To determine the level of leukotriene B4 (LTB4) synthesized and released by synovium of patients with osteoarthritis (OA), and to study the role of lipoxygenase (LO)/cyclooxygenase (COX) products on proinflammatory cytokine and interstitial collagenase (MMP-1) synthesis.
Human OA synovial explants were cultured in the presence of lipopolysaccharide (L) and the ionophores ionomycin (I) and thapsigargin (T) (LIT) for 72 h at 37 degrees C, and LTB4 released into the culture medium was measured in the absence or presence of a COX-2-specific inhibitor, NS-398, or the 5-LO activating protein inhibitor Bay-x-1005. Increasing concentrations of LTB4 (10(-9) to 10(-6) M) were incubated with explants for 24 h at 37 degrees C, and interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in the conditioned medium were quantitated by ELISA. The effect of endogenous eicosanoids on basal and induced levels of IL-1beta, TNF-alpha, and MMP-1 synthesis was examined by incubating explants in the presence of NS-398 and Bay-x-1005. The effect of antiinflammatory cytokines rhIL-4, IL-10, and IL-13 on basal and LTB4 dependent stimulation of IL-1beta/TNF-alpha synthesis was studied under titration conditions.
Physiologically relevant concentrations (10(-10) to 10(-9) mol/l) of LTB4 were produced in the presence of LIT. Bay-x-1005 abrogated LTB4 release, while NS-398 was without effect. LTB4 stimulated IL-1beta and TNF-alpha synthesis with an EC50 of 190 +/- 35 and 45 +/- 9 nmol/l, respectively. Significant concentrations of IL-1beta and TNF-alpha were released (100-200 and 500-600 pg/ml, respectively). Basal and LIT induced IL-1beta and TNF-alpha production were inhibited by Bay-x-1005 in a dose dependent manner, while the addition of NS-398 caused a potent stimulatory effect. The preferential COX-2 inhibitor also induced MMP-1 synthesis in a manner essentially identical to the proinflammatory cytokines. The antiinflammatory cytokine IL-4 blocked LTB4 dependent stimulation of IL-1beta and TNF-alpha synthesis. In contrast, IL-10 markedly stimulated both cytokines when incubated alone or in the presence of LTB4 where the effect was additive.
Endogenous and locally produced eicosanoids regulate proinflammatory cytokine and MMP-1 synthesis under basal and stimulated conditions in vitro, with leukotrienes and prostaglandins having opposite effects in general. The clinical use of antiinflammatory drugs that inhibit eicosanoid synthesis requires an appreciation of their relative capacity to inhibit LO/COX in order to predict their effect on the synthesis of proinflammatory cytokines and matrix metalloproteases. IL-10 stimulated proinflammatory cytokine synthesis in our ex vivo culture system.