Type I interferons might form the link between Toll-like receptor (TLR) 3/7 and TLR4-mediated synovial inflammation in rheumatoid arthritis (RA).

BACKGROUND: Rheumatoid arthritis (RA) has been associated with an increased risk of infections, but the underlying pathways have not yet been identified. Toll-like receptors (TLR) probably play a role in synovial inflammation and may also contribute to the understanding of the role of infections in RA. OBJECTIVES: To investigate if the synovial expression of TLR3 and TLR7 in RA correlates with that of inflammatory cytokines, and to assess whether this has functional consequences for local cytokine production and to study potential links between the TLR3/7 axis and TLR4 in RA synovium. METHODS: Immunohistochemistry was used to study the expression of TLR3, TLR7, interferon alpha (IFNalpha), tumour necrosis factor alpha (TNFalpha) and interleukins IL1beta, IL12, IL17 and IL18 in RA synovium obtained by arthroscopy from 34 patients with RA. Monocytes, monocyte-derived dendritic cells (MoDCs) and RA synovial fibroblasts were stimulated via TLR3 (poly-IC) and TLR7 (loxorubin), after which IL1beta, IL6 and TNFalpha were measured by Luminex bead array technology. Following preincubation with IFNalpha, IL1beta and IL18, TLR3 and TLR7 mRNA expression was assessed using real-time PCR. Cytokine production after preincubation with IFNalpha and subsequent TLR stimulation was measured. RESULTS: Synovial TLR3/7 expression was co-expressed with IFNalpha, IL1beta and IL18, but not with TNFalpha, IL12 and IL17. Stimulation of TLR3/TLR7 on monocytes, MoDCs or synovial fibroblasts led to secretion of type I IFN but no biologically active IL1beta or IL18 could be detected. Type I IFNalpha increased TLR3/7 mRNA expression whereas IL1beta and IL18 did not. In spite of the fact that the mRNA level of TLR4 remained unchanged, IFNalpha enhanced the response to TLR4 agonists, a phenomenon that was clearly more marked in patients with RA. CONCLUSION: Type I interferons are highly co-expressed with TLR3/TLR7 in RA synovium. They enhance TLR3/TLR7-mediated cytokine production and also TLR4-mediated responses.

Dendritic cells from patients with rheumatoid arthritis lack the interleukin 13 mediated increase of Fc gamma RII expression, which has clear functional consequences.

BACKGROUND: Dendritic cell (DC) function is largely tailored by Fc gamma receptors (Fc gamma R) and is critical for every immune response. OBJECTIVE: To compare interleukin (IL) 13 mediated regulation of Fc gamma RII and its related DC function between healthy controls and patients with rheumatoid arthritis (RA). METHODS: DC were derived from peripheral blood mononuclear cells according to standardised protocols. F cgammaRI, II, and III expression and DC phenotype were assessed by FACS analysis. The level of cytokine production and chemokine expression was measured by Luminex and real time quantitative polymerase chain reaction techniques. Antigen uptake capacity was studied by DC fluorescent heat aggregated immunoglobulins and FACS analysis. RESULTS: Replacement of IL4 by IL13 clearly increased the expression of Fc gamma RII on DC from healthy controls (CDC), but had no effect on DC from patients with RA (RADC). The lower production of inflammatory mediators by IL13 CDC upon Fc gamma R mediated triggering suggests that IL13 induces up regulation of specifically Fc gamma RII. RADC co-cultured with IL4 already displayed an inhibitory DC phenotype, but this inhibitory phenotype was not augmented by the addition of IL13. The defective Fc gamma RII regulation was further substantiated by the finding that IL13 CDC increased antigen uptake capacity, whereas IL13 RADC did not. CONCLUSION: IL13 regulates the expression of inhibitory Fc gamma RII in normal subjects but not in RA, potentially resulting in a chronic proinflammatory immune reaction in RA. Unravelling the underlying mechanisms of Fc gamma RII regulation might lead to new therapeutic targets in RA.

The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells.

OBJECTIVE: To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls. METHODS: Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients. RESULTS: TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients. CONCLUSION: TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.