Interleukin-8 (IL-8) has been shown to be a key mediator of immunological reactions in many inflammatory disorders such as respiratory distress syndrome, idiopathic pulmonary fibrosis, rheumatoid arthritis and asthma. As is the case for other proinflammatory mediators, phagocytic leukocytes respond to IL-8 by migrating to sites of inflammation where they become activated and participate in host defense. These functions are mediated via specific cell surface receptors for IL-8. Two subtypes of IL-8 receptors (IL-8RA & IL-8RB) have been described in neutrophils. Both subtypes of IL-8R become desensitized upon IL-8-stimulation. In addition to homologous and heterologous desensitization, IL-8R are cross- desensitized by other chemoattractants. To date, little is known about the mechanism governing IL-8R regulation. Our studies have shown that IL- 8R-mediated responses are the most susceptible to cross-desensitization by other chemoattractants in neutrophils. These studies have also indicated that components(s) distal to the receptor/G-protein may be involved in chemoattractant receptor cross-regulation. The main focus of the present proposal is on the regulation of the IL-8 receptors and the mechanisms by which they undergo desensitization and cross-desensitization. First, we will use a rat basophilic leukemia (RBL-2H3) cell line, a functionally responsive cell line, to stably express epitope-tagged IL-8 receptors (ETIL-8RA and ETIL-8RB) and elucidate the role of agonist-dependent and independent phosphorylation in the regulation of IL-8R-mediated responses. Second, we will use directed mutagenesis and deletion to identify specific amino acid residues of the cytoplasmic domains of the IL-8R involved in modulating receptor functions. Third, we will determine the mechanisms of cross-phosphorylation and desensitization between IL-8 receptors and other chemoattractant receptors by stably coexpressing the receptors in RBL-2H3 cells. And fourth, we will determine the role of phospholipase C beta2 (PLCbeta2) in IL-8 receptor cross-regulation. Understanding the molecular mechanisms involved in the regulation of the IL-8R and the role played by these receptors in the cross-regulation of chemoattractant-mediated inflammatory responses will aid in understanding the control of inflammation as well as the etiology of many inflammatory disorders. These studies will also identify specific targets for the development of therapeutic drugs for the modulation of inflammation.
