Expressions of Fas and FasL mRNA on peripheral blood lymphocytes in Behcet's disease

Abstract

Objective To evaluate the expression of Fas and FasL messenger RNA (mRNA) and their molecules in peripheral blood lymphocytes and its possible implication in Behcet's disease. Methods Blood samples were obtained from 19 patients with active Behcet' s disease and 19 healthy individuals. Total RNA was extracted from peripheral blood lymphocytes. cDNA was prepared from RNA with oligo d(T)18 and M-MuLV reverse transcriptase. The expression of Fas mRNA and FasL mRNA on peripheral blood lymphocytes was determined using fluorescent quantitative polymerase chain reaction (FQ-PCR). The expression of Fas and FasL on peripheral blood T lymphocytes was investigated using flow cytometry and three-color double marked immunofluorescence methods. The monoclonal antibodies used in this study were cy-chrome conjugated mouse anti-human CD4 antibody,FITC conjugated mouse anti-human CD8 antibody,phycoerythrin conjugated mouse anti-human Fas monoclonal antibody,biotinylated mouse anti-human CD95/FasL monoclonal antibody and streptavidin/R-PE. Result The expression of Fas mRNA on peripheral blood lymphocytes in patients with Behcet' s disease ( 1.50 × 106 ± 0. 77 × 106copies/μg)was significantly higher than that seen in controls (0.57 × 106 ± 0.20 × 106copies/μg)(P ＜ 0.05). The expression of FasL mRNA on peripheral blood lymphocytes in these patients (2.00 × 106±2. 25×106copies/μg) was also higher than that seen in controls (0.48×106±0. 35×l06copies/μg) (P ＜0. 01 ). A significantly higher expression of Fas antigen on CD4+ and CD8+ lymphocytes was seen in these patients (26. 20 ± 8. 01 ;9. 68 ± 6. 81, respectively) as compared with that in controls ( 13. 92 ± 5. 19;3. 25 ± 2.69, respectively).However,no significant difference was found in FasL expression on CD4+ T cells and CD8+ lymphocytes between patients and controls. Conclusion A high level of Fas mRNA and FasL mRNA expression and unbalanced expression of Fas and FasL molecules on peripheral blood lymphocytes were observed in the patients with Behcet' s disease. This disturbed expression of Fas and FasL on lymphocytes may be involved in the perpetutation and recurrence of intraocular inflammation seen in Behcet' s disease for these lymphocytes could not properly undergo apoptosis mediated by Fas and FasL interaction. Key words: Fas/FasL; Behcet'; s; disease; quantitative; PCR; flow; cytometer