Platelet activating factor (PAF) and leukotriene B4 (LTB4) formation by human polymorphonuclear neutrophils (PMN) is an important aspect of normal PMN function but overproduction of these lipid mediators has been correlated with tissue damage occurring during acute and chronic inflammation. Activation of phospholipase A2 (PLA2) is the initial step in production of LTB4 and PAF and demonstration that PMN contain two calcium-dependent PLA2/s, a """"""""cytosolic"""""""" enzyme, cPLA2, and a """"""""secretory"""""""" enzyme, sPLA2, has led to intense interest in determining whether one or both are responsible for their formation. This proposal will utilize a permeabilized cell model where cPLA2 is specifically activated in the absence of sPLA2 activity and where both PAF and LTB4 formation occur to determine the actual mechanisms involved in cPLA2 activation. While progress has been made in understanding cPLA2 activation, the question of what mechanism links cell stimulation to cPLA2 activation is still unanswered. In the permeabilized cell model, phospholipase D (PLD) catalyzed production of phosphatidic acid (PA) and diglyceride (DG) are always present when cPLA2 activation occurs, inhibiting PLD activity blocks cPLA2 activation, and adding PA and DG, individually, back to permeabilized cells partially restores cPLA2 activity while a combination of both reconstitutes enzymatic activity. Enhanced PAF and LTB4 formation can be induced in vitro by pretreating PMN with one agonist before stimulation with a second agent by a process called """"""""primed-stimulation"""""""". In the permeabilized cell model increased LTB4 and PAF formation occur when cells are primed with the cytokine, tumor necrosis factor, before stimulation. The increased cPLA2 activity is paralleled by enhanced PA and DG formation and blocking PLD activity in primed cells leads to a parallel decrease in LTB4 and PAF production. This proposal will test the hypothesis that PA and DG function as second messengers in both direct activation of cPLA2 and in enhanced cPLA2 activity occurring in primed-stimulation of human PMN. Studies will utilize PMN permeabilized with Staphylococcus aureus alpha-toxin where cPLA2 is maintained in situ and enzyme activation is induced by addition of calcium, guanine nucleotides, and stimulation with a physiologic agonist. In this model, each agent can be added individually or in combination and effects on enzyme activation monitored.
Specific Aim 1 will focus on determining the interrelationship between cPLA2 activation and PLD activity, will determine the relative importance of PA and DG in inducing cPLA2 activation and will define the molecular species of PA and DG that effect cPLA2 activity.
In Specific Aim 2, requirements for translocation of cPLA2 from cytosol to membrane, for phosphorylation of cPLA2, for activation of the MAP kinase cascade and for activation of protein kinase C will first be determined in the permeabilized PMN, then in cells where PLD activity is blocked and finally in cells where enzyme activity is reconstituted with PA and DG to determine which steps in the signal transduction pathway are effected by PA and DG. This may identify new targets for therapies to combat excessive LTB4 and PAF production occurring during acute and chronic inflammation.
