ESTABLISHMENT OF A MODEL FOR ADHESION OF POLYMORPHONUCLEAR LEUKOCYTE TO SYNOVIAL CELL

AIM: To establish a new and simple method of quantitating adhesion of neutrophil or polymorphonuclear leukocyte(PMN) to rat synovial cell(RSC) using MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphemyl tetrazolium bromide］-stained PMN cell. METHODS: PMN were incubated with MTT, and formed MTT labeled PMN(MTT-PMN) with a blue dye of formazan. After incubation of the MTT-PMN and RSC, non-adhered cells were washed out. Adhered cells were lysed with dimethylsulfoxide and quantitated by measure the absorbance at 540 nm. The concentration and time courses of rhTNF-α and IL-1 which stimulate RSC were studied. According to the positive-cross test, moreover, it were also studied that the inhibitory effects of hydrocortisone (Hy) and meloxicam (Melo) on the adhesion of PMN to RSC which were stimulated by rhTNF-α and IL-1 at 500 U.mL^-1 for 12 h. RESULTS: The absorbance at 540 nm was shown to be well correlated with the cell numbers of MTT-PMN(0.25～2×109 cell.L^-1) adhesion to RSC which were stimulated by 500 U.mL^-1 rhTNF-α and IL-1　for 12 h. Hydrocortisone inhibited the adhesion of PMN to RSC stimulated by 500 U.mL^-1 rhTNF-α and IL-1, the IC₅₀ was 2.6×10-7 mol.L^-1, 2.29×10^-9 mol.L^-1, respectively. But IC₅₀ of meloxicam were 6.1×10^-7 mol.L^-1 and 1.25×10^-6　mol.L^-1, respectively. CONCLUSION: A new and simple method of quantitating adhesion of PMN to RSC was established, by using MTT-stained PMN cell. This method can be used for screening inhibitors of PMN adhesion to RSC.