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The Journal of Immunology, 2002, 169: 1760-1767.
Copyright © 2002 by The American Association of Immunologists

Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients Express Functional IL-15 Receptor Complex: Endogenous IL-15 in Autocrine Fashion Enhances Cell Proliferation and Expression of Bcl-xL and Bcl-21

Mariola Kurowska*, Weronika Rudnicka*, Ewa Kontny*, Iwona Janicka*, Magdalena Chorazy*, Jacek Kowalczewski{dagger}, Maria Ziólkowska*, Sylvie Ferrari-Lacraz2,{ddagger}, Terry B. Strom{ddagger} and Wlodzimierz Maslinski3,*,§

* Department of Pathophysiology and Immunology, and {dagger} Clinic of Orthopaedy, Institute of Rheumatology, Warsaw, Poland; {ddagger} Division of Immunology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and § Department of Experimental and Clinical Physiology, Medical University of Warsaw, Warsaw, Poland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15R{alpha} at mRNA and protein levels. Moreover, we confirmed the presence of IL-2R{beta} and common {gamma}-chains. Interestingly, TNF-{alpha} or IL-1{beta} triggered significant elevation of IL-15R{alpha} chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-xL expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-xL. To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fc{gamma}2a protein exerting properties of specific antagonist to the IL-15R{alpha} chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-xL, and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hallmark of rheumatoid arthritis (RA)4 is leukocytic infiltration of the synovium (1). This inflammatory process stimulates proliferation of apoptosis-resistant fibroblast-like synoviocytes (FLS). As a direct consequence, pannus formation and joint destruction result (2, 3, 4, 5). Intraarticular expression of proinflammatory cytokines, especially TNF-{alpha} and IL-1{beta}, play a key role in the pathogenesis of RA (6). IL-15, first identified as a T cell growth factor (7, 8), is also expressed within the diseased joint.

There are two IL-15 isoforms that differ in their signal sequence, translation efficiency, localization within cells, and tissues (9). The isoform bearing a 48 aa-long signal sequence is localized to the plasma membrane or may be secreted. The other IL-15 isoform, containing a shorter (21 aa) signal sequence, is found within the cytoplasm or nucleus (9, 10, 11). The high-affinity IL-15R is heterotrimeric. The complex contains {beta} and common {gamma}-chain ({gamma}c) subunits that also serve as essential components of the IL-2R. The third receptor component is the IL-15-specific IL-15R{alpha} chain. The shared IL-2/15 {beta} and {gamma}c are responsible for transducing IL-15- and IL-2-triggered intracellular signals. The specificity for IL-15 vs IL-2 binding is provided by unique cytokine-specific, private {alpha}-chain receptor subunits. Monomeric IL-15R{alpha}, but not IL-2R{alpha}, chains provide a cytokine-specific high-affinity (Kd = 10-11 M) binding site. Expression of both IL-2 and IL-2R{alpha} are restricted to activated T cells, while IL-15 and IL-15R{alpha} transcripts have a broader tissue distribution (12, 13).

McInnes et al. (14, 15) suggested that IL-15 may play a primary role in the development of RA. High levels of this cytokine are present in synovial fluid, synovial membrane, and serum isolated from RA patients. IL-15 may increase the number of cells involved in inflammatory reaction in the joint by directly: 1) stimulating migration of neutrophils and T lymphocytes into the joint (14, 16, 17), 2) protecting these cells from apoptosis (18, 19), and 3) triggering the proliferation of memory CD8+ and CD4+ and naive CD8+ T cells (20, 21, 22), and/or indirectly by inducing expression of proinflammatory cytokines TNF-{alpha} (15, 23), IL-1{beta} (23), IL-17 (24), and IL-8 (25, 26), as well as inflammation-inciting free radicals (27). IL-15 may also participate in local bone destruction in RA (28). The role of IL-15 in these processes is supported by evidence showing that administration of soluble IL-15R{alpha} prevents collagen-induced arthritis in mice and effectively reduces inflammation, synovial hyperplasia, and adjacent bone erosion (29).

FLS are believed to actively contribute to the pathogenesis of RA. By aggressive invasion into the cartilage and production of metalloproteinases, these cells directly contribute to the destruction of joint tissue. Moreover, FLS are sources of many factors involved in perpetuation of inflammation: IL-8, IL-6, GM-CSF, PGE2 (3), autocrine growth factor-basic fibroblast growth factor (bFGF; Ref. 30), and angiogenic factor-vascular endothelial growth factor (31). In addition, the phenotype of RA FLS is similar to transformed cells as the phenotype notable for anchorage independent growth, spontaneous expression of protooncogenes, c-myc, c-fos, mutated p53, and expression of antiapoptotic proteins Bcl-2, Bcl-xL, and sentrin-1 (2, 3, 4, 5, 32, 33). Moreover, FLS are important sources of IL-15 in RA joints. Indeed, IL-15 expressed by RA FLS may be responsible for intraarticular T cell activation and expansion (34). The aim of this study was to test the hypothesis that IL-15 is critical to FLS activation and survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

A group of 32 patients who fulfilled the American College of Rheumatology (Atlanta, GA) criteria for the diagnosis of RA and were undergoing knee synovectomy or joint replacement surgery as a normal part of clinical care were included in this study. Subjects were 26 females, 6 males, age 21–78 years, mean ± SEM 55.2 ± 2.4, and duration of disease was 1.5–40 years, mean ± SEM 14.4 ± 1.8.

Cells

Synovial fibroblast cell lines were prepared from synovial samples obtained from RA patients as described previously (35). FLS were used for experiments after three to seven passages.

Expression of mRNA-encoding IL-15, IL-15R{alpha}, IL-2/IL-15R{beta}, IL-2/IL-15R{gamma}, Bcl-2, Bcl-xL, and GAPDH

For the analysis of IL-15, IL-15R{alpha}, IL-2/IL-15R{beta}, and IL-2/IL-15R{gamma} mRNA expression, culture flasks (Nunc, Roskilde, Denmark) were seeded with 0.6–1.5 x 105 synoviocytes/ml for 2–3 days. Then, fresh medium supplemented with rIL-1{beta} (1 ng/ml), rTNF-{alpha} (10 ng/ml), rbFGF (1/5 ng/ml; all from R&D Systems, Abingdon, U.K.), or rIL-2 (250 U/ml; Genzyme Genetics, Framingham, MA) was added, and cells were incubated for an additional 4 h. To test whether exogenous IL-15 modulates the expression of mRNA-encoding Bcl-2 and Bcl-xL, cells were incubated with rIL-15 (25 ng/ml; R&D Systems) in medium containing a low concentration of FCS (0.5%). To determine the role of endogenous IL-15 on the expression of mRNA-encoding Bcl-2 and Bcl-xL directly after seeding, cells were incubated for 24 h in medium containing 2% FCS supplemented with: 1) IL-15DM/Fc{gamma}2a (murine Fc{gamma}2a; 5–10 µg/ml; Ref. 36) or IL-15DM/Fc{gamma}1 (human Fc{gamma}1), a high-affinity receptor site-specific antagonist for IL-15R{alpha}-chain protein (both from Cardion Pharmaceuticals, Boston, MA); 2) relevant isotype controls, murine IgG2a or human IgG1, respectively (both Abs from R&D Systems); 3) goat anti-human IL-15 neutralizing Ab (5 µg/ml); or 4) control total goat IgG (both Abs from R&D Systems). After 24 h, one-third of the culture medium was discarded and fresh medium with supplementary proteins were added for another 24 h. Total RNA extraction, cDNA template preparation, and the RT-PCR were done as previously described (37). Quantitative competitive RT-PCR was used to measure bcl-2, bcl-xL, and GAPDH mRNA expression. In this technique, cellular RNA-derived cDNA was coamplified with the internal control using the pair of primers common for both templates as previously reported (38). Products of competitive bcl-2, bcl-xL, and GAPDH PCRs, separated on 2% agarose gel, were densitometrically scanned using Kodak 1D Image analysis software (Eastman Kodak, Rochester, NY). For every sample, the ratio of tested to internal control product was calculated. Next, each sample was normalized to GAPDH level. The expression of IL-15, IL-15R{alpha} isoforms, IL-2R{beta}, and {gamma}c were measured in simple RT-PCR and analyzed as described above. The rate of amplification of the particular product was within exponential range. The fine details of this system are shown in Table IGo.


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  		Table I. Sequences of used primers and expected product sizes

 
Cell proliferation assay

Proliferation of FLS was assessed by incorporation of tritiated thymidine. For the assay, 96-well flat-bottom culture plates (Nunc) were seeded with 5 x 103 FLS in 0.2 ml of culture medium containing 0.5% FCS for 72 h. Then, the culture medium was removed and replaced for 24 h with fresh medium alone or medium supplemented with 25 ng/ml IL-15 (R&D Systems). To determine the contribution of endogenous IL-15 in the FLS proliferation, cells were incubated with murine IgG2a (2 µg/ml) in medium with 2% FCS for 24 h. Then, medium was replaced with fresh, supplemented with mouse IgG2a (0.5; 2 µg/ml) or antagonist of IL-15R{alpha} (IL-15DM/Fc{gamma}2a) (0.5; 2 µg/ml; Ref. 36), and FLS were cultured for an additional 72 h. [3H]TdR (2 µCi/ml; Amersham Pharmacia Biotech, Little Chalfont, U.K.) was added 18 h before the termination of the cell culture, and radioactivity of the samples were measured as described (35).

Western blotting

To assess spontaneous, or TNF-{alpha} (10 ng/ml) or IL-1{beta} (1 ng/ml) triggered expression of IL-15 protein, synoviocytes (1 x 105/ml) were cultured in medium containing 5% FCS at 37°C for 48 h. Expression of IL-15 was analyzed in cytosolic and membrane protein fractions according to the method previously described (39). Proteins were separated on 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% nonfat milk and incubated with rabbit anti-human IL-15 Ab (2 µg/ml; PeproTech, London, U.K.) overnight at 4°C. The bands were visualized by application of peroxidase-conjugated goat anti-rabbit IgG (dilution, 1/2000; Sigma-Aldrich, St. Louis, MO) and ECL system (Amersham Pharmacia Biotech).

Flow cytometric analysis

For the analysis of IL-15R{alpha} and IL-15 surface expression, cells were stimulated with TNF-{alpha} (10 ng/ml), IL-1{beta} (1 ng/ml), bFGF (1/5 ng/ml), or IL-2 (250 U) for 24 and 48 h. To determine the role of endogenous IL-15 on the expression of Bcl-2, Bcl-xL proteins, and in apoptosis, FLS were cultured with IL-15DM/Fc{gamma}1, goat anti-human IL-15, or relevant isotype control as described above for 48 and 72 h. FLS were dissociated from culture flasks using trypsin/EDTA treatment. To estimate the surface expression of IL-15R{alpha} and IL-15, cells (0.15–0.3 x 106) were washed first in PBS (without Mg2+/Ca2+) buffer containing 1% BSA and 0.06% NaN3, and then with glycine buffer (0.1 M, pH 4.5), followed by incubation with mouse IgG1 (for IL-15) or IgG2a (for IL-15R{alpha}) (1 µg per sample; both IgG from R&D Systems). For detection of IL-15 or IL-15R{alpha}, FLS were incubated with mouse anti-human IL-15 (0.5 µg/ml) (M111; Genzyme Genetics) or IL-15R{alpha} antagonist (IL-15DM/Fc{gamma}2a; 50 ng), or isotype-matched negative control Ab (R&D Systems). Next, cells were stained with PE-conjugated goat anti-mouse IgG (5 µl/sample; DAKO, Glostrup, Denmark). To confirm binding specificity of IL-15DM/Fc{gamma}2a to IL-15R{alpha}, cells were incubated with recombinant human IL-15 (150 ng/sample) for 15 min before their incubation with IL-15R{alpha} antagonist. For intracellular Bcl-2 and Bcl-xL staining, cell aliquots were permeabilized using Cytofix/Cytoperm kit (BD Biosciences, Mountain View, CA) according to the manufacturer’s protocol. Permeabilized FLS were incubated with specific mAb, FITC-labeled anti-Bcl-2 Ab (5 µl/sample; DAKO), or Ab against Bcl-xL (0.5 µg/sample; BD Transduction Laboratories, Lexington, KY) or relevant control Ab (DAKO). For Bcl-xL, a secondary PE-conjugated goat anti-mouse IgG (5 µl/sample; DAKO) was used. For apoptotic cell death analysis, FLS were stained with FITC-conjugated annexin V and propidium iodide according to manufacturer’s instruction (Roche Diagnostic Systems, Mannheim, Germany). All analyses were done using FACSCalibur and CellQuest software (BD Biosciences).

Statistical analysis

Data are expressed as mean ± SEM. Where appropriate, the different groups were tested for statistical significance using paired two-tailed Student’s t tests. Values of p < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of IL-15 expression by proinflammatory cytokines in RA FLS

RA synoviocytes, especially when stimulated with TNF-{alpha} or IL-1{beta}, secrete IL-15 (34). However, it is not known whether FLS express only one or both known IL-15 isoforms: secretory/membrane and cytosolic/nucleus (9), and whether IL-15 protein is present on the FLS surface. To determine the expression of mRNA-encoding IL-15 isoforms in FLS by RT-PCR analysis, the set of primers recognizing specific sequences present in both IL-15 isoforms was used. Resting RA FLS express both IL-15 isoforms (3/3 RA FLS lines). Interestingly, the expression of mRNA-encoding secretory/membrane-bound IL-15 increased after a 4-h stimulation with TNF-{alpha} or IL-1{beta} (450 and 312%, respectively; p < 0.05) (Fig. 1GoA). In contrast, FLS stimulation with bFGF, a known synoviocyte growth factor (30), exerted no effect on IL-15 isoforms mRNA expression (data not shown).



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  		FIGURE 1. Expression of IL-15 in resting and TNF-{alpha}- or IL-1{beta}-stimulated RA FLS. A and B, FLS from RA patients were incubated with TNF-{alpha} (10 ng/ml) or IL-1{beta} (1 ng/ml) or medium alone (lane C) for 4 h followed by total RNA extraction. A, The RT-PCR was performed using a pair of primers recognizing sequences present in cDNA encoding both IL-15 isoforms: 528 bp-long cytosolic and 409 bp-long secretory/membrane-bound protein. B, The level of GAPDH mRNA was used as a control. Representative RT-PCR products of three independent experiments with similar result are shown. C and D, The membrane and cytosolic protein fractions were isolated from the RA FLS as described in Materials and Methods. Proteins (15 µg/lane) were separated on 15% SDS-PAGE. Representative Western blots for membrane (C) and cytosolic (D) protein of four (for TNF-{alpha}) and one (for IL-1{beta}) with similar results are shown.

 
Because posttranscriptional regulation of IL-15 expression is more important (9), the expression of IL-15 protein in the cytosolic and membrane protein fractions isolated from cultured FLS was examined for IL-15 expression by Western blotting technique. As reported previously for other cells (9), in FLS, several bands representing different levels of IL-15 glycosylation were detected. Similar amounts of highly glycosylated IL-15 protein were present in both cytosolic and membrane fractions of nonstimulated RA FLS lines (Fig. 1Go, C and D). However, upon TNF-{alpha} stimulation, a significant increase in IL-15 expression was detected in the membrane (257%; p < 0.05), but not in cytosolic fraction (Fig. 1Go, C and D). In contrast, IL-1{beta} did not affect the IL-15 level in either protein fraction. These data were further confirmed by flow cytometric analysis of surface-expressed IL-15 after extensive washing of receptor-bound IL-15 with low pH glycine buffer. Stimulation with TNF-{alpha}, but not with IL-1{beta}, results in the increase of surface-expressed IL-15 (data not shown). Neither bFGF nor IL-2 enhanced surface-expressed IL-15 (data not shown). Thus, the TNF-{alpha} up-regulated membrane-bound/secretory isoform of IL-15 is likely to dominate the pool of IL-15 produced by FLS.

RA FLS express all receptor chains required for IL-15-triggered signal transduction (IL-15R{alpha}, IL-2/15R{beta}, and IL-2/15R{gamma})

To determine the expression of mRNA encoding for IL-15R{alpha}, the set of primers recognizing extracellular domains of IL-15R{alpha} isoforms was used (13). Using these primers, PCR yields two IL-15R{alpha} chain-related products: 444 and 543 bp, lacking and containing exon 3, respectively. Each of the FLS samples isolated from nine RA patients constitutively expressed mRNA for both IL-15R{alpha} chain isoforms (Fig. 2Go). Moreover, RA FLS expressed both IL-2R{beta} and {gamma}c encoding mRNA (Fig. 3Go). Therefore, FLS express mRNA, encoding all three chains required for the formation of high affinity functional IL-15R complex. Surface-expressed IL-15R{alpha} chain was further analyzed using previously described mutated IL-15 fused to the Fc fragment of murine IgG2a, IL-15DM/Fc{gamma}2a (36), and flow cytometry. Roughly 50% of nonstimulated FLS express IL-15R{alpha} (Fig. 4Go).



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  		FIGURE 2. The effects of cytokines on the expression of mRNA-encoding IL-15R{alpha} isoforms. Analysis of IL-15R{alpha} mRNA expression in FLS: with exon 3 (A) or lacking exon 3 (B). FLS from RA patients were stimulated with TNF-{alpha} (10 ng/ml; n = 3) or IL-1{beta} (1 ng/ml; n = 5), or bFGF (1/5 ng/ml; n = 2) or IL-2 (250 U; n = 2) for 4 h. Total RNA was isolated, and RT-PCRs for IL-15R{alpha} and GAPDH were performed. Data were densitometrically scanned. For every sample, the expression of IL-15R{alpha} was normalized based on GAPDH level. Values are the mean (percentage of nonstimulated control) ± SEM; *, p < 0.05; **, p < 0.02 vs control (paired two-tailed Student’s t test). C, The representative results of RT-PCR products (selected from A and B) are shown. Lane C, Resting control cells.

 


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  		FIGURE 3. Expression of mRNA encoding IL-2R{beta} and {gamma}c in RA FLS. The comparison of IL-2R{beta} (A) or IL-2R{gamma}c (B) mRNA expression in three RA FLS lines and PBMC from a healthy blood donor. Results are shown as RT-PCR products. Twenty microliters (FLS) and 5 µl (PBMC) of cDNA obtained from 1 µg total RNA were used as template. Lanes 1-3, FLS lines from three different RA patients.

 


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  		FIGURE 4. Surface expression of IL-15R{alpha} on resting and cytokines stimulated RA FLS. FLS were stimulated with cytokines as described in Fig. 2Go, but for 48 h. Cells were harvested by trypsin/EDTA treatment, incubated with IL-15DM/Fc{gamma}2a or mouse IgG2a, and stained with PE-labeled goat anti-mouse IgG. IL-15R{alpha} expression in nontreated (A and B) or TNF-{alpha} or IL-1{beta} (A) or IL-2 or bFGF-stimulated cells (B). Representative histograms of three independent experiments with similar results are shown. C and D, Recombinant human IL-15 inhibits binding of IL-15DM/Fc{gamma}2a to TNF-{alpha}-stimulated RA FLS. C, Cells were stained as described above. D, Cells were incubated with recombinant human IL-15 (150 ng) on ice for 15 min before staining with IL-15DM/Fc{gamma}2a. E, The comparison of MFI. Data are expressed as the mean of MFI ± SEM. **, p < 0.008 and *, p < 0.03 vs isotype control, #, p < 0.03 vs spontaneous expression of IL-15R{alpha} (paired two-tailed Student’s t test).

 
TNF-{alpha} and IL-1{beta}, but not bFGF or IL-2, enhance the expression of IL-15R{alpha} in RA FLS

As shown in Fig. 2Go, IL-1{beta} and TNF-{alpha} significantly enhanced the expression of mRNA encoding for both IL-15R{alpha} isoforms in cultured FLS. In contrast, bFGF or IL-2 exerted no stimulatory effect on the IL-15R{alpha} mRNA level. Moreover, TNF-{alpha}, and to a lesser extent IL-1{beta}, enhance surface-expressed IL-15R{alpha}. Both percentage of IL-15R{alpha} positive (Fig. 4GoA) and IL-15R{alpha} chain density (judged by changes of mean fluorescence intensity (MFI); Fig. 4GoE) were elevated. The specificity of IL-15R{alpha} detection was confirmed by preincubation of FLS with 3-fold excess of recombinant human IL-15 for 15 min before staining with IL-15 DM/Fc{gamma}2a. As expected, this procedure resulted in complete blockade of detection of surface-expressed IL-15R{alpha} (Fig. 4Go, C and D).

IL-15 enhances RA FLS proliferation

To determine whether IL-15R complex expressed upon FLS is functional, cells were stimulated with IL-15 in medium containing low FCS (0.5%). As shown in Fig. 5GoA, provision of IL-15 significantly enhanced proliferation of quiescent FLS (176% of unstimulated control).



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  		FIGURE 5. IL-15 stimulates FLS proliferation. A, Exogenous IL-15 stimulates FLS proliferation. FLS from six RA patients were cultured for 72 h in medium with low FCS (0.5%). Then, the culture medium was replaced for 24 h with fresh medium or fresh medium supplemented with IL-15 (25 ng/ml). [3H]TdR was added simultaneously with IL-15. Data are expressed as mean (percentage of nonstimulated control) ± SEM; *, p < 0.02 compared with medium alone (paired two-tailed Student’s t test). B, IL-15R{alpha} antagonist inhibits FLS proliferation. RA FLS were incubated with murine IgG2a (2 µg/ml) in medium with 2% FCS for 24 h, then medium was replaced with fresh, supplemented with mouse IgG2a (0.5; 2 µg/ml) or IL-15DM/Fc{gamma}2a (0.5; 2 µg/ml). Cells were cultured for 72 h. [3H]TdR was added 18 h before culture termination. All data are the average of triplicate samples, and the error bars represent the sample SEM. Similar results were obtained in two independent experiments. #, p < 0.03 vs nontreated cells (medium); *, p < 0.04 vs IgG2a (0.5 µg/ml) treated cells; **, p < 0.009 vs IgG2a (2 µg/ml) treated cells (paired two-tailed Student’s t test).

 
Proliferation of cultured RA FLS depends in part on the autocrine IL-15/IL-15R activation

Nonstimulated FLS express IL-2R{beta} and {gamma}c. Expression of both IL-15 and IL-15R{alpha} is enhanced in the presence of TNF-{alpha} (Figs. 1Go, 2Go, and 4Go). Thus, it is possible that endogenously produced IL-15, in an autocrine fashion, stimulates IL-15Rs providing signals for proliferation and/or preventing cell apoptosis. This hypothesis was tested using IL-15DM/Fc{gamma}2a (36). This protein, exerting properties of high affinity stereo-specific IL-15R antagonist, inhibited FLS proliferation in a dose-dependent manner, while isotype matching control IgG2a Ab did not (Fig. 5GoB). Therefore, our results suggest that FLS-derived IL-15 binds to and activates IL-15Rs expressed on these cells. An IL-15 autocrine pathway helps to drive RA FLS cell proliferation.

Endogenous IL-15 is responsible for enhanced expression of Bcl-xL and Bcl-2 in RA FLS

IL-15, a growth factor, also protects many cell types from apoptotic death (18, 19, 40). To determine whether IL-15 elicits antiapoptotic effects upon RA FLS, the effect of IL-15 on the expression of mRNA encoding antiapoptotic proteins Bcl-2 and Bcl-xL were examined. As expected, FLS express mRNA encoding for both Bcl-xL and Bcl-2 (Fig. 6Go). Exogenously added IL-15 enhanced bcl-xL gene expression in RA FLS (Fig. 6Go, B and C); however, in contrast to PBMC (data not shown), IL-15 did not elevate bcl-2 gene expression (Fig. 6Go, A and C). These results raised the possibility that levels of mRNA-encoding antiapoptotic proteins present in FLS depend on the autocrine IL-15 network. To test this hypothesis, RA FLS were incubated for 48 h in the presence of IL-15DM/Fc{gamma}2a protein or control mouse IgG2a Ab. As illustrated in Fig. 7Go, provision of the IL-15R{alpha} antagonist, in contrast to control IgG2a, significantly inhibited spontaneous expression of mRNA-encoding Bcl-xL and Bcl-2. Similar results were obtained in experiments, where goat anti-human IL-15 neutralizing Ab was used to block biological activities of endogenous IL-15 (Fig. 8Go). The expression of Bcl-xL and Bcl-2 proteins were confirmed using specific Abs and flow cytometric analysis. Indeed, FLS express both tested proteins (Fig. 9Go). As predicted from changes at mRNA levels, FLS cultured in the presence of IL-15R antagonist exert diminished levels of Bcl-2 and Bcl-xL (Fig. 9Go). Similar data were obtained when cells were cultured in the presence of neutralizing anti-IL-15 Abs (data not shown).



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  		FIGURE 6. Exogenous IL-15 triggers significant elevation of mRNA encoding Bcl-xL, but fails to raise the level of bcl-2 in FLS. RA FLS were incubated with IL-15 (25 ng/ml) in medium containing a low level of FCS (0.5%) for 4 h. Total RNA was isolated and competitive RT-PCRs for bcl-2, Bcl-xL, and GAPDH were performed. Data were densitometrically scanned. For every sample, the ratio of tested to internal control product was calculated and normalized to GAPDH level. Data are expressed as the mean (percentage of nonstimulated control) ± SEM of three for bcl-2 (A) and four for Bcl-xL (B) independent experiments. *, p < 0.02 vs control; ns = not statistically significant (paired two-tailed Student’s t test). C, The representative results of competitive RT-PCR products (selected from A and B) are shown. The position and size of internal controls and cDNA derived from cellular mRNA are marked.

 


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  		FIGURE 7. IL-15R{alpha} antagonist inhibits bcl-xL and bcl-2 mRNA expression in RA FLS. RA FLS were cultured in medium alone or supplemented with IL-15DM/Fc{gamma}2a (10 µg/ml) or mouse IgG2a (10 µg/ml) for 48 h. The evaluation of mRNA expression for bcl-2, bcl-xL, and GAPDH was performed as described in Fig. 6Go. Data for bcl-2 (A) and bcl-xL (C) are expressed as the mean (percentage of IgG2a-treated control) ± SEM of four experiments. **, p < 0.0002; *, p < 0.04; ns, not statistically significant (paired two-tailed Student’s t test). B and D, The representative results of RT-PCR products (selected from A and C, respectively) are shown. The position and size of internal control and cDNA derived from cellular mRNA are marked.

 


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  		FIGURE 8. IL-15 neutralizing Ab inhibits bcl-xL and bcl-2 mRNA expression in RA FLS. RA FLS were cultured in medium alone or supplemented with goat anti-human IL-15 (5 µg/ml) or goat IgG (5 µg/ml) for 48 h. Total RNA was isolated and competitive RT-PCRs for bcl-2, bcl-xL, and GAPDH, in the presence of internal controls, were performed. The RT-PCR products from one representative experiment of two with similar results are shown.

 


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  		FIGURE 9. IL-15R{alpha} antagonist decreases Bcl-2 and Bcl-xL protein expression in RA FLS. FLS were cultured in the presence of IL-15DM/Fc{gamma}1 (10 µg/ml) or human IgG1 (10 µg/ml) for 48 h. Cells were harvested by trypsin/EDTA treatment, fixed, permeabilized, and stained with anti-Bcl-2 or anti-Bcl-xL Ab or appropriate isotype control followed by flow cytometric analysis. One representative experiment of three for Bcl-2 (A) and two for Bcl-xL (B) with similar results are shown.

 
Endogenous IL-15 prevents apoptosis of RA FLS

In the next set of experiments, we tested whether autocrine IL-15-triggered elevation of antiapoptotic proteins is reflected by the resistance of FLS to apoptosis. Indeed, enhanced apoptosis (32%) of FLS cultured in the presence of neutralizing anti-IL-15 Abs (Fig. 10GoB) was observed in comparison with FLS cultured in the presence of isotype matching control IgG (11%; Fig. 10GoA). Similar data were obtained when IL-15R{alpha} antagonist was used (data not shown). These results indicate that endogenously produced IL-15 contributes to the antiapoptotic status of RA FLS.



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  		FIGURE 10. Neutralization of endogenous IL-15 induces RA FLS apoptosis. FLS were cultured in the presence of goat IgG (5 µg/ml) (A) or goat anti-human IL-15 (5 µg/ml) (B) for 48 h. Cells were harvested by trypsin/EDTA treatment, stained with FITC-conjugated annexin V and propidium iodide for 15 min. One representative experiment of three with similar results is shown. R1, annexin V positive cells (early apoptosis); R2, annexin V and propidium iodide positive cells (late apoptosis).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have now tested the hypothesis that IL-15 participates in the activation of RA FLS. First, we confirmed recent a report by Harada et al. (34) that RA FLS spontaneously produce IL-15, and that IL-15 expression is further enhanced by TNF-{alpha} and IL-1{beta}. Next, we expanded their findings by analyzing the expression of the two known IL-15 isoforms (9, 11) in RA FLS. In resting RA FLS, both secretory/membrane-bound and cytosolic/nucleus IL-15 isoforms are present at mRNA and protein levels (Fig. 1Go). It was most interesting to note that TNF-{alpha} and IL-1{beta} treatment significantly elevated mRNA levels encoding the secretory/membrane-bound isoform (Fig. 1Go, A and B). In keeping with this observation, we determined at the protein level that TNF-{alpha} stimulation raised the expression of highly glycosylated membrane bound, but not cytosolic, IL-15 isoform (Fig. 1Go, C and D). This was further confirmed by the flow cytometric analysis. However, despite the increased level of mRNA encoding membrane-bound IL-15 upon IL-1{beta} stimulation, there was no rise in membrane-bound IL-15 protein. This apparent discrepancy between levels of IL-1{beta}-triggered IL-15 at mRNA (rise) and protein (no effect) levels led us to the hypothesis that IL-1{beta}, more efficiently than TNF-{alpha}, triggers activation of a specific protease that cleaves surface expressed IL-15 protein. Our preliminary results support this suggestion (M. Kurowska, W. Rudnicka, M. Ziolkowska, and W. Maslinski, manuscript in preparation). Taken together, we provide the evidence that RA FLS express both cytosolic and membrane-bound/secretory forms of IL-15 protein, and that stimulation of FLS by proinflammatory cytokines (TNF-{alpha} or IL-1{beta}) enhance expression of only the latter isoform at mRNA level. In addition, our data correspond well with those of Harada et al. (34), who reported that both TNF-{alpha} and IL-1{beta} increase IL-15 production at the protein level. Based on previous findings showing that IL-15 expressed on the surface of monocytes (41), keratinocytes (42), and dermal fibroblast (43) is biologically active, it is quite possible that cell surface IL-15 expressed upon RA FLS also exerts biological activity.

Next, we examined the expression of mRNA encoding all subunits of the IL-15R complex, i.e., IL-15R{alpha} and IL-2/15 {beta} and {gamma}c. Eight splicing variants of the human IL-15R{alpha} have been identified (44). These include all combinations of exon 2 deletion, exon 3 deletion, and alternative use of exon 7 or 7'. Because all, except exon 2 deletion isoforms bind IL-15 with very high affinity (13, 44), we have chosen PCR primers located in exon 2 (sense) and exon 5 (antisense). Using these primers, amplification of mRNA encoding all IL-15R{alpha} variants that contain exon 2, and thus, bind IL-15, can be detected. All tested RA FLS constitutively express mRNA encoding IL-15R{alpha} isoforms that bind IL-15 (Fig. 2Go). In addition, we confirmed previous findings of Corrigall et al. (45) that RA FLS constitutively express an additional two subunits required for IL-15-triggered signal transduction, i.e., IL-2R{beta} and {gamma}c (Fig. 3Go). Furthermore, we found that proinflammatory cytokines TNF-{alpha} and IL-1{beta}, in contrast to bFGF and IL-2, significantly enhanced the expression of IL-15R{alpha} isoforms at both mRNA and protein levels (Figs. 2Go and 4Go).

The RA joint microenvironment contains high levels of IL-15 (14) and low levels of IL-2 (46). Moreover, the cell surface phenotype of RA FLS is notable for the presence of IL-15R{alpha} (Fig. 4Go), but not IL-2R{alpha} (45). Thus, it seems likely that IL-15, but not IL-2, participates in RA FLS activation. In fact, provision of exogenous IL-15 stimulates RA FLS proliferation (Fig. 5GoA) and enhances the expression of mRNA encoding the antiapoptotic protein Bcl-xL (Fig. 6Go, B and C). Because RA FLS secrete IL-15 and express both surface bound IL-15 and functional IL-15Rs, we tested the hypothesis that an IL-15-dependent autocrine loop contributes to the RA FLS phenotype. Indeed, we found that blockade of the IL-15 pathway using either an IL-15R antagonist or neutralizing anti-IL-15 Abs substantially reduces expression of Bcl-xL and Bcl-2 at mRNA and protein levels ( Fig. 7–9GoGoGo). The latter finding, i.e., inhibition of Bcl-2 expression by anti-IL-15 treatment, indicates that also this antiapoptotic protein is regulated by IL-15. In contrast, exogenous IL-15 failed to increase Bcl-2 expression in RA FLS (Fig. 6Go, A and C). One possible explanation of these somehow opposite results is that endogenous RA FLS-derived IL-15 triggers maximal Bcl-2 expression, and the level of its expression cannot be further enhanced by addition of exogenous IL-15. The role of endogenous IL-15 in FLS activation was further confirmed in experiments where blockade of IL-15 pathway inhibited these cells proliferation (Fig. 5GoB) and increased their apoptosis (Fig. 10Go). The presence of a similar endogenous IL-15/IL-15R{alpha} loop has been postulated as the mechanism of tumor propagation in multiple myeloma (47) and the pathogenesis of psoriasis (42). Recently, Sen and coworkers (48, 49) have postulated that Wnt and Frizzled families, which control cell fate determination during embryogenesis, may contribute to the autonomous RA FLS activation. Our findings are consistent with their observation that normal FLS transfected with wnt-5A expression vector acquired RA FLS-like, persistent activation status manifested by significant production of IL-15 (48, 49).

Taken together, our data provide the evidence that the distinctive phenotype of RA FLS, i.e., persistent activation, spontaneous proliferation, and resistance to apoptosis, is partially related to the autocrine activation of IL-15Rs by FLS-derived IL-15. These results further underline the important role of IL-15 in the pathogenesis of RA.


   Acknowledgments
 
We thank The Foundation for Polish Science for financing the FACSCalibur. IL-15DM/Fc{gamma}2a and IL-15DM/Fc{gamma}1 proteins were gifts from Cardion Pharmaceuticals (Boston, MA).


   Footnotes
 
1 This work was supported by Grant 6PO5A 118 20 from the State Committee for Scientific Research of Poland (to M.K. and W.M.), National Institutes of Health Grant RO1 AI42298 (to T.B.S.), and Swiss National Science Foundation (to S.F.-L.). Presented in part at Annual European Congress of Rheumatology, European League Against Rheumatism, Prague, Czech Republic, June 2001; and 11th International Congress of Immunology, Stockholm, Sweden, July 2001. Back

2 Current address: Division of Immunology and Allergy, University Hospital, Geneva, Switzerland. Back

3 Address correspondence and reprint requests to Dr. Wlodzimierz Maslinski, Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland. E-mail address: zpatiir{at}warman.com.pl Back

4 Abbreviations used in this paper: RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; bFGF, basic fibroblast growth factor; {gamma}c, common {gamma}-chain; MFI, mean fluorescence intensity. Back

Received for publication November 7, 2001. Accepted for publication June 11, 2002.


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