Role of cockroach proteases in airway inflammation

描述(由申请人提供)：蟑螂(CR)暴露是哮喘发展的易感因素。虽然许多空气变应原含有蛋白酶活性，但蛋白酶在调节炎症反应中的作用尚不清楚。我们已经证明蟑螂提取物含有蛋白分解活性，这与协同增加肿瘤坏死因子α介导的白介素8和白介素6的表达有关。此外，我们有初步数据表明，蟑螂蛋白水解酶激活了呼吸道上皮细胞上的蛋白水解酶激活受体(PAR)-2，从而引发了细胞外调节激酶(ERK)激活的信号级联反应。为了了解蟑螂活性蛋白水解酶在体内外调节气道炎症的机制，我们提出了以下具体目标：1.建立CR丝氨酸蛋白酶活性在PARs上的作用及其在促炎反应中的作用。我们将通过RT-PCR、流式细胞术和免疫组织化学检测肿瘤坏死因子α刺激是否调节PAR-2的表达。我们将用活化肽SLIGKV激活PAR-2，并研究其在细胞因子表达中的作用。我们将在Cos-1细胞中过表达PAR-2，并确定其在炎症中的作用。我们将从粗提物中提纯丝氨酸蛋白酶。2.建立介导CR诱导的IL-8调节的下游信号事件。我们有证据表明ERK在CR治疗后被激活。我们将研究转录因子NF-IL6和AP-1在CR诱导的协同作用中的作用。这将通过启动子缺失研究、DMA结合和反式激活研究来完成。我们将确定对IL-8的调节是否需要或足够的核因子-IL6或AP-1。我们将确定ERK和NF-kappa B通路之间是否存在串扰。最后，我们将确定CR提取物是否在转录上调节其他细胞因子/趋化因子。 3.建立蟑螂丝氨酸蛋白酶活性在调节呼吸道炎症和反应性中的体内作用。我们将在治疗前通过体外抑制蛋白水解酶活性来确定蛋白水解酶在调节呼吸道炎症中的作用。我们将利用PAR-2基因敲除小鼠来确定PAR-2在调节呼吸道反应中的作用。最后，我们将使用一种肿瘤坏死因子α中和抗体和肿瘤坏死因子α基因敲除小鼠来研究内源性肿瘤坏死因子α在呼吸道中的作用。

DESCRIPTION (provided by applicant): Cockroach (CR) exposure is a predisposing factor for the development of asthma. While many aeroallergens contain protease activity, the role of proteases in modulating the inflammatory response is unknown. We have shown that cockroach extract contains proteolytic activity which is responsible for synergistically increasing tumor necrosis factor (TNF) alpha-mediated interleukin (IL)-8 and IL-6 expression. In addition, we have preliminary data suggesting that cockroach proteases activate the protease activated receptor (PAR)-2 on airway epithelium to elicits signaling cascade to extracellular regulated kinase (ERK) activation. To understand the mechanism by which active cockroach proteases modulate airway inflammation in vitro and in vivo, we propose the following Specific Aims: 1. Establish the role of CR serine protease activity on PARs and its effects on the pro-inflammatory response. We will examine whether TNF alpha stimulation regulates PAR-2 expression by RT-PCR, flow cytometry and immunohistochemistry. We will activate PAR-2 with the activating peptide SLIGKV and investigate its role in cytokine expression. We will overexpress PAR-2 in Cos-1 cells and determine its role in inflammation. We will purify the serine protease from the crude CR extract. 2. Establish the downstream signaling events mediating CR-induced regulation of IL-8. We have evidence that ERK is activated following CR treatment. We will examine the role of transcription factors NF-IL6 and AP-1 on CR-induced synergy. This will be done by promoter deletion studies, DMA binding and transactivation studies. We will determine if NF-IL6 or AP-1 is required or sufficient for the regulation of IL-8. We will determine if crosstalk occurs between ERK and NF-kappa B pathways. Finally we will determine if CR extract transcriptionally regulates other cytokines/chemokines. 3. Establish an in vivo role for cockroach serine protease activity in modulating airway inflammation and reactivity. We will determine the role of proteases in modulating airway inflammation by ex vivo inhibition of the protease activity prior to treatment. We will establish a role for PAR-2 in modulating airway responses using PAR-2 knockout mice. Finally, we will investigate the role of endogenous TNF alpha in the airways using a TNF alpha neutralizing antibody and TNF alpha knockout mice.