He Wendy, Pelletier Jean-Pierre, Martel-Pelletier Johanne, Laufer Stefan, Di Battista John A
Osteoarthritis Research Unit, Hôpital Notre-Dame, Centre Hospitalier de l'Université de Montréal, Québec, Canada.
J Rheumatol. 2002 Mar;29(3):546-53.
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.
测定骨关节炎（OA）患者滑膜合成和释放白三烯B4（LTB4）的水平，并研究脂氧合酶（LO）/环氧化酶（COX）产物对促炎细胞因子和间质胶原酶（MMP-1）合成的作用。
将人OA滑膜外植体在脂多糖（L）、离子载体离子霉素（I）和毒胡萝卜素（T）（LIT）存在的条件下于37℃培养72小时，在不存在或存在COX-2特异性抑制剂NS-398或5-LO激活蛋白抑制剂Bay-x-1005的情况下，测定释放到培养基中的LTB4。将浓度递增的LTB4（10⁻⁹至10⁻⁶M）与外植体在37℃孵育24小时，通过酶联免疫吸附测定法（ELISA）定量条件培养基中的白细胞介素1β（IL-1β）和肿瘤坏死因子-α（TNF-α）。通过在NS-398和Bay-x-1005存在的情况下孵育外植体，研究内源性类花生酸对IL-1β、TNF-α和MMP-1基础合成水平及诱导合成水平的影响。在滴定条件下，研究抗炎细胞因子重组人IL-4、IL-10和IL-13对IL-1β/TNF-α基础合成及LTB4依赖性刺激合成的影响。
在LIT存在的情况下产生了生理相关浓度（10⁻¹⁰至10⁻⁹mol/l）的LTB4。Bay-x-1005消除了LTB4的释放，而NS-398没有作用。LTB4刺激IL-1β和TNF-α的合成，其半数有效浓度（EC50）分别为190±35和45±9nmol/l。释放出了显著浓度的IL-1β和TNF-α（分别为100 - 200和500 - 600pg/ml）。Bay-x-1005以剂量依赖性方式抑制基础及LIT诱导的IL-1β和TNF-α产生，而添加NS-398则产生强烈的刺激作用。优先COX-2抑制剂也以与促炎细胞因子基本相同的方式诱导MMP-1合成。抗炎细胞因子IL-4阻断LTB4依赖性的IL-1β和TNF-α合成。相反，IL-10单独孵育或在LTB4存在下孵育时显著刺激这两种细胞因子，其作用是相加的。
内源性和局部产生的类花生酸在体外基础和刺激条件下调节促炎细胞因子和MMP-1的合成，白三烯和前列腺素通常具有相反的作用。抑制类花生酸合成的抗炎药物的临床应用需要了解它们抑制LO/COX的相对能力，以便预测它们对促炎细胞因子和基质金属蛋白酶合成的影响。在我们的体外培养系统中，IL-10刺激促炎细胞因子合成。
