Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis

Abstract

Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis-trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28-50 nM). Estimated concentrations of the SF-derived cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r = 0.91, P < 0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an approximately 18-kD protein band reacted with polyclonal antibodies that recognize cyclophilin A and B, but not with antibodies specific for cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human cyclophilin A. The finding is unexpected since cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.

Figure 1

Time course of the peptidyl prolyl cis-trans isomerase activity. (Triangles) Cyclophilin B added (25 nM final concentration); (filled circles) 50 μl synovial fluid from a patient with RA (patient WA, left knee); (open squares) same as filled circles, but 20 μM FK 506 included; (open circles) Same as filled circles, but 20 μM cyclosporin A included; (filled squares) 50 μl synovial from a patient with OA (patient HU) added; (+) no synovial fluid added, i.e., substrate cleavage proceeding in the absence of isomerase (control). Note that the last three curves coincide.

Figure 2

Correlation of cyclophilin-like protein concentration with total cell counts in synovial fluids of RA patients (filled circles) and with percentage of neutrophils (open triangles).

Figure 3

SDS–polyacrylamide gel electrophoresis of partially purified cyclophilin A from synovial fluids of patients (KL and AA) with RA. Lane 1, recombinant cyclophilin B; lane 2, recombinant cyclophilin A; lane 3, pooled fractions of gel filtration chromatography containing the synovial cyclophilin activity. A Gel (20%) stained with Coomassie brillant blue. (B) Immunoblot developed with antibodies which recognize cyclophilins A and B. Positions of marker proteins (M) are indicated.