Demonstration of granzyme A and perforin messenger RNA in the synovium of patients with rheumatoid arthritis

Objective. To examine the gene expression of 2 highly specific markers of cytotoxic T lymphocyte (CTL) activation, the serine protease granzyme A and the poreforming protein perforin, in synovial tissue of patients with rheumatoid arthritis (RA), and to compare the findings with those in osteoarthritis (OA) synovial tissue.

Methods. Snap-frozen synovial tissue specimens from 9 patients with RA and 5 patients with OA were examined. The number of CTL that expressed granzyme A or perforin messenger RNA was determined by in situ hybridization using nonradioactive riboprobes for granzyme A and perforin, and by a novel in situ reverse transcriptase technique. The signals were visualized by an immunogold–silver immunohistochemistry technique and compared with immunohistochemical labeling of T and B cells. Additional double-labeling was achieved using anti–type IV collagen, anti-macrophage (anti-CD68), anti–T lymphocyte (anti-CD45RO), anti–B lymphocyte (anti-CD20), and anti–natural killer cell (anti-CD56) antibodies in an alkaline phosphatase–anti–alkaline phosphatase assay.

Results. Granzyme A and perforin messenger RNA (mRNA) was observed in CTL in synovial specimens from all of the RA patients, whereas in specimens from OA patients only a few, single cells with a positive mRNA signal for these molecules could be detected. In the RA specimens, the number of lymphocytes showing a positive mRNA signal for granzyme A or perforin varied from 10% to 50%, reflecting the recent findings of other investigators studying synovial fluid.

Conclusion. Our results demonstrate that gene expression of at least 2 CTL products, granzyme A and perforin, is up-regulated in the synovium of patients with RA compared with that in the synovium of patients with OA. These molecules presumably play an important role not only in lymphocyte-mediated cytotoxicity, but also in facilitating the migration of blood-born mononuclear cells through the vascular basement membrane into the rheumatoid synovium.