title: Studies on chemoattractant activity of rheumatoid synovial fluid for human lymphocytes in vitro
Creators: name: Al-Mughales, Jamil A. Al-wali
Publisher: name: University of Glasgow
Year: 1996

description: The present research was carried out to investigate the chemoattraction activity of rheumatoid and non rheumatoid synovial fluids for human lymphocytes separated from peripheral blood, synovial tissue and synovial fluid. The phenotyping of locomotor cells in response to these fluids was also studied. Established procedures were used to separate lymphocytes from blood, synovial tissue and synovial fluid. The level of the chemotactic factors in the synovial fluid was measured by commercial and in-house developed methods. The inhibitory effect of anti-inflammatory drags on the lymphocyte locomotion was also studied. The chemoattractant activity of synovial fluid for human lymphocytes was investigated using the following methods: i. A Polarization assay which measures the shape change from spherical or round to a polarized shape (i.e from immotile to a motile shape) following stimulation with chemoattractants. ii. Collagen gel invasion, which measures the migration of lymphocytes into collagen gels- containing chemoattractants. Two methods were used for phenotyping the cells responding to the synovial fluids (I) APAAP which allowed the detection of lymphocyte surface markers in stained cytospin preparations, (ii) FACS which allowed the detection of cell surface markers of lymphocytes recovered from collagen gels after collagenase digestion. In addition methods used to measure the levels of chemotactic factors in the synovial fluid, were (I) Commercial single antibody sandwich ELISA kits (R&amp;D) which measured IL-2, IL-8, MIP-1α and MCP-I, (ii) In-house developed multiple antibody sandwich ELISA which measured IL-15 in the fluids. The ability of synovial fluids from patients with rheumatoid (n=35) and other arthritides (n=18) to attract lymphocytes from peripheral blood of normal subjects, from rheumatoid synovia, and from joint fluids, was studied. The majority of synovial fluids from 29 rheumatoid arthritis patients were strongly attractive for blood lymphocytes which had been cultured overnight. Three out of five fluids from OA also attracted lymphocytes but to a lesser extent than RA fluids. In addition four of seven fluids from other inflammatory arthritides also gave high responses Rheumatoid synovial tissue lymphocytes responded to synovial fluids without a requirement for a period of culture. In contrast lymphocytes derived from rheumatoid and other synovial fluids were completely unresponsive to locomotor stimulants. Most of the responding cells from blood mononuclear cell fractions were T lymphocytes and the CD45RO isotype was attracted preferentially. Rheumatoid synovial fluids contained IL-8 , IL-15, MIP-1α and MCP-1 at levels in the nanogram range, sufficient to attract lymphocytes, but levels of IL-2 were too low to exert a chemoattractant effect. In contrast the levels of chemotactic factors in OA fluids were low and these fluids also showed less activity in attracting lymphocytes. The activity of the fluids could not be abolished by treatment with antibodies to IL-8, IL-2, MTP-1α, MCP-1 or IL-15 tested individually, but combinations of these antibodies inhibited most of the activity, suggesting that attraction of lymphocytes by the fluids is due to a combination of attractants. The accumulation of lymphocytes within the synovial fluids was not correlated with any single chemotactic factor mentioned above, suggesting that such accumulation is due to combined chemoattractants. In the present study it was also observed that neutrophils separated from normal blood gave a strong chemotactic response to the synovial fluids. In contrast neutrophils separated from the synovial fluid were immotile, suggesting that these cells had an intrinsic defect or that their locomotion was selectively blocked by synovial fluid chemotactic inhibitors. Moreover there was no correlation between IL-8 or levels of any other single cytokine and the accumulation of these cells in the fluids, indicating the possibility of multiple chemotactic factor involvement. The manipulation of the locomotion activity of lymphocytes in vitro in response to synovial fluid was studied using anti-inflammatory drugs. It was demonstrated that NSAIDs (including Aspirin, Ibuprofen and indomethacin), DMARDs (including gold, D-penicillamine and primaquine) and cytotoxic drugs including rapamycin and cyclophosphamide had no inhibitory effect on lymphocyte locomotion. On the other hand cyclosporin A and Glucocorticosteroids (including dexamethasone, prednisone and prenisolone) showed a significant inhibitory effect.