The role of various surfaces in the phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) was studied. Uptake of both opsonized and unopsonized staphylococci on the surface of a monolayer of human venous endothelial cells was compared with uptake on an inert plastic surface, with an assay that uses radiolabeled bacteria. Uptake of unopsonized S. aureus was threefold higher on the endothelial cell surface than on the plastic surface and was followed by efficient killing of the phagocytosed staphylococci. Uptake of unopsonized S. aureus on endothelial cells was not inhibited by treatment of the PMN with pronase or 2-deoxy-D-glucose and was only partially inhibited by cytochalasin B treatment of the PMN. The supporting effect of endothelial cells on the phagocytosis of unopsonized S. aureus was not due to opsonization of the bacteria by immunoglobulin or complement from the endothelial cell surface, nor to coating with fibronectin.
dissolved in dimethyl sulfoxide (J. T. Baker Chemicals, Deventer, The Netherlands); CDNB and NDGA were dissolved in ethanol, and all reagents were further diluted to the 1447 on July 31, 2020 by guest http://iai.asm.org/ Downloaded from
Phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN) on the surface of endothelial cells is accompanied by adherence of the PMN to the endothelial surface and detachment of the endothelial cells from the culture monolayer. We studied the role of the leucocyte adherence-related glycoproteins (Leu-CAM: Mo1/LFA-1/150,95 or CD11a-c-CD18 complex) in these processes. Phagocytosis of S. aureus induced increased expression of the common beta chain (CD18) of Leu-CAM as demonstrated by flow cytometric analysis of PMN treated with a monoclonal antibody (MoAb) (CLB-LFA-1/1) directed against CD18 and fluorescein isothiocyanate (FITC)-conjugated anti-MoAb. This same MoAb also inhibited the increased adherence of the PMN to the endothelial cells which occurs during phagocytosis. Blocking of adherence during phagocytosis with MoAb CLT-LFA-1/1 had no effect on the detaching activity of the PMN on the endothelial cells. We conclude that adherence of PMN to endothelial cells during phagocytosis of S. aureus is mediated by the Leu-CAM complex. Adherence through the Leu-CAM, however, is not necessary for endothelial damage by the phagocytosing PMN.
When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.
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