Alterations In Pulmonary Immune Function And Host Resistance In COX Null Mice

我们正在研究环氧合酶在基础肺功能和对环境因素的肺反应中的作用。在基线时，COX-1基因缺失小鼠的肺前列腺素E_2水平低于野生型或COX-2基因缺失小鼠，但在基础肺功能或肺组织病理学方面，两种基因型之间没有显著差异。在过敏原(卵清蛋白)致敏暴露后，COX-1基因缺失和COX-2基因缺失小鼠的肺炎症指数明显高于野生型小鼠。COX-1基因缺失小鼠呼吸道嗜酸性粒细胞数量增加，CD3CD4淋巴细胞(Th细胞)数量增加。来自过敏性COX-1缺失呼吸道的肺泡巨噬细胞表现出激活的生化和形态证据。过敏性COX-1基因缺失小鼠的支气管肺泡灌洗液(BALF)中Th2细胞因子IL-4、IL-5和IL-13水平显著升高，LTB4和半胱氨酰白三烯水平升高，趋化因子TARC和嗜酸性粒细胞趋化因子水平升高。COX-1基因缺失小鼠的这些变化与BALF IgE水平升高和MUC5AC产生粘蛋白分泌增加有关。此外，过敏性COX-1和过敏性COX-2缺失小鼠肺组织中黏附分子VCAM-1和ICAM-1的表达均增加。过敏性COX-1基因缺失的小鼠肺顺应性降低，变应原诱导的支气管收缩增加，对吸入乙酰甲胆碱表现出高反应性。我们还研究了COX基因的干扰对其他环境相关因素的肺反应的影响，包括吸入内毒素(细菌脂多糖)、五氧化二钒和流感病毒。在脂多糖暴露后，所有小鼠都表现出更强的支气管收缩和乙酰甲胆碱高反应性；然而，与野生型对照相比，COX-1缺失和COX-2缺失小鼠的这些变化要明显得多。有趣的是，在脂多糖暴露后，不同基因型的BALF细胞或肺组织病理学没有显著差异。因此，COX-1和COX-2的平衡在调节吸入性内毒素的生理性反应而不是炎症反应中起重要作用。在五氧化二钒(V2O5)暴露后，COX-2基因缺失的小鼠，而不是COX-1基因缺失的小鼠，急性肺部炎症增加，并发展成更多的肺纤维化(肺组织羟脯氨酸增加和三色染色增强)。我们还利用肺部流感传染性模型来评估宿主抵抗力，并确定COX-1基因缺失和COX-2基因缺失小鼠对病毒感染的先天或获得性免疫反应是否存在缺陷。体重和体温变化证明，与野生型和COX-1基因缺失小鼠相比，感染导致COX-2基因缺失小鼠的病情不那么严重。COX-2基因缺失小鼠的死亡率显著降低。COX-1基因缺失的小鼠BAL液中炎症反应增强，促炎细胞因子提前出现，而COX-2基因缺失小鼠的炎症反应和细胞因子反应减弱。然而，与野生型和COX-1基因缺失小鼠相比，COX-2基因缺失小鼠的肺病毒滴度显著升高。感染后COX-1缺失组前列腺素E_2水平降低，而COX-2缺失组半胱氨酸白三烯水平升高。因此，COX-1和COX-2的缺失导致宿主对流感感染的反应不同，这些差异与感染后前列腺素和白三烯的产生改变有关。COX基因缺陷小鼠的反应因环境刺激而异。最近，我们开发了肺特异性高表达人COX-1的转基因小鼠(小鼠CC10启动子驱动)。虽然没有观察到基础呼吸或肺力学参数的差异，但COX-1转基因小鼠的支气管肺泡灌洗液前列腺素E2含量高于野生型小鼠，并且对吸入乙酰甲胆碱的呼吸道反应性降低。在卵清蛋白诱导的过敏性呼吸道炎症模型中，在过敏性野生型和COX1转基因小鼠的肺中观察到COX-2蛋白的上调。此外，过敏性小鼠肺泡灌洗液中的细胞总数、嗜酸性粒细胞含量和炎性细胞因子含量以及吸入乙酰甲胆碱后的气道反应性均未观察到基因型差异。为了消除COX-2上调可能产生的混杂效应，将COX-1转基因小鼠培育成COX-2缺失背景。在这些小鼠中，COX-1转基因的存在并没有改变过敏原诱导的炎症反应，但显著地减轻了过敏原诱导的气道高反应性，这与降低气道白三烯水平是一致的。总而言之，这些数据表明，COX-1的过度表达减弱了基础条件下的气道反应性，但不影响过敏性气道炎症。我们目前正在开发在II型细胞中过表达COX-2的转基因小鼠(SPA启动子驱动)，以检测COX-2在远端呼吸道中的作用。我们还开发了在肺中选择性敲除COX-2的小鼠(Clara细胞，II型肺泡细胞)，以确定全身或局部前列腺素的生物合成是否在调节肺对环境因素的反应中起重要作用。最后，我们正在研究COX-1和COX-2在体外和体内肺T细胞分化中的作用。

We are investigating the role of cyclooxygenases in basal lung function and in the pulmonary response to environmental agents. At baseline, lung prostaglandin E2 levels are lower in COX-1 null mice compared to either wild type or COX-2 null mice, but there are no significant differences in basal lung function or in lung histopathology between the genotypes. Following allergen (ovalbumin) sensitizationexposure, lung inflammatory indices are significantly greater in COX-1 null and COX-2 null mice compared to wild type mice. Airways of allergic COX-1 null mice have increased numbers of eosinophils and increased numbers of CD3CD4 lymphocytes (Th cells). Alveolar macrophages from allergic COX-1 null airways show biochemical and morphologic evidence of activation. Bronchoalveolar lavage fluid (BALF) from allergic COX-1 null mice contains significantly higher levels of the Th2 cytokines IL-4, IL-5 and IL-13, increased levels of LTB4 and the cysteinyl leukotrienes, and increased levels of the chemokines TARC and eotaxin. These changes in the COX-1 null mice are associated with increased BALF IgE levels and increased MUC5AC productionmucin secretion. Moreover, expression of the adhesion molecules VCAM-1 and ICAM-1 are increased in the lungs of both allergic COX-1 and allergic COX-2 null mice. Allergic COX-1 null mice have reduced lung compliance, increased allergen-induced bronchoconstriction and display hyperresponsiveness to inhaled methacholine. We have also examined the effects of disruption of COX genes on the pulmonary responses to other environmentally relevant agents including inhaled endotoxin (bacterial lipopolysaccharide, LPS), vanadium pentoxide, and influenza virus. Following LPS exposure, all mice exhibit increased bronchoconstriction and methacholine hyperresponsiveness; however, these changes are much more pronounced in both the COX-1 null and COX-2 null mice relative to wild type controls. Interestingly, there are no significant differences in BALF cells or lung histopathology between the genotypes following LPS exposure. Thus, the balance of COX-1 and COX-2 is important in regulating the physiologic but not the inflammatory responses to inhaled LPS. Following vanadium pentoxide (V2O5) exposure, COX-2 null mice, but not COX-1 null mice, have increased acute lung inflammation and develop more lung fibrosis (increased lung hydroxyproline and enhanced trichrome staining). We have also utilized a pulmonary influenza infectivity model to evaluate host resistance and to determine if there are defects in innate or adaptive immune responses to viral infection in COX-1 null and COX-2 null mice. Infection induced less severe illness in COX-2 null mice in comparison to wild type and COX-1 null mice as evidenced by body weight and body temperature changes. Mortality was significantly reduced in COX-2 null mice. COX-1 null mice had enhanced inflammation and earlier appearance of pro-inflammatory cytokines in the BAL fluid, whereas the inflammatory and cytokine responses were blunted in COX-2 null mice. However, lung viral titres were markedly elevated in COX-2 null mice relative to wild type and COX-1 null mice. Levels of prostaglandin E2 were reduced in COX-1 null airways whereas cysteinyl leukotrienes were elevated in COX-2 null airways following infection. Thus, deficiency of COX-1 and COX-2 leads to contrasting effects in the host response to influenza infection, and these differences are associated with altered production of prostaglandins and leukotrienes following infection. The response of COX-deficient mice varies depending on the environmental stimulus. More recently, we developed transgenic mice with lung-specific overexpression of human COX-1 (murine CC10 promoter driven). Whereas no differences in basal respiratory or lung mechanical parameters were observed, COX-1 transgenic mice had increased bronchoalveolar lavage fluid prostaglandin E2 content compared to wild type littermates and exhibited decreased airway responsiveness to inhaled methacholine. In an ovalbumin-induced allergic airway inflammation model, comparable upregulation of COX-2 protein was observed in the lungs of allergic wild-type and COX 1 transgenic mice. Furthermore, no genotype differences were observed in allergic mice in total cell number, eosinophil content and inflammatory cytokine content of bronchoalveolar lavage fluid, or in airway responsiveness to inhaled methacholine. To eliminate the presumed confounding effects of COX-2 upregulation, COX-1 transgenic mice were bred into a COX-2 null background. In these mice, presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly attenuated allergen-induced airway hyperresponsiveness, coincident with reduced airway leukotriene levels. Collectively, these data indicate that COX-1 overexpression attenuates airway responsiveness under basal conditions but does not influence allergic airway inflammation. We are currently developing transgenic mice with overexpression of COX-2 in type II cells (SPA promoter driven) to examine the role of COX-2 in the distal airway. We are also developing mice with selective knockout of COX-2 in the lung (Clara cells, type II alveolar cells) to determine if systemic or local biosynthesis of prostaglandins is important in regulating the lung response to environmental agents. Finally, we are studying the role of COX-1 and COX-2 in differentiation of lung T-cells in vitro and in vivo.