During the last two decades, ample evidence has been obtained that osteoclasts, the multinucleated calcified-matrix resorbing giant cells of bone, which form by fusion of mononuclear precursor cells, are of hematogenous origin. The evidence stems from experiments done in parabionts of labeled animals (1), studies on osteopetrotic animals and humans (2-4), and quail-chick and mouse-quail transplantation experiments (5-7). Essentially, these in vivo studies have shown that osteoclasts derive from bone marrow or other hematopoietic tissues and have indicated mononuclear phagocytes as the most likely candidates for the precursor cells which fuse to form an osteoclast. However, in vitro evidence for the direct transformation of monocytes and/or tissue macrophages into bone-resorbing osteoclasts is still lacking, although it has long been known that cultured macrophages can form foreign-body giant cells in vitro by fusion (8-10).Several in vitro studies have dealt with the destruction of calcified bone matrix by mononuclear phagocytes; for this work, use was made of human peripheral blood monocytes (11, 12) or rodent macrophages (13) in combination with devitalized bone particles. Monocytes and macrophages were able to resorb mineral in a contactmediated fashion, but did not form cells with the morphological characteristics of osteoclasts (13). Recent investigations, however, point to the importance of interactions between bone-forming and -resorbing cells during osteoclastic bone resorption (14). This means that studies done on devitalized bone without viable bone-forming cells might be of limited value with respect to the formation of osteoclasts and osteoclastmediated bone resorption.We recently found (7) that early removal of the perichondrium-periosteum from embryonic mouse long-bone primordia prevents the formation of osteoclasts during organ culture of such bones. In mouse-quail transplantation studies, such stripped bone rudiments are invaded by quail osteoclasts, but mouse osteoclasts are not formed because the stripping procedure has removed the osteoclast precursor cells.In the present study, stripped live bone rudiments were used to assess the capacity of various populations of mononuclear phagocytes to form osteoclasts in vitro. Stripped bone rudiments were co-cultured with embryonic liver as well as with * Present address:

Bacterial infection coincides with migration of leucocytes from the circulation into the bacterium-infected tissue. Recently, we have shown that endothelial cells, upon binding and ingestion of Staphylococcus aureus, exhibit proinflammatory properties including procoagulant activity and increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression on the cell surface, resulting in hyperadhesiveness, mainly for monocytes. The enhanced extravasation of monocytes to bacterium-infected sites is facilitated by the local production of chemotactic factors. From another study we concluded that the locally produced chemokine MCP-1 is important in the recruitment of monocytes to the peritoneal cavity in a model of bacterial peritonitis. In the present study we investigated whether cultured human endothelial cells after infection with bacteria produce and release MCP-1, which in turn stimulates monocyte chemotaxis. We observed that endothelial cells released significant amounts of MCP-1 within 48 h after ingestion of S. aureus. This was dependent on the number and the virulence of the bacteria used to infect the endothelial cells. The kinetics as well as the amount of MCP-1 released by S. aureus-infected endothelial cells differed markedly from that released by endothelial cells upon stimulation with IL-1beta. Supernatant from S. aureus-infected or IL-1beta-stimulated cells promoted monocyte chemotaxis which was almost entirely abrogated in the presence of neutralizing anti-MCP-1 antibody, indicating that most of the chemotactic activity was due to the release of MCP-1 into the supernatant. Our findings support the notion that endothelial cells can actively initiate and sustain an inflammatory response after an encounter with pathogenic microorganisms, without the intervention of macrophage-derived proinflammatory cytokines.

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Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.

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