Interleukin-8 (L-8) and the IL-8 family of proteins are among the most potent and effective neutrophil (PMN) chemoattractants known. They are important inflammatory mediators in the lung. They exert their chemotactic effect on neutrophils via two cell surface receptors. Identification of the receptor binding domains of IL-8 is important to both understanding the molecular mechanisms of lung inflammation and to the design of reagents to block inflammatory process mediated by the IL-8 family of proteins. Our GOALS in these studies are to identify the domains of the IL-8 molecule involved in PMN chemotaxis-receptor recognition, and to develop effective and specific inhibitors of PMN chemotaxis mediated by the IL-8 family. We have cloned and expressed in mammalian cells the two PMN receptors which bind IL-8. Our expressed receptor clones are functional with respect to ligand binding signal transduction. These are tools that are critical to the development of receptor-specific antagonists. We have purified and cloned the cDNAs for two porcine homologues of the IL-8 family which are potent chemoattractants for porcine PMN, but they have discrepant activity for human PMN. Together with the primary structures and relative potencies of the other IL-8 family members, these porcine homologues help identify domains that are conserved in the chemotactically potent family members but either absent or poorly conserved in the single chemotactically inactive family member, IP-10. Finally, we have modelled the 3-dimensional structure of IP-10 and found it superimpossible on the solution-structure of IL-8, suggesting that differences in its primary sequence account for its inactivity. These observations provide important clues to the receptor binding and signalling sites of IL-8. Our OBJECTIVES are 1.) to construct and express chimeras of the IL-8 molecule with the structurally related but chemotactically inactive IP-10 molecule in order to identify the specific domains involved in receptor binding and signaling. 2.) to synthesize peptides based on the results of objectives #1 and examine their ability to agonize or antagonize receptor binding and signal transduction in the expressed IL-8 receptor clones; 3.) to examine the biological activity of selected chimeric proteins and synthetic peptides as agonists or antagonists of PMN chemotaxis in in-vitro and in-vivo assays; and 4.) to examine the effects of selected peptide inhibitors of PMN chemotaxis in an animal model of acute lung inflammation. These studies will help to define the specific sites of interaction of IL-8 with its cognate receptors and identify potentially useful inhibitors of IL-8 function. Specific inhibitors of the PMN chemotactic function of IL-8 will be an important tool for studying the role of IL-8 in disease and may prove useful in limiting some types of lung inflammation.
