Association of the microsatellite in the 3' untranslated region of the CD154 gene with rheumatoid arthritis in females from a Spanish cohort: a case-control study

Abstract

CD40-CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.

Figure 1

Genotype frequencies of the CD154 microsatellite in healthy females and those with rheumatoid arthritis. (a) Seventy-nine female patients with rheumatoid arthritis (RA) were compared with 56 healthy females (P_c = 0.483). Genotypes represented fewer than five times are not included. *P = 0.012. (b) The frequency for carriers of two (24/24), one (24/X) or zero (X/X) alleles of 24CAs, where X represents any allele different from 24CAs. One hundred and forty female patients with RA were compared with 80 healthy females (P_c = 0.026). **P = 0.042, ***P = 0.012.

Figure 2

Expression of CD154 mRNA in T lymphocytes according to CD154 genotype. T lymphocytes from patients with rheumatoid arthritis (RA; 24CAs group, n = 9; non-24CAs group, n = 11) and controls (24CAs group, n = 3; non-24CAs group, n = 3), obtained after in vitro expansion of peripheral blood mononuclear cells, were stimulated for 6 or 24 hours with anti-CD3 plus anti-CD28; after this, mRNA decay assays were performed as indicated in the Materials and methods section. (a) mRNA half-life, t_1/2, in T lymphocytes. (b) mRNA molecules per μg of total RNA in CD4⁺ T lymphocytes. *P < 0.05, **P < 0.005, comparing the 24CAs group with the non-24CAs group.

Figure 3

Kinetics of surface expression of CD154 in stimulated T lymphocytes (a) CD154 kinetic expression in CD4⁺ T lymphocytes from patients with rheumatoid arthritis (RA) after stimulation with anti-CD3/anti-CD28, according to CD154 genotype. Peripheral blood mononuclear cells from patients with RA (24CAs group, n = 19; non-24CAs group, n = 16) were stimulated in vitro with anti-CD3/anti-CD28 for 24, 48, 72, and 92 hours. After this, the surface expression of CD154 was measured by flow cytometry. The graph shows the median percentage of CD154 expression in CD4⁺ T lymphocytes from each group at the indicated times. *P = 0.046, comparing the 24CAs group with the non-24CAs group. (b) Cytometry histograms showing CD154 staining (thick line) compared with non-specific staining (isotype control mAb, thin line) in CD4⁺ T lymphocytes from representative patients with RA (upper panel, 24CAs; lower panels, non-24CAs), after 24 hours (left panels) and 48 hours (right panels) of stimulation with anti-CD3/anti-CD28. PE, phycoerythrin.