Interleukin (IL)-33 induces the release of pro-inflammatory mediators by mast cells.

IL-33 (or IL-1F11) was recently identified as a ligand for the previously orphaned IL-1 family receptor T1/ST2. Previous studies have established that IL-33 and T1/ST2 exert key functions in Th2 responses. In this study, we demonstrate that IL-33 induces the production of pro-inflammatory mediators in mast cells. IL-33 dose and time-dependently stimulated IL-6 secretion by P815 mastocytoma cells and primary mouse bone marrow-derived mast cells (BMMC). This effect was dependent on T1/ST2 binding. In addition, IL-33 also induced IL-1beta, TNF-alpha, MCP-1, and PGD2 production in BMMC. By RNase protection assay, we demonstrated that IL-33 increased IL-6 and IL-1beta mRNA expression. These effects of IL-33 appeared to occur independently of mast cell degranulation, The results of this study show for the first time that IL-33, a novel member of the IL-1 family of cytokines, stimulates the production of pro-inflammatory mediators by mast cells in addition to its effect on T helper 2 responses. These findings open new perspectives for the treatment of inflammatory diseases by targeting IL-33.

Influence of cytokine inhibitors on concentration and activity of MMP-1 and MMP-3 in disc herniation.

INTRODUCTION: Spontaneous resorption of disc herniation (DH) after sciatica is well documented. The matrix metalloproteinases (MMP)-1 and MMP-3 are enzymes potentially involved in this process. Glucocorticoid injections are commonly used for treatment, and other anti-inflammatory molecules like tumor necrosis factor (TNF) inhibitors are under clinical investigation. However, little is known about the effect of these molecules on DH resorption. METHODS: DH tissue was harvested from patients undergoing surgery for sciatica. Samples were thoroughly washed. Diced explants were cultured ex-vivo in 1) 0.5 ml Dulbecco's modified Eagle's medium (DMEM) 10% fetal calf serum (FCS), (controls), 2) recombinant interleukin 1 receptor antagonist (IL-1Ra), (100 ng/ml), 3) dexamethasone (10E-5 M), or 4) TNF inhibitor monoclonal antibody (10 microg/ml). Supernatants were harvested at 48 hours and frozen. Immunocapture activity assays determined total MMP activity, active MMP levels and pro-MMP levels. RESULTS: Fourteen DH tissue samples were analysed. Levels of all forms of MMP-3 were higher than the respective levels of MMP-1(P < 0.01). In particular, the median (interquartile range [IQR]) total MMP-3 level was 0.97 (0.47 - 2.19) ng/mg of tissue compared to 0.024 (0.01 - 0.07) ng/mg of total MMP-1 level (P < 0.01). Incubation with IL-1Ra, dexamethasone, or TNF inhibitors significantly decreased levels of all forms of MMP-3 (P < 0.05). Dexamethasone significantly decreased the ratio of active MMP-3 to total MMP-3 activity. A significant inhibitory effect of dexamethasone was observed only on active MMP-1, while IL-1 and TNF inhibitor had no significant effect on any form. CONCLUSIONS: MMP-3 appears to play a greater role than MMP-1 in DH resorption. Dexamethasone, IL-1-Ra and TNF inhibitor decreased active MMP-3, indicating that the clinical use of these drugs may affect the resorption of DH under certain conditions.

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes.

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.

Pre-interleukin-1alpha expression reduces cell growth and increases interleukin-6 production in SaOS-2 osteosarcoma cells: Differential inhibitory effect of interleukin-1 receptor antagonist (icIL-1Ra1).

Interleukin-1 receptor antagonist (IL-1Ra) inhibits the classical biological effects of interleukin (IL)-1 by preventing its binding to IL-1 receptors. There are, however, four different isoforms of IL-1Ra, of which three are intracellular (icIL-1Ra1, 2, 3). Due to their localization, icIL-1Ras cannot interact with cell surface IL-1 receptors and have been suggested to carry out specific functions inside cells. The recent description of nuclear effects of the IL-1alpha precursor (preIL-1alpha) led to the hypothesis that icIL-1Ra variants might antagonize the intracellular actions of preIL-1alpha. The aim of this study was to investigate effects of preIL-1alpha and icIL-1Ra1 in stably transfected SaOS-2 cells. Expression of preIL-1alpha significantly decreased cell growth and this effect was not reversed by addition of exogenous IL-1Ra, suggesting an intracellular mode of action. Transfection of SaOS-2 cells with icIL-1Ra1 did not affect their growth. Furthermore, co-expression of icIL-1Ra1 did not reverse the growth inhibitory effect of preIL-1alpha. In contrast, the production of IL-6 induced by preIL-1alpha was decreased in icIL-1Ra1 co-transfected cells. In conclusion, expression of preIL-1alpha decreased the growth of SaOS-2 cells, likely by an intracellular mechanism. Co-expression of icIL-1Ra1 did not antagonize this effect, indicating that intracellular effects of preIL-1alpha are not necessarily susceptible to inhibition by icIL-1Ra1.

Production of interleukin-1 receptor antagonist by human articular chondrocytes.

Interleukin-1 receptor antagonist (IL-1Ra) is a natural IL-1 inhibitor possessing anti-inflammatory properties. IL-1Ra is produced as different isoforms, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, icIL-1Ra2 and icIL-1Ra3), derived from the same gene. We examined the production of IL-1Ra species by cultured human articular chondrocytes in response to various cytokines. The levels of IL-1Ra were undetectable in culture supernatants of untreated cells, but were significantly increased by IL-1beta. Cell lysates contained very low levels of IL-1Ra, even in response to IL-1beta, suggesting that chondrocytes produce predominantly sIL-1Ra. IL-6, which had no effect on its own, enhanced the effect of IL-1beta, while dexamethasone prevented the response. We observed by RT-PCR that IL-1beta and IL-6 induced primarily the production of sIL-1Ra mRNA. Furthermore, IL-1beta alone or combined with IL-6 increased the levels of nascent unspliced sIL-1Ra mRNA, suggesting that sIL-1Ra expression is regulated at the transcriptional level. Reporter gene assays in immortalized chondrocytes, C-20/A4, consistently showed increased sIL-1Ra promoter activity in response to IL-1beta and IL-6. In conclusion, human articular chondrocytes produce sIL-1Ra in response to IL-1beta and IL-6. The production of sIL-1Ra by chondrocytes may have a protective effect against articular inflammatory and catabolic responses.

The active metabolite of leflunomide, A77 1726, increases the production of IL-1 receptor antagonist in human synovial fibroblasts and articular chondrocytes.

Leflunomide is an immunomodulatory agent used for the treatment of rheumatoid arthritis. In this study, we investigated the effect of A77 1726 - the active metabolite of leflunomide - on the production of IL-1 receptor antagonist (IL-1Ra) by human synovial fibroblasts and articular chondrocytes. Cells were incubated with A77 1726 alone or in combination with proinflammatory cytokines. IL-1Ra production was determined by ELISA. A77 1726 alone had no effect, but in the presence of IL-1beta or tumour necrosis factor-alpha it markedly enhanced the secretion of IL-1Ra in synovial fibroblasts and chondrocytes. The effect of A77 1726 was greatest at 100 micromol/l. In synovial fibroblasts and de-differentiated chondrocytes, A77 1726 also increased IL-1beta-induced IL-1Ra production in cell lysates. Freshly isolated chondrocytes contained no significant amounts of intracellular IL-1Ra. A77 1726 is a known inhibitor of pyrimidine synthesis and cyclo-oxygenase (COX)-2 activity. Addition of exogenous uridine did not significantly modify the effect of A77 1726 on IL-1Ra production, suggesting that it was not mediated by inhibition of pyrimidine synthesis. Indomethacin increased IL-1beta-induced IL-1Ra secretion in synovial fibroblasts and de-differentiated chondrocytes, suggesting that inhibition of COX-2 may indeed enhance IL-1beta-induced IL-1Ra production. However, the stimulatory effect of indomethacin was consistently less effective than that of A77 1726. A77 1726 increases IL-1Ra production by synovial fibroblasts and chondrocytes in the presence of proinflammatory cytokines, and thus it may possess chondroprotective effects. The effect of A77 1726 may be partially mediated by inhibition of COX-2, but other mechanisms likely concur to stimulate IL-1Ra production.

Primary human articular chondrocytes, dedifferentiated chondrocytes, and synoviocytes exhibit differential responsiveness to interleukin-4: correlation with the expression pattern of the common receptor gamma chain.

Interleukin (IL)-4, which exhibits potent anti-inflammatory activities, is of potential therapeutic value in destructive arthropathies. To further define the response of human joint cells to IL-4, we analyzed the ability of this cytokine to modulate the effects of IL-1beta and growth factors. Freshly isolated chondrocytes, dedifferentiated chondrocytes, and synoviocytes were treated with IL-4 before determination of nitric oxide (NO) and collagenase production in response to IL-1beta, or before proliferation assays in presence of IL-1beta, platelet-derived growth factor (PDGF), or transforming growth factor (TGF)-beta. IL-4 downregulated IL-1beta induced NO production in dedifferentiated chondrocytes and inhibited IL-1beta induced collagenase release, as well as IL-1beta and growth factor induced proliferation in dedifferentiated chondrocytes and synoviocytes. In contrast, IL-4 had no effect in freshly isolated primary chondrocytes and in cartilage explants. The lack of response to IL-4 in primary chondrocytes was associated with impaired signal transduction, as indicated by markedly decreased IL-4 dependent tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-6. It also correlated with differences in the expression pattern of IL-4 receptor (IL-4R) subunits during chondrocyte dedifferentiation. Indeed, whereas the IL-4Ralpha and IL-13Ralpha' subunits were expressed in all cell types, expression of the common receptor gamma chain was restricted to freshly isolated chondrocytes. In conclusion, IL-4 downregulated IL-1beta-induced catabolic events and cell proliferation in dedifferentiated chondrocytes and synoviocytes, but had no effects in freshly isolated chondrocytes. The difference in IL-4 responsiveness between primary and dedifferentiated chondrocytes correlated with changes in proximal signaling events and in the expression pattern of IL-4R subunits during cell dedifferentiation.