The recently described IL-33 acts as a pro-inflammatory cytokine, inducing the expression of multiple responses in the target cells. Although a nuclear localization of IL-33 has been described, its exact functional relevance is presently unknown. The present study was conducted to analyze the effects of IL-33 on the TNF-α induced synthesis of the pro-inflammatory mediators IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the pro-destructive molecules matrix metalloproteinase-1 (MMP-1), MMP-3, and TIMP-1 of rheumatoid arthritis synovial fibroblast (RA-SFs) using RNA overexpression and silencing. TNF-α significantly induced IL-33 mRNA expression and protein synthesis in RA-SFs. TNF-α-induced IL-33 protein expression was mediated via p38 signaling. Immunohistochemistry for IL-33 clearly showed that nuclear translocation of IL-33 was induced in TNF-α stimulated RA-SFs. IL-33 overexpression enhanced TNF-α-induced pro-inflammatory and pro-destructive functions in RA-SFs. IL-33 silencing significantly downregulated TNF-α-induced pro-inflammatory functions, whereas TNF-α-induced pro-destructive functions were less influenced by IL-33 silencing. This study identifies IL-33 as a critical regulator/enhancer of TNF-α-induced functions in RA-SFs, pointing to a central role of this cytokine in the perpetuation of pro-inflammatory and pro-destructive processes in rheumatoid arthritis (RA) and other inflammatory and degenerative diseases.

Objective

To examine the relative importance of tumour necrosis factor‐receptor 1 (TNF‐R1) and TNF‐R2 and their signalling pathways for pro‐inflammatory and pro‐destructive features of early‐passage synovial fibroblasts (SFB) from rheumatoid arthritis (RA) and osteoarthritis (OA).

Methods

Cells were stimulated with tumour necrosis factor (TNF)α or agonistic anti‐TNF‐R1/TNF‐R2 monoclonal antibodies. Phosphorylation of p38, ERK and JNK kinases was assessed by western blot; proliferation by bromodesoxyuridine incorporation; interleukin (IL)6, IL8, prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)‐1 secretion by ELISA; and MMP‐3 secretion by western blot. Functional assays were performed with or without inhibition of p38 (SB203580), ERK (U0126) or JNK (SP600125).

Results

In RA‐ and OA‐SFB, TNFα‐induced phosphorylation of p38, ERK or JNK was exclusively mediated by TNF‐R1. Reduction of proliferation and induction of IL6, IL8 and MMP‐1 were solely mediated by TNF‐R1, whereas PGE2 and MMP‐3 secretion was mediated by both TNF‐Rs. In general, inhibition of ERK or JNK did not significantly alter the TNFα influence on these effector molecules. In contrast, inhibition of p38 reversed TNFα effects on proliferation and IL6/PGE2 secretion (but not on IL8 and MMP‐3 secretion). The above effects were comparable in RA‐ and OA‐SFB, except that TNFα‐induced MMP‐1 secretion was reversed by p38 inhibition only in OA‐SFB.

Conclusion

In early‐passage RA/OA‐SFB, activation of MAPK cascades and pro‐inflammatory/pro‐destructive features by TNFα is predominantly mediated by TNF‐R1 and, for proliferation and IL6/PGE2 secretion, exclusively regulated by p38. Strikingly, RA‐SFB are insensitive to p38 inhibition of MMP‐1 secretion. This indicates a resistance of RA‐SFB to the inhibition of pro‐destructive functions and suggests underlying structural/functional alterations of the p38 pathway, which may contribute to the pathogenesis or therapeutic sensitivity of RA, or both.

Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.