Background The role of T-lymphocytes (LT) in the pathogenesis of rheumathoid arthritis (RA) is demonstrated, but the site of T-cell activation is still unknown. The differentiation and maturation of LT took place either in the peripheral blood (PB) and synovial fluid (SF). This process can be followed using the isoforms of the leucocyte common antigen (CD45). Cells expressing CD45RA demonstrate functional characteristics of “naive cells” and those expressing CD45RO of “memory cells”. Following stimulation, naive cells lose CD45 RA and gain CD45 RO with a transitional stage of dual CD45RA and CD45RO positivity. In RA, the LT from the PB express primarily CD 45RA whereas most LT found in the SF express CD45RO. Expression of HLA-DR is increased on LT found in SF. The population of dual positive LT cells CD45RA+CD45RO+ is expanded in SF, supporting the hypothesis that the LT are activated within the joint.
Objectives Using monoclonal antibodies labelled with fluorochromes and the flow-cytometry technique we tried to evidentiate the most important modification of lymphocytes subtypes in PB and SF in patients with chronic arthritis.
Methods The study was performed on 29 cases: 19 with RA in the stage II and III of evolution; 8 cases with ankylosing spondylitis and 2 cases with knee ostheoarthritis used as control. Paired samples of PB and SF were obtained from all the patients. From these, nucleated cells were prepaired (after erithrocyte lysis in PB) and mononuclear cells in SF after density gradient centifugation. The cells from both sources were labelled with monoclonal antibodies conjugated with fluorochromes in three colour staining (CD45/CD14/CD3, CD3/CD16+56/CD45, CD3/CD19/CD45, CD45RA/CD45RO/CD3, CD45RA/HLA-DR/CD3).
After two washing steps, the cells were analysed whithin a flow-cytometer (FACSCalibur, BD) using the CellQuest (BD) and Paint-a-Gate (BD) software.
Results From the SF we isolated a greater percent of CD14+, CD19+ and CD3+ cells compared with PB. We found in patients with RA a higher report CD4/CD8 in SF than in the PB; and many of the CD3+ CD8+ cells showed a citotoxic phenotype. Among the patients with RA the median percent of cells expressing CD45RA+CD45RO+HLA-DR+ phenotype was 2% in PB respectively 25% in SF; for the CD45RA+CD45RO-HLA-DR- phenotype 25% in PB and 0 in SF;CD45RA-CD45RO+HLA-DR- 24% in PB and 26% in SF;CD45RA-CD45RO+HLA-DR+ 5% in PB and 30% in SF; CD45RA+CD45RO+HLA-DR- 25% in PB and 0 in SF. The HLA-DR expression was 6,1% of LT in PB in RA, predominantly associated with CD45RO expression.
Conclusion Our results confirmed the previous studies reports that described the presence of dual CD45RA+CD45RO+ LT population in PB and SF of patients with RA. The LT dual positive CD45RA and CD45RO population which did not express HLA-DR in PB made up 25% of LT from PB in RA. In SF all dual positive LT coexpress HLA-DR. These results sustained the hypothesis that LT are in a more activated state in SF than in PB.
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