The objective of this study was to define the role of oxidants and lipid mediators in the leukocyte-endothelial cell adhesion and albumin leakage elicited in rat mesenteric venules by ischemia-reperfusion (I/R). Intravital fluorescence microscopy was used to monitor leukocyte adherence and emigration, platelet-leukocyte aggregation, mast cell degranulation, and albumin leakage after release of a 20-min arterial occlusion. I/R elicited large increases in leukocyte-endothelial cell adhesion and albumin leakage. These responses were significantly attenuated in venules treated with either superoxide dismutase, oxypurinol (an inhibitor of xanthine oxidase), lodoxamide (a mast cell stabilizer), WEB-2086 (a platelet-activating factor antagonist), or SC-41930 (a leukotriene B4-receptor antagonist) but not by U-74006F (an inhibitor of lipid peroxidation). Platelet-leukocyte aggregates and mast cell degranulation induced by I/R were also attenuated by administration of either superoxide dismutase or lodoxamide. These results support the hypothesis that oxidants produced, in part, by xanthine oxidase promote the formation (by mast cells and endothelial cells) of platelet-activating factor and leukotriene B4, which recruit and activate leukocytes in postischemic venules. The adherent and emigrated leukocytes then mediate the increased albumin extravasation observed in the postcapillary venules.

We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by "'Tc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/ CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CDMlb (Mac-i) showed partial inhibition of hemorrhage, while mAb against CDMla (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition. (J. Clin. Invest. 1992.89:259-272.)

Several laboratories, including our own have reported the synthesis and activity of certain low relative molecular mass inhibitors of mammalian serine proteases, especially human leukocyte elastase (HLE, EC 3.4.21.37), an enzyme whose degradative activity on lung elastin has been implicated as a major causative factor in the induction of pulmonary emphysema, and which is present in the azurophil granules of human polymorphonuclear leukocytes (PMN). Normally, these granules fuse with phagosomes containing engulfed foreign material (such as bacteria), and HLE, in combination with other lysosomal enzymes, catabolizes the particles. Under certain pathological conditions, however, PMN become attached to host protein (elastin fibres, basement membrane, connective tissue, immune complexes), and in response to this adherence, the granules may fuse with the PMN outer membrane and release their contents, including HLE, directly onto the tissue. Besides emphysema, HLE may also contribute to the pathogenesis of disease states such as adult respiratory distress syndrome, and its potential involvement in rheumatoid arthritis makes HLE inhibitors of considerable interest. It is known that cephalosporin antibiotics (for example, cephalothin (compound I, Table 2)) are acylating inhibitors of bacterial serine proteases which help synthesize the cell wall by performing a transpeptidation reaction on a peptidyl substrate bearing a D-Ala-D-Ala terminus. We now report that neutral cephalosporins (that is, compounds not bearing a free carboxyl at position C-4) can be modified to become potent time-dependent inhibitors of HLE.