Grant: Discovery Grants Program - Individual

Inflammation is a host's innate defense response to biological, chemical or physical insults. Inflammation is induced through infections with bacteria, viruses and parasites. Inflammation underlies many diseases in organs such as the liver (e.g., hepatitis), joints (e.g., arthritis) and lungs (e.g., pneumonia and asthma). Inflammation is characterized by the activation of defense cells such as neutrophils and monocytes while in the blood stream. Activated cells such as neutrophils move from the blood into the organs to fight bacteria. Neutrophils migrate from the blood vessels through interactions with the endothelial cells. Endothelial cells line the inside of the walls of blood vessels. Activated neutrophils kill bacteria through production of chemicals such as free oxygen radicals. Excessive migration of activated neutrophils into the tissues results in significant tissue damage from free oxygen radicals and other chemicals produced by the neutrophils. The tissue damage caused by products of neutrophils, if excessive, contributes to morbidity and mortality. In order to maintain balance and insure that neutrophils do more good than harm, we need to maintain the anti-microbial actions of the neutrophils, and at the same time, fine-tune their migration into the tissues to prevent tissue damage. This can be potentially accomplished through control of migration of the neutrophils. Neutrophil migration occurs through various steps such as slowing, rolling on the endothelium, firm adhesion with the endothelium and finally migration out of the blood vessels. One of the very early events in the migration of neutrophils into the tissues is the interaction between the neutrophils and the endothelium. This interaction is facilitated through the formation of projections (called pseudopods) on the neutrophil membrane through reorganization of cell skeleton proteins such as F-actin. The mechanisms that lead to the cytoskeletal changes in neutrophils are not fully understood. I propose to study the roles of CD34 and podocalyxin (PC) proteins in these processes to eventually discover molecular mechanisms to control neutrophil migration. The studies include evaluation of the mechanisms that influence the expression of CD34 and PC in neutrophils and endothelial cells as well as the study of the role of these proteins in the interactions of neutrophils with the endothelium. The studies will be conducted with an array of in vitro and in vivo methods to investigate the cell morphology, molecules and outcomes: confocal and electron immunocytochemistry; western blots; flow cytometry; endothelial and neutrophil cultures; mouse models of inflammation in cremaster muscle and lungs. Taken together, my trainees and I will conduct studies that range from isolated cultured neutrophil and endothelial cells to two different animal models of inflammation to understand the fundamental nature of the neutrophil migration. The data from these studies are expected to show hitherto unknown roles for CD34 and PC in the formation of pseudopods on neutrophils, which facilitate interactions with endothelial cells lining the inside walls of the blood vessels. Because these initial membrane contacts are critical for the eventual migration of neutrophils, these data will advance our knowledge and contribute towards the development of new molecular management models of inflammatory diseases for animals and humans.