Donald W. Lawrence, Jr., Ph.D.

Dissertation research performed under the guidence of Dr. Katherine B. Pryzwansky

ABSTRACT
    Neutrophils are the first line of defense against bacterial infections. In response to chemical mediators produced during inflammation, neutrophils adhere to, and then migrate across the endothelial layer to sites of inflammation. The second messenger cyclic 3’,5’-guanosine monophosphate (cGMP) has been implicated as a positive regulator of human neutrophil adhesion. This dissertation was designed to advance the knowledge of cGMP-mediated regulation of human neutrophil adhesion and spreading. The following data are presented: (i) A rapid, but transient increase in intracellular cGMP levels was observed in adhering and spreading neutrophils. This increase in cGMP levels was significantly elevated prior to neutrophil spreading. In neutrophil suspensions, cross-linking of b2 integrin receptors caused a rapid elevation in cGMP levels, suggesting that integrin engagement and clustering are involved in the activation of guanylyl cyclase. (ii) Low concentrations of 8-Br-cGMP induced rapid shape changes and an increase in the numbers of neutrophils adhered to serum proteins. This increase in adhesion by 8-Br-cGMP was not due to an upregulation of cell surface adhesion receptors. The soluble guanylyl cyclase inhibitor, LY83583, inhibited shape changes during neutrophil adherence, supporting the hypothesis that elevations in cGMP are required for changes in cell shape. (iii) During neutrophil adherence there was a transient phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a substrate of cGMP-dependent protein kinase (G-kinase). VASP phosphorylation occurred during the early stages of neutrophil spreading and was coincidental with elevations in cGMP levels. Adhesion-induced VASP phosphorylation was attenuated by a specific G-kinase inhibitor. (iv) By immunofluorescence microscopy VASP was localized to punctate structures at a plane below the nucleus. Collectively, these data suggest that cGMP is a positive regulator of the initial stages of neutrophil adhesion and spreading, and that the regulation of these processes may involve the phosphorylation of VASP by G-kinase.