RESUMEN

Cartilage is a specialized connective tissue. It contains few cells into an extracell matrix. The matrix mainly constituents are collagen and proteoglycans. Its degradation depends on synoviocytes activity, that secrete metalloproteases, agents to proteoglycans catabolism. There are two types of synoviocytes: macrophagics (type "A:') and fibroblastics (type "B"). The proteoglycan destruction can be LT-dependent or LT-independent. The aim of this work is synoviocytes function ex vívo study, free immune system influence. In order to do it, heparinized synovial fluid samples were obtained from 6 osteoarthritic (OA) and 6 arthritic (RA) both sex untreated patients, diagnosed according ACR criteria, which disease duration was longer than 6 months. Patients average age was 70 +/- 2 years. Control samples were synovial fluid from traumatic arthritis or non inflammatory bone-muscle pathology. Synovial fluid was centifugated at 1500 g for 30 minutes to isolate synoviocytes. Sediment containing cells was 6 hs incubed with Dulbecco-Eagles media, that has HEPES Gibco (26 mM); NaHCO3 (0.5 g/I); glutamine (2 mM), streptomicine (100 mg/l), G-penicillin (1 U/ml); anphotericine B (2.5 mg/l). Cells calification and viability were cytopathologically determined. Before and after incubation, collagenase activity was measured by ELISA-double-sandwich, using 10 micrograms/ml monoclonal anti-MMPs in phosphate-buffer-saline. The antigen-antibody complex production with inespecific proteins was blocked by bovine seric albumine. Streptavidin peroxidase was added and washed with 2,2,azin,di(3-ethyl-benztazoilinsuiphonic) acid to develop color. The link of labeled antibody by absorbance at 410 nm was determined in ELISA-spectrophothometer. RA patients earlier MMPs synoviocytes production was 1373 +/- 115 ng/ml. Then 6 hs incubation 2143 +/- 132 ng/ml was reached. The increase (56%) had high significance (p < 0.0001). OA earlier MMPs cells production was 276 +/- 23 ng/ml, but after incubation it reached 542 +/- 47 ng/ml. (96% increased with highly significativa difference too: p < 0.0001). Microscopic study was carried out before and after incubation, and shows a lot of synoviocytes with plenty of cytopiasme when the collagenase leveis were highest. On the contrary, when low MMPs production by synovial fluid, as no incubated osteoarthritic material, a few cells containing picnotics nucleous were observed. Significant quantitative differences in AR and OA enzymatic secretion were observed. Although in rheumatoid arthritic MMPs leveis synoviocytes production were 4.6 times than OA levels, after 6 hs incubation percentage of increase in OA cells secretion was highest. Described results confirm MMP-1 synthesis by synoviocytes, and these levels correlate with inflammation, more pronounced in acute (RA) than chronic pathology (OA). Synoviocyte incubation let us to test disease changes in synovial fluid according to cells number and phagocytic activity. Authors agree to assert that synovial fluid may reflect what is happening in an articular cartilago, because SF provides markers of joint disease. MMPs are giving information about pathways involved in OA and RA cartilage degradation.

Asunto(s)

RESUMEN

Cartilage is a specialized connective tissue. It contains few cells into an extracell matrix. The matrix mainly constituents are collagen and proteoglycans. Its degradation depends on synoviocytes activity, that secrete metalloproteases, agents to proteoglycans catabolism. There are two types of synoviocytes: macrophagics (type [quot ]A:) and fibroblastics (type [quot ]B[quot ]). The proteoglycan destruction can be LT-dependent or LT-independent. The aim of this work is synoviocytes function ex vívo study, free immune system influence. In order to do it, heparinized synovial fluid samples were obtained from 6 osteoarthritic (OA) and 6 arthritic (RA) both sex untreated patients, diagnosed according ACR criteria, which disease duration was longer than 6 months. Patients average age was 70 +/- 2 years. Control samples were synovial fluid from traumatic arthritis or non inflammatory bone-muscle pathology. Synovial fluid was centifugated at 1500 g for 30 minutes to isolate synoviocytes. Sediment containing cells was 6 hs incubed with Dulbecco-Eagles media, that has HEPES Gibco (26 mM); NaHCO3 (0.5 g/I); glutamine (2 mM), streptomicine (100 mg/l), G-penicillin (1 U/ml); anphotericine B (2.5 mg/l). Cells calification and viability were cytopathologically determined. Before and after incubation, collagenase activity was measured by ELISA-double-sandwich, using 10 micrograms/ml monoclonal anti-MMPs in phosphate-buffer-saline. The antigen-antibody complex production with inespecific proteins was blocked by bovine seric albumine. Streptavidin peroxidase was added and washed with 2,2,azin,di(3-ethyl-benztazoilinsuiphonic) acid to develop color. The link of labeled antibody by absorbance at 410 nm was determined in ELISA-spectrophothometer. RA patients earlier MMPs synoviocytes production was 1373 +/- 115 ng/ml. Then 6 hs incubation 2143 +/- 132 ng/ml was reached. The increase (56

) had high significance (p < 0.0001). OA earlier MMPs cells production was 276 +/- 23 ng/ml, but after incubation it reached 542 +/- 47 ng/ml. (96

increased with highly significativa difference too: p < 0.0001). Microscopic study was carried out before and after incubation, and shows a lot of synoviocytes with plenty of cytopiasme when the collagenase leveis were highest. On the contrary, when low MMPs production by synovial fluid, as no incubated osteoarthritic material, a few cells containing picnotics nucleous were observed. Significant quantitative differences in AR and OA enzymatic secretion were observed. Although in rheumatoid arthritic MMPs leveis synoviocytes production were 4.6 times than OA levels, after 6 hs incubation percentage of increase in OA cells secretion was highest. Described results confirm MMP-1 synthesis by synoviocytes, and these levels correlate with inflammation, more pronounced in acute (RA) than chronic pathology (OA). Synoviocyte incubation let us to test disease changes in synovial fluid according to cells number and phagocytic activity. Authors agree to assert that synovial fluid may reflect what is happening in an articular cartilago, because SF provides markers of joint disease. MMPs are giving information about pathways involved in OA and RA cartilage degradation.