Low-Level Production of Interleukin-13 in Synovial Fluid and Tissue from Patients with Arthritis

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 85, No. 2, November, pp. 210–220, 1997 Article No. II974441

Low-Level Production of Interleukin-13 in Synovial Fluid and Tissue from Patients with Arthritis1 James M. Woods,* G. Kenneth Haines,† Manisha R. Shah,* Ghazi Rayan,‡ and Alisa E. Koch*,§ *Department of Medicine and †Department of Pathology, Northwestern University Medical School, and §Department of Veterans’ Affairs, Chicago Health Care System, Lakeside Division, Chicago, Illinois 60611; and ‡Hand and Microsurgery Center, Oklahoma City, Oklahoma 73112

Rheumatoid arthritis (RA) is a chronic, aggressive disease characterized by inflammatory cells in the synovial tissue (ST) and synovial fluid (SF). Interleukin (IL)-13 inhibits the production of proinflammatory cytokines, chemokines, and hematopoietic growth factors by activated human monocytes. The aim of this study was to determine the production of IL-13 in various forms of arthritis. The presence of IL-13 in RA was found to be low, in that 18 of 26 RA SF samples and 10 of 14 RA peripheral blood (PB) samples had nondetectable levels (£12 pg/ml). Similar low levels were found in SF and PB from patients with osteoarthritis (OA) and other arthritides. In contrast, RANTES, IL-8, monocyte chemotactic protein-1, and soluble P-selectin were found at levels of 13-, 120-, 1200-, and 2000fold excess of IL-13, respectively. Mononuclear cells isolated from RA SFs did not produce significant levels of IL-13 in culture (£12 pg/ml) but were able to do so when stimulated with phytohemagglutinin. Likewise, tissue explants from RA synovium cultured for 24 or 48 hr with or without serum did not produce appreciable quantities of IL-13 (£12 pg/ml). Immunohistochemical data were in accordance with this result in that antigenic IL-13 was not detected on the majority of RA, OA, and normal (NL) ST cells. These results demonstrate a paucity of IL-13 within the joints of RA, OA, NL, and other arthritic patients by comparison with levels of other cytokines. q 1997 Academic Press Key Words: interleukin-13; rheumatoid arthritis; synovium.

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic disease characterized by proliferating fibroblasts, mononuclear 1 This work was supported by NIH Grants AR30692 and AR41492 (A.E.K.), funds from the Veterans’ Administration Research Service (A.E.K.), the Dr. Ralph and Marion Falk Challenge Prize of the Arthritis Foundation, Illinois Chapter (A.E.K.), an Arthritis Foundation Fellowship (J.M.W.), and a Northwestern Memorial Hospital Grant (J.M.W.).

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cells (MNCs), and endothelial cells which infiltrate the synovial tissue (ST) and synovial fluid (SF) (1). The inflamed ST aggressively invades cartilage and bone, destroying the joints’ ability to function (2, 3). Although many factors are involved in synovial inflammation, cytokines have emerged as regulatory factors of particular importance (2). The end effect of joint destruction in RA may be the result of an imbalance between the proinflammatory cytokines which perpetuate the influx of cells and the anti-inflammatory cytokines and cytokine inhibitors (3). One of these cytokines with many anti-inflammatory actions, interleukin (IL)-13, has been cloned and characterized in humans (4–6). There are a large number of activated T cells in the RA ST (7). The importance of these cells in synovitis has recently been underscored by the demonstration that synovial T cells from RA patients can cause inflammatory arthritis in severe combined immunodeficient mice (8). The division of helper T (Th) cells into Th0, Th1, and Th2 cells is based primarily on the lymphokines synthesized by cloned Th cells. Th1 cells primarily produce IL-2 and interferon-g (IFN-g), Th2 cells synthesize and secrete IL-4, IL-5, and IL-13, and Th0 cells can produce lymphokines of both Th1 and Th2 types (9). In many aspects, human IL-13 is similar to IL-4. These similarities include the tandem arrangement and organization of their genes, the structure of their gene products, shared biological functions, and the use of receptors with common structural elements (4, 10– 13). A comparison of IL-13 and IL-4 similarities and differences has been reviewed (4, 10, 14). IL-13 is mainly produced by activated T helper cells (15, 16), while other cell types also synthesize IL-13 mRNA and protein in vitro (17–19). Although IL-13 is mainly a product of T cells, it is noteworthy that the cytokines IL-8, RANTES, and MCP-1 can all be produced by T lymphocytes as well as numberous other cell types. IL-13 elicits a broad spectrum of biological responses, the majority of which are anti-inflammatory. These include altering the phenotype of monocytes (14, 20, 21)

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as well as the production of IL-1a (14), IL-1b (14, 22, 23), IL-6 (14, 23), tumor necrosis factor (TNF)-a (14, 22, 23), the chemokines macrophage inflammatory protein-1a (14) and IL-8 (14, 23), and the hematopoietic growth factors granulocyte/macrophage-colony-stimulating factor (14) and granulocyte-colony-stimulating factor (14) by lipopolysaccharide (LPS)-stimulated monocytes. IL-13 has additional anti-inflammatory effects on monocytes and neutrophils (PMNs), increasing the production of IL-1 receptor antagonist (IL-1ra), a naturally occurring competitive inhibitor of IL-1a and IL-1b (14, 24–26). Also, IL-13 acts on macrophages to inhibit their nitric oxide production and Fc receptor expression (27). In autoimmune diseases, IL-13 modulates the production of rat macrophage IL-1b and TNFa in vitro while suppressing experimental autoimmune encephalomyelitis without inducing adverse systemic effects in vivo (28). Another interesting effect of IL-13 involves the production of the regulated on activation normal T expressed and secreted (RANTES) chemokine induced by TNF-a and IFN-g in human umbilical vein endothelial cells (HUVECs). Expression of RANTES in HUVECs is significantly inhibited by IL-13 (29). IL-13 also exerts regulatory effects on various hematopoietic progenitor cells (30–32). In addition, IL-13 may mediate a Th2dependent effect on vascular endothelium, since IL-13 selectively stimulates HUVECs to express functional cell surface VCAM-1 (26, 33). While not appearing to have effects on T cells, IL-13 binding to its receptor on B cells is one required costimulant in the induction of B cell proliferation and isotype switching to IgG4 and IgE classes (34–38). Taken together, these data suggest that IL-13 may be a key modulator of inflammation in an autoimmune disease such as RA because of its broad capacity to act in an anti-inflammatory manner and to inhibit the production of proinflammatory cytokines, chemokines, and hematopoietic growth factors thought to be involved with the pathogenesis of RA. We hypothesized that IL-13 levels in RA patients would be low, concomitant with elevated quantities of proinflammatory factors that IL-13 inhibits. To test this, we analyzed IL13 quantities in SF and peripheral blood (PB) from RA, osteoarthritis (OA), and other arthritic patients and compared them to quantities of another interleukin, two chemokines, and a soluble cellular adhesion molecule (CAM) determined in the same samples. We determined the production of IL-13 by RA ST explants as well as MNCs isolated from RA SFs. We utilized immunohistochemistry to compare the numbers of IL13 immunopositive cells in RA, OA, and normal ST samples. We also examined the quantity of IL-13 produced in vitro from RA ST PMNs, normal PB PMNs,

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and RA fibroblasts under stimulated and unstimulated conditions. MATERIALS AND METHODS

Patient Population SF and PB specimens were obtained from patients diagnosed with RA, OA, and other arthritic diseases during therapeutic arthrocentesis. ST was obtained from RA and OA patients undergoing total joint replacement. Normal STs were obtained from fresh autopsies or amputations. STs used for immunohistochemistry were snap-frozen in OCT (Miles Laboratories, Elkhart, IN) and stored at 0807C until sectioned. Cultured PMNs were isolated from RA SF and normal PB. Fibroblasts were isolated from fresh RA ST (see below). RA and OA patients met the criteria established by the American College of Rheumatology and all specimens were obtained with Institutional Review Board approval (39–41). Isolation of Human RA ST Fibroblasts and Preparation of Conditioned Medium (CM) ST was minced and digested in a dispase, collagenase, DNase solution for 2 hr at 377C as described (42, 43). ST fibroblasts were cultured in RPMI 1640 media with 10% FBS and gentamicin. Cells were used at passage 3 or older at which point they were a homogeneous population of fibroblasts. RA ST fibroblasts (1.2 1 105 cells/ml) were cultured in serum-free RPMI in the presence or absence of various immune modulators for 24 hr at 377C. These included TNF-a (34 ng/ml), IL-1b (30 ng/ml), or LPS (1 mg/ml). These concentrations of cytokines have been used previously in our laboratory to induce production of IL-8 (44) and growth-related gene product a (45) by ST fibroblasts. After 24 hr, supernatants were harvested and assayed for IL-13 by enzyme-linked immunosorbent assay (ELISA). Preparation of CM from PMNs and T Cells PMNs were isolated from SF and PB using Ficoll– Hypaque density gradient centrifugation. Cells (1 1 107 cells/ml) were incubated for 24 hr in serum-free RPMI or in the presence of 1 mg/ml LPS at 377C. Supernatants were collected after 24 hr and used for IL-13 ELISAs. T cells were isolated as described previously (46). Briefly, PB MNCs were obtained from healthy donors by centrifugation of heparinized blood over Ficoll–Hypaque gradients. CD4/ cells were selected via anti-CD4 mAb-coupled magnetic beads (Dynal Inc., Oslo, Norway) and rested overnight to detach from beads. One million T cells were stimulated with anti-CD3 (OKT3)

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from culture supernatant diluted 1:100 and with antiCD28 (clone 9.3) from ascites diluted 1:105 for up to 3 days in 24-well plates. CM was removed at 24 and 72 hr and frozen at 0207C until assayed. Preparation of CM from RA ST Explants and RA SF MNCs ST from RA patients were minced and cultured in 24well plates (0.25 g tissue/ml media) using RPMI 1640 media and gentamicin with or without 10% FBS. CM was removed from the wells at 24 or 48 hr. Each time point was performed in triplicate per patient. MNCs were isolated from heparinized RA SFs using Ficoll–Hypaque followed by Sepracell-MN (Sepratech Corp., Oklahoma City, OK) as described previously (47). Unstimulated control MNCs were compared to those incubated in the presence of 1 mg/ml phytohemagglutinin (PHA). Supernatants were removed at 24, 48, and 72 hr for analysis of IL-13 by ELISA. Immunohistochemistry ST sections 7-mm thick were cut and mounted onto Vectabond (Vector Laboratories, Burlingame, CA)coated glass slides and stored at 0807C until used for staining. Sections were fixed for 20 min in 4% paraformaldehyde and phosphate-buffered saline (PBS), then washed in PBS with 1% bovine serum albumin (PBS/BSA, used for all washes). Sections were fixed in acetone for 10 min at 47C and rehydrated in PBS/BSA. Immunostaining was carried out using the Vector ABC-peroxidase kit (Vector Laboratories). Briefly, sections were blocked with 10% rabbit serum for 20 min at 377C. Serum was removed and replaced with 10 mg/ ml rat anti-human IL-13 (clone JES10-5A2, Biosource International, Camarillo, CA) in a humid chamber overnight at 47C. After two washes, sections were incubated with biotinylated rabbit anti-rat IgG for 20 min at 377C. Sections were then washed and incubated with ABC reagent (peroxidase-labeled avidin biotin complex) for 20 min. After an additional wash, the reaction was initiated by addition of the substrate diaminobenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Slides were counterstained with Harris’ hematoxylin and mounted in Accu-mount 60 (Baxter, Deerfield, IL). An irrelevant isotype-matched rat IgG antibody (PharMingen, San Diego, CA) was used as a negative control. Various cell types in the ST including synovial lining cells, interstitial macrophages, and blood vessels/endothelial cells were identified by immunohistochemical staining reactions and/or morphologic features. Immunostaining was graded for each ST component by the frequency of staining on a scale of 0– 100%, where 0% indicates no staining and 100% represents all cells being immunoreactive. Each slide was

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FIG. 1. IL-13 levels were measured by ELISA assays in SFs from 49 patients: 26 RA, 11 OA, and 12 with other arthritic diseases. These other diseases included 5 with psoriatic arthritis, 3 with Reiter’s syndrome, 1 with mixed connective tissue disease, 1 with pseudogout, 1 with ankylosing spondylitis, and 1 with polymyositis. IL-13 levels were also quantified in PB from 22 patients: 14 RA, 5 OA, and 3 other arthritic diseases, including 2 with systemic lupus erythematosus and 1 with psoriatic arthritis. RA, rheumatoid arthritis; OA, osteoarthritis; Oth., other arthritic patients; SF, synovial fluid; PB, peripheral blood; n, number of samples.

evaluated by a single observer without knowledge of patient diagnosis, and selected sections were analyzed by two additional observers. ELISAs for IL-13, IL-8, sP-Selectin, RANTES, and MCP-1 IL-13 levels were determined in SF, PB, and cell culture-conditioned medium samples using an immunoassay kit for human IL-13 from Biosource International according to the procedure of the manufacturer. IL-13 was detected accurately §12 pg/ml. While both of the antibodies for ELISA and immunohistochemistry were purchased from the same manufacturer, each procedure used different antibodies which likely recognized different epitopes of IL-13. Commercially available ELISA kits for human sP-selectin (Bender MedSystems, Vienna, Austria) and RANTES (CYTImmune Sciences, College Park, MD) were also used in accordance with the manufacturer’s protocol. ELISAs for antigenic MCP-1 (48, 49) and IL-8 (44) were measured using a double-ligand method as described in detail previously. RESULTS

IL-13 levels were measured in SFs from 49 patients, including 26 with RA, 11 with OA, and 12 with other arthritic diseases. These other diseases included 5 with psoriatic arthritis, 3 with Reiter’s syndrome, 1 with mixed connective tissue disease, 1 with pseudogout, 1

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with ankylosing spondylitis, and 1 with polymyositis (Fig. 1). All SF IL-13 measurements were low in that 69% of RA, 82% of OA, and 75% of other patients were at or below the limit of detection of the ELISA (£12 pg/ml). IL-13 levels were also quantified in PB from 22 patients including 14 diagnosed with RA, 5 with OA, and 3 with other arthritic diseases, including 2 with systemic lupus erythematosus and 1 with psoriatic arthritis. Low levels were again detected, with 71% of RA patients, and all OA and other patients being at or below the limit of detectibility. As an additional positive control, we included SF samples spiked with recombinant human IL-13 and found IL-13 detectable by ELISA in these fluids (data not shown). The highest level of IL-13 (240 pg/ml) was found in the SF of a patient diagnosed with polymyositis. To be certain that these low levels were not the result of protein degradation in the SFs chosen, we compared IL-13 levels to those of the chemokines RANTES, IL8, and MCP-1, as well as a soluble CAM, sP-selectin, in the same samples (Fig. 2). We quantitated IL-13, as well as RANTES, IL-8, and MCP-1 levels in the same four patients and found these chemokines present at 13-, 120-, and 1200-fold higher levels than IL-13, respectively (Fig. 2A). In SFs from six additional patients, we quantitated both IL-13 and sP-selectin and found the CAM to be in 2000-fold excess of IL-13 (Fig. 2B). Concentrations of IL-8, MCP-1, and sP-selectin in these SFs were found to be consistent with levels reported previously (44, 48, 50). To identify if RA synovium could produce appreciable amounts of IL-13, supernatants were collected from 24and 48-hr cultured tissue explants. RA ST tissue explants produced £12 pg/ml, if any, detectable IL-13 in their supernatants when cultured in serum-free media (Fig. 3). The same result was demonstrated when cultured with 10% FBS (data not shown). Purified T cells from normal patients were used as a positive control and were stimulated with the combination of anti-CD3 and anti-CD28. Although anti-CD3 has been shown to negatively influence human IL-13 production (51) this is still an applicable positive control in that cells stimulated in this way produced ú1500 pg/ml and ú2300 pg/ml of IL-13, after 24 and 72 hr, respectively (Fig. 3). IL-13 production was also assayed in CM generated from MNCs isolated from RA SFs (Fig. 4). Similar to tissue explants, most samples were at or below the limit of detection of the ELISA when MNCs were not stimulated. We did find, however, that RA SF MNCs were capable of producing IL-13 when stimulated with 1 mg/ml PHA (Fig. 4) or the combination of phorbol myristate acetate (PMA) (1 ng/ml) and anti-CD28 (3 ml ascites/ml media; data not shown). Immunohistochemistry was utilized to detect antigenic IL-13 in RA, OA, and NL STs (Fig. 5). Cell types

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FIG. 2. SF IL-13 levels were compared with levels of RANTES, IL-8, MCP-1, and sP-selectin. Results are expressed as the mean { standard error of the mean (SEM). (A) RANTES, IL-8, and MCP-1 levels were determined to be present at 13-, 120-, and 1200-fold higher levels than IL-13, respectively. (B) In SFs from six additional patients, sP-selectin was present in 2000-fold excess of IL-13. Concentrations of IL-8, MCP-1, and sP-selectin in these SFs were consistent with levels reported previously. n, number of samples.

that were analyzed included synovial lining cells, macrophages, endothelial cells, and fibroblasts. Three out of 10 RA patient tissues examined had synovial lining that was immunopositive for IL-13, while only 2 of 10 had immunopositive macrophages. Similarly, 2 of 10 OA patients showed immunostaining in the synovial lining while only 1 of 10 had immunopositive macrophages. In the NL group, no patient displayed immunoreactive IL-13 in any cell type. Endothelial cells and fibroblasts were not immunopositive for IL-13 in any group of patients tested. To better identify if cells other than lymphocytes could produce IL-13 in the RA joint, we analyzed RA SF PMN and RA ST fibroblast CM. RA SF PMNs were cultured for 24 hr under unstimulated conditions (Fig. 6). These PMNs averaged greater than 95% purity, while eosinophils, not lymphocytes, were the main constituent of the õ5% of other cells. We found that PMNs

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from RA SFs were capable of producing small quantities of IL-13 (48.9 { 9.4 pg/ml), while the stimulation of these cells with LPS did not significantly alter this (data not shown). In addition, we isolated PMNs from NL PB and likewise found them capable of producing small amounts of IL-13 (32.2 { 8.6 pg/ml), while incubation of the cells with LPS did not significantly increase or decrease this level (data not shown). Similarly, IL-13 levels were evaluated in conditioned medium from RA ST fibroblasts cultured for 24 hr (Fig. 6). These experiments were performed in the presence or absence of IL-1b, TNF-a, or LPS. Fibroblasts produced very low levels of IL-13, if at all, regardless of the conditions assayed. Under all conditions, at least four of the six fibroblast CM were at or below the detection limit of the ELISA. FIG. 4. MNCs were isolated from RA SFs and their CM were analyzed by IL-13 ELISA. Unstimulated control MNCs were compared to those stimulated with 1 mg/ml PHA. CM were collected at 24, 48, and 72 hr for analysis. n, number of samples.

DISCUSSION

In this study we investigated the levels of IL-13 in RA ST and SF. Our results indicate that IL-13 is not consistently present at detectable levels in the joints of RA patients. This is demonstrated by our findings that SFs from the majority of RA, OA, and other arthritis patients had IL-13 levels at or below 12 pg/ml. In addition, PB from RA, OA, and other arthritis patients also demonstrated IL-13 too low to be detected in the

FIG. 3. CM from RA ST explants and NL PB-purified T cells were assayed for IL-13. RA ST explants were cultured in serumfree medium. Twenty-four- and 48-hr CM were assayed for IL-13 by ELISA. Purified T cells from NL PB were cultured in the presence of 10% human serum, anti-CD3 (1:100 dilution of a hybridoma supernatant), and anti-CD28 (9.3, 1:105 dilution of ascites fluid). Twentyfour- and 72-hr CM were assayed for IL-13 by ELISA. n, number of patients.

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majority of samples. This finding is consistent with negligible levels of IL-13 determined in PB from patients with perennial allergic rhinitis (52) as well as PB from NL individuals (53). In addition, our findings of minimal IL-13 in RA synovium are consistent with those of Kotake and co-workers who, in preliminary studies, reported rarely detecting IL-13 mRNA in synovial specimens from early arthritis patients (54). Cultured ST explants from RA patients likewise demonstrated minute production of IL-13. ELISAs on CM from RA SF MNCs further confirmed this result. Immunostaining for antigenic IL-13 also suggests that this lymphokine is present in synovium from a minority of patients. When we compared levels of this lymphokine with other known cytokines in the same SFs to determine the integrity of these fluids, we found the quantity of IL-13 to be very low. These comparisons were made with levels of RANTES, IL-8, MCP-1, and sP-selectin, which were found at quantities comparable to those previously reported (44, 48, 50). While the techniques utilized rely heavily upon antibody-based detection of IL-13, these methods are reliable for detection of proteins and we are confident that our positive controls reaffirm our findings. For comparison of IL-13 with other T-cell-produced cytokines in RA, our results can be compared with those found when studying IL-4 and IL-10 production. Similar to our findings, Miossec and co-workers have reported the virtual absence of IL-4 in RA SF (55). Likewise, IL-4 was not produced by unstimulated rheumatoid synovial membrane (55). Additionally, SF MNC culture supernatants were not found to contain IL-4 protein by ELISA assay (56) nor were IL-4 mRNA transcripts present (57). IL-10 in humans is mainly the

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FIG. 5. Antigenic IL-13 was detected using immunohistochemistry on frozen STs from RA, OA, and NL groups. Cell types analyzed included synovial lining cells, macrophages, endothelial cells, and fibroblasts. (A) RA ST lining cells immunostained with negative control rat IgG antibody (1708). (B) RA ST lining cells from most patients did not express antigenic IL-13 (1708). (Inset) IL-13-immunopositive RA ST lining cells (1708). Only 3 of 10 RA patients, 2 of 10 OA patients, and 0 of 6 NL patients were IL-13 immunopositive. (C) RA ST macrophages immunostained with negative control rat IgG antibody (1708). (D) RA ST macrophages from most patients were IL-13 negative (1708). (Inset) IL-13-immunopositive RA ST macrophages (1708). Only 2 of 10 RA, 1 of 10 OA, and 0 of 6 NL patients were IL-13 immunopositive. (E) RA ST vessels immunostained with negative control rat IgG antibody (1708). (F) RA ST vessels did not express antigenic IL-13 (1708).

product of activated CD4/ and CD8/ T cells, while other activated cell types have also been shown to produce this cytokine. In contrast to our findings for IL13, plentiful IL-10 has been demonstrated in SF and

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serum from RA patients (58–61). While not detecting IL-10 in nine RA SFs, Katsikis and co-workers found IL-10 protein in RA synovial membrane cultures without extrinsic stimulation as well as by immunohistol-

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FIG. 6. RA SF PMNs were cultured for 24 hr and their CM were analyzed by IL-13 ELISA. RA SF PMNs (1 1 107 cells/ml medium) produced small quantities of IL-13 (48.9 { 9.4 pg/ml, mean { SEM.). PMNs isolated from NL PB likewise produced small amounts of IL13 (32.2 { 8.6 pg/ml). RA ST fibroblasts were cultured in serum-free medium (1.2 1 105 cells/ml media). Twenty-four-hour conditioned medium was assayed for IL-13 by ELISA in the presence or absence of IL-1b (30 ng/ml), TNF-a (34 ng/ml), or LPS (1 mg/ml). RA, rheumatoid arthritis; SF, synovial fluid; PMNs, neutrophils; NL, normal; PB, peripheral blood; LPS, lipopolysaccharide; IL-1b, interleukin1b; TNF-a, tumor necrosis factor-a; n, number of samples.

ogy in all RA tissue sections examined (62). Thus, by comparison with reported values of two mainly T-cellproduced cytokines, our findings on IL-13 appear to be similar to those of IL-4, and low by comparison with reports on IL-10. RA joints have an abundance of many factors which IL-13 inhibits. In light of our results, it is possible that the low levels of IL-13 may contribute to the unregulated secretion of inflammatory monokines present in various forms of arthritis. Despite the small quantities of this lymphokine, the determinations made in this study utilized immunologic methods which quantitate levels of IL-13 and do not reflect the bioactivity of the protein. It remains to be determined in vivo whether small quantities of IL-13 can significantly inhibit excessive monokine production and inflammatory infiltration. This is certainly a possibility, in that in another autoimmune disease, antibodies to the lymphokine IFN-g can significantly improve the insulitis in NOD mice, despite the minute quantities of IFN-g present (63). Most studies have shown that T cell products are rarely detected at appreciable levels in rheumatoid joints, despite the abundance of T lymphocytes which morphologically and phenotypically appear activated in synovium (7, 64–67), although exceptions do exist (58, 62, 68). For example, IFN-g, which is abun-

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dantly produced by stimulated T cells in vitro, is absent or barely detectable in ST and SF (69, 70). Likewise, T-cell-produced IL-3 mRNA cannot be detected in RA ST (71), while IL-2 levels in RA SF are also negligible (70–72). In contrast to the negligible amounts of IL-13 found in our study, Isoma¨ki et al. recently reported much higher IL-13 levels in RA SFs, averaging greater than 1300 pg/ml (73). This finding indicates that levels of this anti-inflammatory lymphokine exceed RA SF levels of the proinflammatory cytokines IL-1 (59, 74–76) and TNF-a (59, 74, 75, 77) in a disease characterized by inflammation. Possible differences between the reports could in part be explained by differences in ELISA assays used. It is possible that SFs contain an inhibitor of IL-13 that remains undetermined. It is possible that antibodies used in our ELISA recognize an epitope that may be masked by an inhibitor. However, we feel this is unlikely in light of our studies demonstrating low IL-13 levels by RA ST explants and RA SF MNCs. Further, our assay detected IL-13 in CM of NL PB T cells. Stimulation with PHA or PMA/anti-CD28 showed that RA SF MNCs are capable of producing IL-13. Nonetheless, inhibitors for the T-cell-produced cytokines IL-2 (78) and IL-4 (55) have been detected in SF. Another possible explanation for the difference in results is that large amounts of IL-13 are produced and could be absorbed by activated cells. Despite the fact that the present study cannot rule out this possibility, immunohistochemical detection of IL-13 did not detect the lymphokine at significant levels on other cells types. PMA/ anti-CD28 were chosen as stimulatory agents because this combination has previously been used to stimulate IL-13 production by T lymphocytes (51). Our choice of PHA was based on its mitogenic ability on T lymphocytes and our data demonstrating that PHA increases RA SF MNCs IL-13 represent a novel finding. Analysis of immunostaining for IL-13 demonstrated a paucity of antigenic IL-13. Our findings are in agreement with a previous report that IL-13 could not be identified using standard immunohistochemical techniques, but required fixation with paraformaldehyde (68). Observing the few RA synovial specimens that were immunopositive, IL-13 appeared to be localized to the synovial lining and to macrophage-like cells. Of the RA and OA STs in which we detected IL-13-positive macrophages (2 of 10 RA and 1 of 10 OA patients) these STs also had IL-13-positive synovial lining cells. In each group there was one patient with IL-13-positive synovial lining that did not exhibit IL-13-immunostained macrophages. IL-13 is mainly produced by activated T cells (15, 16), although recent in vitro studies also demonstrate its production by activated mast cells (18) and basophils (19), but not stimulated PMNs or eosinophils (18).

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To determine if other cell types in the joint were capable of producing IL-13, PMNs and fibroblasts were cultured and their CM was analyzed by ELISA. Fibroblasts were chosen because of the proliferative and activated phenotype they display in the thickened synovial lining of RA patients. PMNs were examined because they are the predominant cell type in RA SF (1). In addition, analysis of the combination of PMNs and MNCs from RA SF indicates that we have analyzed greater than 90% of cell types that comprise the SF. LPS was chosen because of its ability to stimulate fibroblasts to secrete a wide variety of cytokines. IL-1b and TNF-a were chosen as stimulants for fibroblasts because of their suspected key roles and presence in RA as well as their ability to activate fibroblasts through their respective receptors. Fibroblasts did not produce detectable quantities of IL-13 (õ12 pg/ml), while PMN CM contained only very low levels. The same low-level production of IL-13 was demonstrated in CM from NL PB PMNs. From this we conclude that neither fibroblasts nor PMNs appear to be significant contributors of IL-13 in the RA joint. Recently IL-13 was shown to significantly suppress LPS-induced IL-1b production by SF and PB MNCs from patients with inflammatory arthritis (22). In addition, IL-13 significantly inhibited LPS-induced TNF-a production from PB MNCs from these patients, but not from their SF MNCs (22). Many studies demonstrate that IL-1b and TNF-a play pivotal roles in RA, as well as many other inflammatory monokines that have been previously mentioned (79). IL-13’s anti-inflammatory effects include the regulation of many of the monokines involved in RA. Our findings indicate that there is a minimal amount of IL-13 in SF and ST from RA, OA, and other arthritic patients. These data suggest that IL-13 is found in the joint in small quantities, which may not be optimal for inhibiting the production of the macrophage-derived proinflammatory cytokines. It is possible that the lack of T-cell-derived lymphokines, including IL-13, demonstrated in the synovitis of various types of arthritis may represent a functional loss of regulatory control over cytokines secreted by synovial macrophages and fibroblasts. Perhaps IL-13 is found at higher levels in SF of normal individuals, and from this location it may act on lining cell macrophage receptors, keeping control of inflammatory cytokines such as IL-1b and TNF-a. The incidence and severity of collagen-induced arthritis in mice has been significantly reduced utilizing Chinese hamster ovary fibroblasts engineered to secrete IL-13 and IL-4 (80), which underscores the importance of their regulatory ability. Thus, strategies focusing on increasing production of the antiinflammatory lymphokines may prove beneficial in attenuating the arthritic disease process.

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Received March 28, 1997; accepted with revision July 18, 1997

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