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The extravasation of leukocytes from the lumen of the vessel to a site of inflammation requires specific binding events. The interaction of leukocytes with endothelium, via specific receptors, may provide intracellular signals that activate extravasating cells. In the present study, we have investigated the production of chemokines, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) during monocyte: endothelial cell interactions. Both unstimulated and interferon-gamma (IFN-gamma)-prestimulated human umbilical vein endothelial cells (HUVEC) produced low constitutive levels of IL-8 and MCP-1. The addition of enriched monocytes with unstimulated HUVEC resulted in synergistic increases in production of both IL-8 and MCP-1. Monocytes cultured with IFN-gamma-preactivated HUVECs demonstrated little additional increase in IL-8 and MCP-1 production in coculture assays compared with unstimulated HUVEC. Northern blot analysis paralleled the protein data, demonstrating upregulated expression of IL-8 and MCP-1 mRNA in stimulated and unstimulated coculture assays. Culture of enriched monocytes and endothelial cells in transwells demonstrated no increases in IL-8 or MCP-1, indicating the necessity for cellular contact for chemokine production. In previous investigations, we have demonstrated that increased monocyte-derived MIP-1 alpha production was induced by intracellular adhesion molecule-1 (ICAM-1) interactions on activated HUVECs. In contrast, addition of anti-ICAM-1 monoclonal antibodies (MoAbs) did not diminish the production of IL-8 and MCP-1 in the present study. Furthermore, neither antibodies to IL-1 nor tumor necrosis factor (TNF) diminished the production of either IL-8 or MCP- 1. However, when soluble matrix proteins were added to the coculture to block cellular interactions, the chemokine protein and mRNA levels were significantly decreased. IL-8 production was decreased by both soluble collagen and fibronectin, whereas MCP-1 was decreased by only soluble collagen, suggesting differential activation pathways. These results indicate that IL-8 and MCP-1 production are increased during monocyte and endothelial cell interactions in part due to matrix protein binding mechanisms. This mechanism may serve a role in cell activation, production of chemokines, as well as extravasation and recruitment of additional leukocytes during inflammatory responses.

The extravasation of leukocytes from the lumen of the vessel to a site of inflammation initially requires a specific binding event followed by migration of the cells through the endothelial cell layer into the inflammatory foci. The interaction of leukocytes with the endothelium via specific receptors may provide intracellular signals that activate the cells. In the present study we have investigated the production of MIP-1 alpha, a mononuclear cell chemotactic protein, during monocyte:endothelial cell interactions. Neither unstimulated nor interferon (IFN)-stimulated human umbilical vein endothelial cells (HUVECs) produced substantial MIP-1 alpha protein. However, the addition of enriched monocyte populations with unstimulated HUVECs resulted in the production of MIP-1 alpha. Monocytes cultured with IFN-gamma-activated HUVECs showed an additional increase in MIP-1 alpha production. Immunohistochemical analysis demonstrated that the monocyte was the cellular source of MIP-1 alpha production in this coculture system. The mechanism of MIP-1 alpha expression was further assessed by determining the role of adhesion molecules in the regulation of MIP-1 alpha production during monocyte:HUVEC interactions. To attenuate the increased production of MIP-1 alpha by the monocyte:HUVEC interaction, anti-adhesion molecule monoclonal antibodies (MoAbs) were added to the cultures. Addition of anti-ICAM-1 neutralizing MoAbs significantly inhibited the production of MIP-1 alpha, whereas neutralizing anti-VCAM-1 MoAbs failed to block MIP-1 alpha production. Furthermore, MIP-1 alpha production was induced in monocytes cultured on ICAM-1-coated plates. These results indicate an intimate relationship between leukocyte-endothelial cells, adhesion molecule, and the expression of the monocyte-derived chemokine MIP-1 alpha during cellular adhesion. This mechanism may serve an important role in cell activation and recruitment of leukocytes during the initiation of an inflammatory response.