Elastase-mediated fibrinogenolysis by chemoattractant-stimulated neutrophils occurs in the presence of physiologic concentrations of antiproteinases.

Plasma levels of the HNE-derived fibrinopeptide A alpha 1-21 reflect in vivo enzyme activity. To provide a possible explanation for the presence of circulating A alpha 1-21 in individuals with normal plasma antiproteinase concentrations we investigated whether PMN-associated HNE is more resistant to inhibition than the free enzyme. PMN were stimulated to migrate across 125I-fibrinogen-coated nitrocellulose filters in response to 10(-7) M FMLP, and the extent of fibrinogenolysis was determined by measuring release of A alpha 1-21 and 125I-labeled fibrinogen degradation products. The fibrinogenolytic activity of migrating PMN was then compared with that of free HNE present in PMN lysates or secreted by PMN stimulated with FMLP. Whereas the fibrinogenolytic activity of soluble HNE was completely inhibited by low concentrations (1%) of plasma or serum and macromolecular antiproteinase (alpha 1 proteinase-inhibitor and soybean trypsin-inhibitor), even in the presence of undiluted plasma or serum the activity of the migrating PMN was incompletely blocked (81-85%). Further, concentrations of alpha 1 proteinase-inhibitor and soybean trypsin-inhibitor that totally inhibited free HNE activity also incompletely blocked (88-89%) the fibrinogenolytic activity of migrating PMN, indicating that FMLP-stimulated PMN demonstrate significant fibrinogenolytic activity in the presence of antiproteinases as small as 20,000 mol wt. A specific low molecular weight HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl), however, totally blocked PMN-mediated fibrinogenolysis without affecting intracellular HNE activity, HNE secretion from PMN, or PMN migration in response to FMLP. These findings support the hypothesis that PMN migrating on a fibrinogen-coated surface form zones of close contact with fibrinogen, thus preventing access of plasma antiproteinases to HNE released at the cell-substrate interface. The occurrence of this phenomenon in vivo would explain the presence of circulating A alpha 1-21 in individuals with normal antiproteinase concentrations

Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes