Interleukin-10 (IL-10) Polymorphism And Selected Cytokines In Malaysian Rheumatoid Arthritis Patients

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation and synovitis, which will often lead to the destruction of cartilage and bones. A large number of cytokines are detected in rheumatoid synovium and they have been classified into two groups according to their inflammatory properties i.e. pro- and anti-inflammatory cytokines. Although the effect of pro- and anti-inflammatory cytokines are more clearly defined in animal models, their role in the pathogenesis of RA in humans is still poorly understood. Interleukin-10 (IL-10) is an anti-inflammatory cytokine and has been reported to exert a protective role in animal models of RA. The human IL-10 gene promoter region is polymorphic and three proximal single nucleotide polymorphisms (SNPs) at positions -1082G>A, -824C>T and -597C>A have been reported. These SNPs are found in the putative regulatory region, which could affect the transcription of the human IL-10 gene and this in turn could affect the production of IL-10. We compared the frequencies of the SNPs in the human IL-10 gene promoter between RA patients and control subjects. The SNPs in the human IL-10 gene promoter were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The results from the PCR-RFLP were validated by DNA sequencing. The differences of the SNP genotype, allele or haplotype frequencies observed between RA patients and control subjects were found to be statistically not significant. The SNPs in the human IL-10 gene promoter have also been shown to be statistically correlated with the clinical measures of RA such as deformity, production of rheumatoid factor and early age of RA onset. The levels of IL-10 produced by Concanavalin A (Con-A)-stimulated peripheral blood leucocytes (PBL) were obtained from RA patients and control subjects using ELISA. The -1082G wild-type allele was associated with higher IL-10 protein production compared to the -1082A mutant allele. Interleukin-10 production was significantly lower in RA patients compared to control subjects. We also measured the levels of interferon-gamma (IFN-γ) and Interleukin-4 (IL-4) production in conjunction with IL-10 production. There was significantly higher production of IFN-γ in RA patients than control subjects. In contrast, significantly lower production of IL-4 was observed in these patients. The results confirm that RA is a TH1-driven clinical condition and that TH2 cytokines are important in the control of RA. The selective expression of Interleukin-12 beta-2 receptor (IL-12β2R) mRNA and soluble CD30 (sCD30) protein were analysed to study the basis of TH1/TH2 immune response in RA. The IL-12β2R mRNA expression was analysed using reverse-transcription-polymerase chain reaction (RT-PCR) method while the sCD30 levels were determined by ELISA. There was no significant difference observed on the expression of the IL-12β2R mRNA between RA patients and control subjects. However, mean sCD30 level was significantly higher in RA patients than control subjects. Soluble CD30 level was significantly lower in RA patients with active disease than those with their disease under control.