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.
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