Pulmonary surfactant protein A enhances the host-defense mechanism of rat alveolar macrophages.

The effects of surfactant, surfactant lipids, and surfactant protein A (SP-A) on the surface phagocytosis of [3H]thymidine-labeled Staphylococcus aureus (SAE) by rat alveolar macrophages were studied. Alveolar macrophages only ingest SAE when the bacteria are opsonized with rat serum prior to incubation with alveolar macrophages. Preincubation or "opsonization" of the bacteria with surfactant did not result in phagocytosis by the macrophages. However, preincubation of the macrophages with surfactant increased the phagocytosis of rat serum-opsonized bacteria by approximately 70% when compared to the control macrophages. The factor present in surfactant causing the stimulation of the phagocytosis is probably SP-A. Preincubation of macrophages with human SP-A enhanced the phagocytosis to the same extent as whole surfactant, whereas preincubation with surfactant lipids had no effect on the phagocytosis. The SP-A-induced enhancement of the phagocytosis is time, temperature, and concentration dependent. Phagocytosis of opsonized SAE by alveolar macrophages was maximal after 15 min of incubation and at an SP-A concentration of 1 micrograms/ml. No phagocytosis occurred at 0 degrees C. In addition, whole surfactant and SP-A induce a lucigenin-dependent chemiluminescence response in alveolar macrophages. The chemiluminescence response is initiated after 15 min of incubation and reaches a maximum after 30 min. The concentration of SP-A needed for an optimal response is in the same order of magnitude as the concentration needed for maximal enhancement of the phagocytosis of SAE by alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Internalization of antithrombin III by cultured human endothelial cells and its subcellular localization.

The presence of antithrombin III was demonstrated in cultured human endothelial cells derived from the umbilical cord by using immunofluorescence, immunoelectron microscopy studies, and an enzyme-linked immunosorbent assay (ELISA) specific for antithrombin III. Immunofluorescence studies indicated the presence of antithrombin III in granule-like structures in the endothelial cell. Immunoelectron microscopy studies performed with ultrathin cryosections of endothelial cells showed a colocalization of antithrombin III and a lysosomal marker protein in low electron dense organelles, indicating a lysosomal localization of antithrombin III. By using the ELISA, 77 +/- 40 ng (n = 8) antithrombin III was quantitated in 10(6) endothelial cells. Immunoprecipitation studies performed with metabolically labeled cultured human endothelial cells indicated that antithrombin III was not synthesized by the cells. Endothelial cells cultured in antithrombin III-depleted human serum did not contain antithrombin III, as was measured by ELISA. Internalization studies performed with radiolabeled purified antithrombin III and antithrombin III-thrombin complexes indicated that endothelial cells internalize antithrombin III when it is complexed to thrombin. Antithrombin III alone was not internalized by the endothelial cells.

High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization.

The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

The binding of high molecular weight kininogen to cultured human endothelial cells.

Binding of 125I-labeled high molecular weight kininogen (high Mr kininogen) to cultured human endothelial cells derived from the umbilical cord was demonstrated. The binding was time-dependent, specific, and saturable and required the presence of zinc ions. Maximal binding of 125I-labeled high Mr kininogen was observed at 50 microM Zn2+. Calcium ions inhibited the Zn2+-dependent binding of 125I-labeled high Mr kininogen to endothelial cells. In the presence of 3 mM CaCl2 the total binding of 125I-labeled high Mr kininogen was significantly decreased, and a concentration of 100 microM Zn2+ was then required for the binding of 125I-labeled high Mr kininogen to the cells. Higher calcium concentrations did not further decrease the binding of 125I-labeled high Mr kininogen. Analysis of the binding data by the LIGAND computer program indicated 3.2 X 10(6) binding sites per cell for high Mr kininogen with an apparent Kd of 35 nM at 50 microM ZnCl2 and 1 mM CaCl2. Binding of high Mr kininogen also occurred at physiological plasma Zn2+ concentrations. Saturation of the high Mr kininogen binding sites at 25 microM ZnCl2 occurred at 80 nM of high Mr kininogen with 2.8 X 10(6) molecules of high Mr kininogen bound per cell. At 10 microM ZnCl2, the high Mr kininogen binding sites appeared to be saturated at 130 nM with 1.6 X 10(6) molecules bound per cell. In addition it was demonstrated that endothelial cells internalize 125I-labeled high Mr kininogen, since 125I-labeled high Mr kininogen was detected in solubilized endothelial cells after the cell-bound 125I-labeled high Mr kininogen had been removed with dextran sulfate.

Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation.

To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal individuals bound 650 125I-2.28 molecules/platelet, whereas thrombin-activated platelets bound 12,600 molecules/platelet with high affinity (4.6 nmol/L). Immunoelectrophoretic analysis revealed that 2.28 reacted with a 53,000-mol wt protein. Immunocytochemistry showed that the antigen is located in a special subclass of platelet granules in unstimulated platelets and is exposed on the surface of thrombin-activated platelets. Double-labeling studies with immunogold labels disclosed simultaneous localization of 2.28 binding sites and cathepsin D in the same granules both in megakaryocytes and endothelial cells, thereby indicating that the antigen may be localized in lysosomes. By using flow cytofluorometry, in vivo platelet activation was studied in patients undergoing cardiac surgery with cardiopulmonary bypass. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed after extracorporeal perfusion. The percentage of 2.28-positive platelets in the circulation was 3.9% +/- 2.7% (SD) in controls (n = 20), 5.5% +/- 3.0% in patients (n = 10) before cardiopulmonary bypass surgery, 24.6% +/- 13.5% after the bypass, and 8.5% in two patients with acute deep venous thrombosis. These data indicate that 2.28 may serve as a useful probe of in vitro and in vivo platelet activation.