Neutrophils mediate tissue injury in two classical models of experimental pathology: the Arthus lesion(1903) and the Shwartzman phenomenon (1927).
The aim of this proposal is to explain in the language of 1989 how vascular injury induced by immune complexes and complement (the Arthus lesion) differs from the induced by endotoxin, cytokines and complement (the Shwartzman phenomenon). We will study neutrophil/endothelial cell interactions in vitro to test novel hypotheses of """"""""triggering"""""""" and activation"""""""" of neutrophils. We will determine the functional effects on human neutrophils and cytoplasts of """"""""defensive"""""""" molecules release from activated vs. resting endothelial cells. A variety of endothelial cells (eg HUVEC) will be exposed to endotoxin or cytokines and the release measured of a) prostacyclin b) adenosine c) endothelium-derived relaxation factor (EDRF, or NO) and d) sphingosine (an endogenous inhibitor of O2 generation). The effects of these molecules will be studied on neutrophils or cytoplasts activated by chemoattractants or immune complexes with respect to a) heterotypic adherence (to HUVEC), b) cell-cell aggregation, c) disaggregation d) chemotaxis in several systems, and e) release of O-2 H2 O2 and lysosomal enzymes. We will also expose neutrophils activated by chemoattractants and immune complexes to PGI2 adenosine, NO and sphingosine. Stimulus/response coupling will be examined with attention to receptors for iC3b and immune complexes (FcRII,FcRIII), cytosolic pH and free calcium (pH, and Cai) 45Ca fluxes, membrane viscosity, membrane potential (delta psi), crosslinking of cytoskeletal proteins, microtubule assembly, phospholipid remodelling, and protein phosphorylation. We will compare the susceptibility of resting HUVEC with cells activated by endotoxin or cytokines (IL-1, TNF and IFN) to killing by neutrophils exposed to chemoattractants (C5a, LTB, FMLP). We will study whether immune complexes present a) in the bulk phase b) on the HUVEC monolayer, or c) in the subphase, provoke neutrophil-mediated cell injury despite release of """"""""defensive"""""""" molecules from HUVEC. Finally, using marine sponge cells and human neutrophils, we will study whether 20.4 and other cis unsaturated fatty acids activate a novel GTP-binding protein the function of which is mimicked by phospatidic acid. These studies will employ novel liposomal methods for phospholipid presentation. Thus we will be able to define the roles played by cytokine activated endothelial cells (the Shwartzman model) and immune complexes (the Arthus model) in neutrophil-mediated blood vessel injury.
Showing the most recent 10 out of 59 publications
