Alice R. Johnson

Effect of platelet activating factor on leukocyte-endothelial cell interactions

The proinflammatory effects of platelet activating factor (PAF) that impact upon tissue inflammation were studied in vitro using the adherence of human neutrophils to endothelium and the increase in macromolecule permeability of endothelial monolayers. PAF produced both a time- and dose-dependent increase in neutrophil-endothelial cell adhesion. The adhesion promoting properties observed were primarily due to an effect of PAF on endothelium and not on neutrophils and was independent of endothelial cell cyclooxygenase products. The PAF receptor antagonist kadsurenone inhibited the adhesion response suggesting endothelial surface PAF receptors are involved in these responses. Whereas PAF alone did not alter endothelial cell barrier properties, leukocyte activation (neutrophil and platelets) with PAF produced significant increases in 125I-albumin clearance across endothelial monolayers. These studies suggest that PAF has potent proinflammatory effects and that it can play a significant role in the endothelial response to injury.

Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor.

Neutral endopeptidase 24.11 (NEP/CALLA/ CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemo‐ tactic peptide M‐formylmethionyl‐leucyl‐phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested—interleukin‐1 (IL‐1), IL‐6, and IL‐8, granulocyte colony‐stimulating factor, and granulocyte‐ macrophage colony‐stimulating factor (GM‐CSF) —GM‐ CSF provided the most consistent increase in surface NEP activity. Low concentrations (10−9‐10−7 M) of GM‐ CSF increased NEP activity in a time‐ and concentration‐ dependent manner to more than 225% that of control (phosphate‐buffered saline‐treated) cells. Cytofluorome‐ try of cells stained with a fluorescent antibody to CD10 indicated that GM‐CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM‐CSF on NEP activity was enhanced still further by simultaneous exposure to IL‐1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid up‐ regulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.

Detection and analysis of neutral endopeptidase from tissues with substrate gel electrophoresis

Neutral endopeptidase from human or bovine tissues retains enzymatic activity following electrophoresis and immobilization in polyacrylamide gels. Infiltration of the gel with a fluorogenic substrate permits identification of the active enzyme by fluorescence associated with a distinct protein band. This technique both separates and identifies the enzymatically active species from a crude cell membrane fraction or from partially purified extracts that contain contaminating proteins. Enzymatic activity is quantitated by photographing the fluorescent bands and scanning the negatives with a laser densitometer. Because as little as 25 ng of enzyme can be detected by this method, it could be used where the amount of material is limited.

Inhibition of factor Xa-mediated procoagulant activity of human lung fibroblasts and pleural mesothelial cells.

Extravascular fibrin deposition characterizes diverse forms of lung and pleural injury. Fibrin formation in these compartments is locally potentiated by the assembly and expression of the prothrombinase procoagulant complex (factors Xa, Va and II) at the surface of human lung fibroblasts and pleural mesothelial cells. We sought to identify structural domains on factor Xa that mediate expression of prothrombinase activity by these cells. In order to accomplish this objective, we used panels of monoclonal antibodies (MoAbs) to factor X to block prothrombinase assembly and function on the surface of cultured human lung fibroblasts and pleural mesothelial cells. Of 30 factor X MoAbs that recognized native factors X and Xa, 10 completely inhibited factor Xa function (prothrombin activation), and five others neutralized Xa function without affecting cell-binding, presumably by blocking the prothrombin binding site. Western blots showed that these inhibitory MoAbs reacted with the Xa heavy-chain. One MoAb that recognized the factor Xa light-chain blocked prothrombin activation at the factor Va binding site. Our results indicate that prothrombinase activity at the surface of lung parachymal or pleural cells can be blocked by MoAbs that interact with either the heavy- or light-chain of factors X. Antibodies that neutralize cell surface-expressed prothrombin activation offer a potential means to arrest pericellular fibrin formation in the lung and pleural space.

Genetic Regulation and Expression of Elastase in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a gram-negative organism, is a frequent cause of lung infections in cystic fibrosis (CF) or in debilitated and immunosuppressed individuals. Elastase secreted by P. aeruginosa not only promotes entry of the bacteria through the respiratory tract but also contributes to the pulmonary damage during infection. Chronic Pseudomonas infections are commonly difficult to resolve even with antibiotic therapy. Therefore, the genetic regulation of virulence factors, such as elastase, is of primary concern.