Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: Novel inflammatory role of this cell type
Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: Novel inflammatory role of this cell type
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DOI：
10.1038/sj.bjp.0700963
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发表时间：
1997-03-01
影响因子：
7.3
通讯作者：
Mitchell, JA
中科院分区：
文献类型：
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作者：
Belvisi, MG;Saunders, MA;Mitchell, JA
1环氧合酶（考克斯）是将花生四烯酸转化为前列腺素H-2（PGH（2））的酶，前列腺素H-2可进一步代谢为调节各种气道功能的前列腺素类。考克斯至少有两种亚型。考克斯-1组成型表达，而考克斯-2响应促炎刺激而表达。前列腺素类在生理和病理生理条件下由肺中的许多细胞类型产生。然而，人气道平滑肌（HASM）细胞中不同考克斯亚型的调节尚未确定。2使用考克斯-1和考克斯-2的特异性抗体，通过Western印迹分析测定考克斯-1和考克斯-2蛋白。通过使用考克斯-2 cDNA探针的北方印迹分析来评估考克斯-2 mRNA水平。考克斯的活性通过放射免疫测定法测定内源性或外源性花生四烯酸转化为三种代谢产物PGE（2）、血栓素B-2或6-酮PGF（1 α）的转化率。3在对照培养条件下，HASM细胞表达考克斯-1，但不表达考克斯-2蛋白。然而，细胞因子的混合物（白细胞介素-1 β（IL-1 β）、肿瘤坏死因子α（TNF α）和干扰素γ（IFN γ），各为10 ng ml（-1））诱导考克斯-2 mRNA表达，在12 h时达到最大值，并被地塞米松（1 μ M;在细胞因子前30 min加入）抑制。此外，在细胞因子处理后24小时检测到考克斯-2蛋白，并且该蛋白的表达也被地塞米松（1 μ M）和环己脲（10 μ g/ml;在细胞因子处理前30分钟加入）抑制。4在24小时内测量，未处理的HASM细胞释放低量或不可检测量的所有考克斯代谢物。用细胞因子混合物孵育细胞（IL-1 β、TNF α、IFN γ各10 ng ml（-1），持续24 h）引起PGE（2）和6-酮-PGF（1 α）的积累。5在考克斯-2代谢花生四烯酸内源性储存的实验中，用IL-1 β和TNF α联合处理HASM细胞引起类似的PGE释放（2）6在其他设计用于直接测量考克斯-2活性的实验中，用细胞因子处理细胞24小时，然后加入含有外源花生四烯酸（30 μ M，15分钟）的新鲜培养基，然后测量PGE（2）。IL-1 β和TNF α增加了考克斯-2活性，并且三种细胞因子联合产生了额外的小幅增加。7这些发现表明，考克斯-2表达增加与暴露于促炎细胞因子的HASM细胞过度释放前列腺素密切相关。这些数据表明，气道平滑肌细胞除了其收缩功能外，还作为参与产生介质的炎性细胞发挥作用，所述介质可能有助于在疾病如哮喘中观察到的炎症反应。
1 Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H-2 (PGH(2)) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined.2 COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE(2), thromboxane B-2 or 6-ketoPGF(1 alpha) by radioimmunoassay.3 Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) each at 10 ng ml(-1)) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 mu M; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 mu M) and cyclohexamide (10 mu g ml(-1); added 30 min before the cytokines).4 Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1 beta, TNF alpha, IFN gamma each at 10 ng ml(-1) for 24 h) caused the accumulation of PGE(2) and 6-keto-PGF(1 alpha).5 In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1 beta in combination with TNF alpha caused a similar release of PGE(2) to that when the three cytokines were given in combination.6 In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 mu M for 15 min) after which PGE(2) was measured. IL-1 beta and TNF alpha increased COX-2 activity and an additional small increase was produced by the three cytokines in combination.7 These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.