Human polymorphonuclear leukocytes (PMN) have previously been shown to bind to aggregates of fibrin and to fibrinogen-coated surfaces. During their interactions with fibrinogen-coated surfaces, PMN make such close contact with the surface that a portion of the secreted elastase activity is protected from macromolecular protease inhibitors in the surrounding medium. Here we show that the receptor on PMN that mediates this interaction is complement receptor type 3 (CR3; CD11b/CD18), a molecule previously identified as a receptor for the complement protein fragment C3bi. Monoclonal antibodies against CR3 that block the binding of C3bi also block the binding of PMN to fibrinogen-coated surfaces and the formation of a protected compartment. The region of fibrinogen recognized by CR3 lies at the carboxyl terminus of the y chain, since peptides based on this sequence effectively inhibit the binding of PMN to fibrinogen-coated surfaces. These peptides also block the binding of C3bi-coated erythrocytes to CR3, thus indicating that a single binding site is used for binding both C3bi and fibrinogen. Sequence analysis shows strong structural similarity between this region of fibrinogen and other known ligands of CR3. These studies thus indicate that CR3 functions as a receptor not only for C3bi but also for fibrinogen.Several observations suggest that polymorphonuclear leukocytes (PMN) bind specifically to fibrin and may be involved in dissolution of clots. PMN can be found concentrated in fibrin deposits in vivo (1, 2), aggregates of fibrin are phagocytosed by PMN in vivo (1, 2), and radioiodinated aggregates of fibrin bind avidly to PMN in vitro (3). Recently, we have observed that during migration across fibrinogen-coated surfaces in response to chemotactic stimuli, PMN release elastase that degrades the fibrinogen (4). Degradation of surface-bound fibrinogen by elastase is completely inhibited by the low molecular weight inhibitor MeO-Suc-Ala2-ProVal-CH2Cl (AAPVCK) but degradation cannot be completely inhibited by high molecular weight inhibitors such as soybean trypsin inhibitor (SBTI). The elastase appears to be protected from inhibitory proteins in the surrounding medium by sequestration in a compartment formed between the PMN and the substrate similar to the compartment that forms between macrophages and substrates coated with IgG or the complement protein C3 (5). This supposition is strengthened by the observation that fluoresceinated fibrinogen is lost from the substrate in patches that correspond to the margins of spread PMN (S.D.W., unpublished observations).Here we examine the receptors on PMN that mediate adhesion to fibrinogen and the formation of the protected compartment. We find that adhesion to fibrinogen-coated surfaces is inhibited by peptides derived from the carboxyl terminus of the 'y chain of fibrinogen. This is the same region that is recognized by the Peptides. Peptide L10 (LGGAKQAGDV), based on residues 402-411 of the y chain of human fibrinogen, was synthesized by Peninsula Laborato...

Abstract. 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++] This process can be initiated by PMN in response to soluble chemoattractants or by endothelial cells (EC) in response to cytokines such as interleukin 1 OL-1) and tumor necrosis factor (8, 29). Cell surface molecules including the CD11/CD18 complex of proteins on the PMN surface (12) and intercellu-

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.

The mechanism or mechanisms by which polymorphonuclear leukocytes (PMN) penetrate junctions between neighboring endothelial cells (EC) to traverse endothelial barriers remain unresolved. We report that chemoattractant-stimulated PMN induce a coordinate increase in both phosphorylation of serine 19 and threonine 18 of EC myosin regulatory light chains and isometric tension generation by EC monolayers. Unstimulated PMN had no effect on either parameter. These findings, coupled with our previous report (Huang et al., J. Cell Biol. 120: 1371-1380, 1993) that chemoattractant-stimulated PMN cause a rise in EC cytosolic free Ca2+, provide strong presumptive evidence that myosin light chain kinase is the EC enzyme responsible for initiating myosin light chain phosphorylation, EC contraction, and isometric tension generation in response to chemoattractant-stimulated PMN. We suggest that, by inducing phosphorylation of EC cytoskeletal proteins, chemoattractant-stimulated PMN induce EC to open their intercellular junctions, thereby facilitating transendothelial movement of these leukocytes.

Human neutrophil elastase (HNE)' has been implicated in the pathogenesis of a wide variety of human diseases (1). In the past, it was difficult to measure HNE activity in vivo because the enzyme rapidly interacts with its major plasma inhibitor, a, proteinase-inhibitor (2), which complexes and inactivates the free enzyme with an estimated rate constant of association of 6.5 x 10'/M-s (3).Recently, Weitz et al. (4) developed a sensitive assay to measure in vivo HNE activity . This assay makes use of the capacity of HNE to cleave the Val (Aa2l )-Glu (Aa22) bond at the NH2-terminal region of the Aa chain of fibrinogen, thus releasing the fibrinopeptide A-containing fragment Aal-21 . This peptide can be measured in plasma by RIA and its level reflects in vivo HNE activity (4). Using this assay, higher Aa1-21 levels were found in plasma of cigarette smokers than in plasma of nonsmokers (5). Further, individuals with congenital deficiency of a, proteinase-inhibitor had values of plasma Aal-21 considerably higher than those in smokers (4). Increased Aal-21 levels in patients lacking antiproteinase were expected . However, the presence of circulating Aal-21 in individuals with normal plasma concentrations of antiproteinase was puzzling given the rapidity of the interaction between HNE and a, proteinase-inhibitor.In vitro studies of proteolysis by polymorphonuclear leukocytes (PMN) suggest an explanation for the presence of Aal-21 in the plasma of normal individuals. These studies show that enzymes released from stimulated PMN can degrade a variety of susceptible macromolecular substrates in the presence of antiproteinases (6-9), thereby raising the possibility that cell-associated proteinases are more resistant to inhibition than are the free enzymes. To examine this hypothesis, we