IL10 in Reactive Arthritis

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IL-10 has received increasing interest in the last years as a potentially immunosuppressive cytokine. While upregulation of such a cytokine would be wanted in assumed autoimmune diseases such as rheumatoid arthritis we hypothesized that it might contribute to bacterial persistence in ReA, possibly by downregulation of the Thl-cytokines IFNy and TNFa.

To investigate this question further we performed a serie of experiments. First, we could show that Chlamydia trachomatis specific CD4+ T cells clones derived from the synovial fluid from patients with Chlamydia-induced ReA were capable to secrete besides IFNy also IL-10 upon in vitro stimulation with whole Chlamydia.9 Subsequently, synovial fluid mononuclear cells (MNC) from 9 ReA patients due to Chlamydia trachomatis (4 patients), Yersinia enterocolitica (4 patients), or Salmonella enteritidis (1 patient) were stimulated in vitro with the responsible bacterium and cytokines were measured in the supernatant by ELISA.6 SF MNC produced high amounts of IFNy and IL-10, relatively less TNFa, and no detectable IL-4. These amounts were significantly higher than cytokine production by SF MNC stimulated with LPS and than cytokines secreted by PB MNC derived from healthy controls upon stimulation with the same whole bacteria. Interestingly, the amount of IL-10 was relatively the highest compared to IFNy and TNFa resulting in a IL-10/IFNy ratio of 2 (±1.9) and in a IL-10/TNFa ratio of 14 (±11) (Fig. 1). These ratios were strikingly high compared with the results found in the joints of 6 patients with Lyme arthritis (after stimulation of these SF MNC with Borrelia burgdorferi), in which the mean ratio was 0.6 (±0.4) for IL-10/IFNy and 1.2 (±1.1) for IL-10/TNFa (Fig. 1).6 Other researchers could also show in patients with Chlamydia-induced and other ReA that a relative high amount of IL-10 mRNA, as measured by PCR,10 and a low amount of IFNy, as detected by immunohistology,7 are present. When peripheral blood MNC from ReA were stimulated in vitro with mitogens a significant lower amount of TNFa was found in ReA compared to rheumatoid arthritis. In the same study, IL-10 levels were found to be higher in ReA than in RA, although this difference was not significant.8

In contrast to the ReA-associated bacteria, a Th2 response seems to be necessary for the elimination of Borrelia burgdorferi.11 Thus, these data indicate that the presence of the "wrong" Th2/Th3-response in the joint of ReA-patients and the "wrong" Th1-response in the joint of Lyme arthritis patients contributes to the persistence of these bacteria.

We further investigated whether IL-10 would have a regulatory role in the production of IFNy and TNFa produced by SF-derived MNC. We could indeed show that IL-10 is most likely responsible for the inhibition of IFNy and TNFa secretion within the ReA joint, because neutralizing endogenous IL-10 in culture (SF MNC stimulated with the responsible bacterium in vitro) with anti-IL-10 resulted in enhanced seceretion of IFNy and TNFa. The addition of exogenous IL-10 further reduced secretion of both cytokines.6 Interestingly, the enhanced

Cytokine ratio

y

-A-,

1L-I0/IFN7 IHO/TNFa

□ Reactive Arthritis □ Lyme Arthritis

Figure 1. Ratio of IL-10/TNFa and IL-10/IFNy- secreted by synovial fluid mononuclear cells stimulated with the triggering bacteria in case of reactive arthritis or with Borrelia burgdorferi in case of Lyme arthritis for 24 hours amd measured in the supernatant by ELISA (modified from Yin et al, 1997).6

effect of anti-IL-10 could be reduced by adding neutralizing antibody against IL-12, indicating that IL-10 suppressed IFNy and TNFa secretion by inhibiting IL-12 synthesis. It has been shown before that the Th1 inhibitory effect of IL-10 might be mediated by suppression of IL-12.12

While in these studies we used whole bacteria for in vitro stimulation of MNC we next asked the question whether the cytokine secretion pattern, including IL-10, is dependent on which immunodominant antigen is used for stimulation of CD4+ T cells. We performed a detailed analysis of the response of T cell clones derived from synovial fluid from a patient with Yersinia-induced ReA which were specific for the heat shock protein (hsp) 60 from Yersinia.13 Yersinia hsp60 specific CD4+ T cells were raised which were specific for different hsp60-derived antigens. Four clones specific for the same peptide were used for further analysis of the cytokine secretion pattern. Most interestingly, only one clone produced, in addition to IFNy, significant amounts of IL-10. A longer peptide induced a greater production of IL-10 than the 12 amino acid long core peptide and an altered peptide (single AA substitution at position 10) induced the lowest secretion of IL-10.13

While in this study antigen specific T cell clones were used we applied subsequently the technique of antigen specific flow cytometry to investigate to capacity ofT cell subpopulations to produce IL-10 upon antigenic stimulus. 4 For this method whole SF cells or whole PB are stimulated with antigen in vitro for 6 hours. For the last 4 hours the secretion inhibitor brefeldin A is added for accumulation of intracellular cytokines and the cytokine positive cells are quantified by flow cytometry. Besides antibodies specific for the IFNy and IL-10 we used also antibodies binding to the cell surface marker CD4 allowing us to quantify the percentage of CD4+ T cells secreting IL-10 or IFNy alone or together. In 2 patients with Chlamydia-induced ReA the percentage of IL-10-positive CD4+ T cells was between 0.10 and 0.23% while the percentage of IFNy-positive CD4+ T cells was between 0.5 and 2.5% after in vitro stimulation with the Chlamydia-specific 'major outer membrane protein' (MOMP) or hsp 60 (Fig. 2) Interestingly, there were nearly no IFNy/IL-10-double-positive T cells (Fig. 2).

These data clearly indicate that CD4+ T cells (and maybe also others, such as macrophages, which were not further differentiated in our experiments using whole MNC) produce IL-10 upon stimulation both with whole ReA-associated bacteria and with immunodominant bacteria derived from these bacteria. The production of IL-10 seems to be different for different T cell clones recognizing the same antigen and seems also to be dependent on the antigen.

Figure 2. Stimulation ofwhole synovial fluid with different recombinant proteins from Chlamydia trachomatis. After staining for T cell surface markers and intracellular cytokines, a gate for CD4+ T cells was set. Double staining was performed for IL-10 and IFNy. The percentage of the IFNy single positive cells (left upper quadrant), the IFNy/IL-10 double positive cells (right upper quadrant), and the IL-10 single-positive cells (right lower quadrant) of the CD4+/CD69+ T cell subpopulation are indicated. Reproduced with permission from Thiel et al, 2000. ©2000 Wiley Publishers.14

Figure 2. Stimulation ofwhole synovial fluid with different recombinant proteins from Chlamydia trachomatis. After staining for T cell surface markers and intracellular cytokines, a gate for CD4+ T cells was set. Double staining was performed for IL-10 and IFNy. The percentage of the IFNy single positive cells (left upper quadrant), the IFNy/IL-10 double positive cells (right upper quadrant), and the IL-10 single-positive cells (right lower quadrant) of the CD4+/CD69+ T cell subpopulation are indicated. Reproduced with permission from Thiel et al, 2000. ©2000 Wiley Publishers.14

This flexibility of the IL-10-response offers the possibility for therapeutic manipulations. Therefore, an effective elimination of bacteria might be inhibited by IL-10 which can be overcome by exogenuous IL-12.

Subsequently, colleagues from Germany and Finland addressed the question whether differences in the production of IL-10 in ReA patients might be genetically determined. In this study 85 finish ReA patients and 62 HLA-B27-positive Finish controls were investigated. From genomic DNA, IL-10 microsatellites G and R and IL-10 promotor polymorphisms at positions -1087 and -524 were typed by polymerase chain reaction, automated fragment length analysis, and restriction fragment digestion. There was a significant decrease in the promoter alleles G12and G10 in the ReA group compared with the HLA-B27-positive controls indicating that these alleles might have a protective effect for the occurrence of ReA.15 Although it is not clear at the moment whether these alleles are associated with a higher production of IL-10 this data raises the possibility that the relative increase of IL-10 found in ReA might, at least partially, be genetically determined.

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