkine/CX3CL1 ex- 16. Takagi, N., M. Mihara, Y. Moriya, N. Nishimoto, K. Yoshizaki, T. Kishimoto, pression in human vascular endothelial cells in culture. Exp. Cell Res. 269:35. Y. Takeda, and Y. Ohsugi. 1998. Interleukin-6 receptor blockage ameliorates 41. Oh, J. W., N. J. Van Wagoner, S. Rose-John, and E. N. Benveniste. 1998. Role joint disease in murine collagen-induced arthritis. Arthritis Rheum. 41:117. of IL-6 and the soluble receptor in inhibition of VCAM-1 gene expression. J. Im- 17. Imazeki, I., H. Saito, M. Hasegawa, H. Shinkura, T. Kishimoto, and Y. Oshugi. munol. 161:4992. 1998. IL-6 functions in cynomolgus monkeys blocked by a humanized antibody 42. Wipke, B. T., and P. M. Allen. 1998. Essential role for neutrophils in the initi- to human IL-6 receptor. Int. J. Immunopharmacol. 20:345. ation and progression of a murine model of rheumatoid arthritis. J. Immunol. 18. Mihara, M., M. Kotoh, N. Nishimoto, Y. Oda, E. Kumagai, N. Takagi, 167:1601. K. Tsunemi, Y. Ohsugi, T. Kishimoto, K. Yoshizaki, and Y. Takeda. 2001. Hu- 43. Pope, R. M. 2002. Apoptosis as a therapeutic tool in rheumatoid arthritis. Nat. manized antibody to human interleukin-6 receptor inhibits the development of Rev. Immunol. 2:1. collagen arthritis in cynomolgus monkeys. Clin. Immunol. 98:319. 44. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. 19. Choy, E. H. S., D. A. Isenberg, T. Garrod, S. Farrow, Y. Ioannou, H. Bird, 45. Gong, J., L. G. Ratkay, J. D. Waterfield, and I. Clark-Lewis. 1997. An antagonist N. Cheung, B. Williams, B. Hazleman, R. Price, et al. 2002. Therapeutic benefit of monocyte chemoattractant protein-1 (MCP-1) inhibits arthritis in the MRL-lpr of blocking interleukin-6 activity with an anti-interleukin-6 receptor monoclonal mouse model. J. Exp. Med. 186:131. antibody in rheumatoid arthritis. Arthritis Rheum. 46:3143. 20. Jones, S. A., S. Horiuchi, N. Topley, N. Yamamoto, and G. M. Fuller. 2001. The 46. Suzuki, H., K. Yasukawa, T. Saito, M. Narazaki, A. Hasegawa, T. Taga, and soluble interleukin-6 receptor: mechanisms of production and implications in T. Kishimoto. 1993. Serum-soluble interleukin-6 receptor in MRL-lpr mice is ele- disease. FASEB J. 15:43. vated with age and mediates the interleukin-6 signal. Eur. J. Immunol. 23:1078. 21. Jones, S. A., and S. Rose-John. 2002. Soluble receptors in cytokine biology: the 47. Teague, T. K., P. Marrack, J. W. Kappler, and A. T. Vella. 1997. IL-6 rescues agonistic properties of the sIL-6R/IL-6 complex. Biochim. Biophys. Acta resting mouse T-cells from apoptosis. J. Immunol. 158:5791. 1592:251. 48. Atreya, R., J. Mudter, S. Finotto, J. Mu¨llberg, T. Jostock, S. Wirtz, M. Schu¨tz, 22. Heinrich, P. C., I. Behrmann, G. Muller-Newen, F. Schaper, and L. Graeve. 1998. B. Bartsch, M. Holtmann, C. Becker, et al. 2000. Blockade of interleukin-6 trans Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflam- Biochem. J. 334:297. mation: evidence in Crohn disease and experimental colitis in vivo. Nat. Med. 6:583. 23. Kotake, S., K. Sato, K. J. Kim, N. Takahashi, N. Udagawa, I. Nakamura, 49. Salmon, M., D. Scheel-Toellner, A. P. Huissoon, D. Pilling, N. Shamsaadeen, A. Yamaguchi, T. Kishimoto, T. Suda, and S. Kashiwazaki. 1996. Interleukin-6 and A. D. D’Angeac, P. A. Bacon, P. Emery, and A. Akbar. 1997. Inhibition of T-cell soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis pa- apoptosis in rheumatoid synovium. J. Clin. Invest. 99:439. tients are responsible for osteoclast-like cell formation. J. Bone Miner. Res. 11:88. 50. Atsumi, T., K. Ishihara, D. Kamimura, H. Ikushima, T. Ohtani, S. Hirota, 24. Mihara, M., Y. Moriya, T. Kishimoto, and Y. Oshugi. 1995. Interleukin-6 (IL-6) H. Kobayashi, S.-J. Park, Y. Saeki, Y. Kitamura, and T. Hirano. 2002. A point induces the proliferation of synovial fibroblastic cells in the presence of soluble mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 IL-6 receptor. Br. J. Rheumatol. 34:321. causes autoimmune arthritis. J. Exp. Med. 196:979. 25. Franchimont, N., S. Rydziel, A. M. Delaney, and E. Canalis. 1997. Interleukin-6 51. Peters, M., S. Jacobs, M. Ehlers, P. Vollmer, J. Mu¨llberg, E. Wolf, G. Brem, and its soluble receptor cause a marked induction of collagenase 3 expression in K.-H.M. zum Bu¨schenfelde, and S. Rose-John. 1996. The function of the soluble rat osteoblastic cultures. J. Biol. Chem. 272:12144. interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor 26. Flannery, C. R., C. B. Little, C. E. Hughes, C. L. Curtis, B. Caterson, and transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6. S. A. Jones. 2000. IL-6 and its soluble receptor augment aggrecanase-mediated J. Exp. Med. 183:1399. proteoglycan catabolism in articular cartilage. Matrix Biol. 19:549. 52. Tanaka, M., M. Kishimura, S. Ozaki, F. Osakada, H. Hashimoto, M. Okubo, 27. Hurst, S. M., T. S. Wilkinson, R. M. McLoughlin, S. Jones, S. Horiuchi, M. Murakami, and K. Nakao. 2000. Cloning of novel soluble gp130 and detection N. Yamomoto, S. Rose-John, G. M. Fuller, N. Topley, and S. A. Jones. 2001. of its neutralizing autoantibodies in rheumatoid arthritis. J. Clin. Invest. 106:137.