Platelet-endothelial cell adhesion molecule-1 (PECAM-1)-deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane.

Studies with neutralizing antibodies have indicated roles for platelet-endothelial cell adhesion molecule-1 (PECAM-1) in leukocyte migration through the endothelium and the perivascular basement membrane. Because some of these findings have been contentious, this study aimed to explore the role of PECAM-1 in leukocyte migration by analyzing leukocyte responses in interleukin 1beta (IL-1beta)- and tumor necrosis factor-alpha (TNFalpha)-activated cremasteric venules of PECAM-1-deficient mice using intravital and electron microscopy. Although no differences in levels of leukocyte rolling flux or firm adhesion were observed, a delay in leukocyte transmigration in response to IL-1beta, but not TNFalpha, was detected in PECAM-1-deficient mice. Electron microscopy indicated that this delay occurred at the level of perivascular basement membrane. To address the cytokine specificity of PECAM-1 dependence, in vitro experiments demonstrated that TNFalpha, but not IL-1beta, could induce rapid adhesion of murine neutrophils to protein-coated surfaces, suggesting that TNFalpha elicited leukocyte transmigration in wild-type mice via direct stimulation of leukocytes. In summary, the results suggest a regulatory role for PECAM-1 in leukocyte migration through the perivascular basement membrane, a role that appears to be cytokine-specific and associated with the ability of the cytokine to stimulate rapid neutrophil adhesion.

Interleukin 10 regulates cellular responses in monocyte/endothelial cell co-cultures.

Adhesive interactions between monocytes and vascular endothelial cells increase the expression of the inflammatory genes, tissue factor (TF) and E-selectin, thus contributing to the inflammatory process. In this study, we have shown that these responses could be regulated by the immunomodulatory cytokine interleukin 10 (IL-10). IL-10 reduced TF generation in monocyte/endothelium co-cultures (64. 3 +/- 3.3% reduction, P < 0.01, n = 4) by acting directly on monocytes, whereas IL-4 inhibited TF expression in both monocytes and endothelium. Similarly, IL-10 reduced the induction of endothelial E-selectin by monocytes (100% reduction at 21 h), but had no effect on cytokine-induced E-selectin expression. IL-10 itself was not able to induce E-selectin protein or mRNA in endothelial cells. IL-10 mRNA was detected in monocytes after 6 h co-culture with endothelial cells, and was sustained for up to 30 h. Finally, IL-10 significantly reduced the adhesion of monocytes to endothelium (45% reduction), which may account in part for the inhibitory actions of IL-10. We conclude that IL-10 has an anti-inflammatory effect on monocyte/endothelium interactions, and may itself be produced as a result of such interactions.

Monocytes stimulate expression of the Bcl-2 family member, A1, in endothelial cells and confer protection against apoptosis.

We have investigated the molecular mechanisms underlying the ability of peripheral blood monocytes to block apoptosis induction in endothelial cells. Monocytes stimulated the expression of the bcl-2 homologue A1 in serum-starved endothelial cells after 6 h of coincubation, with elevated A1 levels persisting for up to 21 h. IL-1 and TNF also stimulated A1 expression at 6 h, but A1 transcript levels fell by 21 h. Direct cellular contact with monocytes was required for stimulation of A1 mRNA in endothelial cells. Stimulation of endothelial cell A1 mRNA by monocytes was not inhibited by anti-beta2 integrin Abs, but anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) mAb reduced A1 transcript levels at 21 h. Studies employing either TNF on its own, or anti-TNF in endothelium/monocyte cocultures showed that TNF plays a role in the early (6-h) stimulation of A1, but is less important for the sustained elevation of A1 levels at 21 h. Serum-starved endothelial cells demonstrated increased survival and decreased apoptosis after coculture with monocytes. IL-10 reduced A1 mRNA expression in, as well as survival of, endothelial cells that were cocultured with monocytes. In comparison with A1, Bcl-2 was expressed at low levels and was up-regulated by monocytes only at 21 h, while neither Bax nor Bcl-xL levels were altered by monocytes. The interaction of monocytes with endothelium during the course of an inflammatory reaction may provide survival signals to endothelial cells.

Monocytes induce E-selectin gene expression in endothelial cells: role of CD11/CD18 and extracellular matrix proteins.

E-selectin is an endothelium-specific inducible adhesion molecule which binds several inflammatory cell types, including neutrophils, monocytes, natural killer cells and a subset of memory T cells. E-selectin is important in the initial rolling interaction of these cells on inflamed endothelium. The transient kinetics of E-selectin induction in vitro contrast with in vivo observations of prolonged expression of this adhesion molecule in chronic inflammation. This raises the possibility that signals generated within inflammatory tissues are more complex than the agonists used to activate endothelial cells in vitro. We investigated whether adhesive interactions with extravasating monocytes are able to provide activating signals that can induce E-selectin expression on endothelium, and prolong the response to cytokine stimulation. We report that co-culture with monocytes led to transcriptional activation of the E-selectin gene in endothelial cells, and marked enhancement of the response to substimulatory concentrations of interleukin-1. In addition, the presence of monocytes resulted in prolonged up-regulation of E-selectin. Induction of E-selectin by monocytes was inhibited when cell contact between monocytes and endothelium was prevented (80 +/- 8% inhibition, p < 0.001, n = 4). Monoclonal antibody (mAb) against tumor necrosis factor (TNF) was able to abolish 57.2 +/- 9.7% of the response (p < 0.01, n = 4). The ability of adherent monocytes to induce sustained E-selectin expression in endothelial cells could not be reproduced either by supernatants harvested from monocytes cultured for 18 h, or by maximal concentrations of TNF. The induction of E-selectin in monocyte/endothelium co-cultures was inhibited by mAb to CD11b, but not by those directed against VLA-4 or L-selectin. Extracellular matrix molecules may also play a role in adhesion-dependent cellular activation, as inclusion of soluble collagen type I led to significant reduction in E-selectin expression in monocyte/endothelium co-cultures. We conclude that adhesive interactions between monocytes and endothelial cells provide a source of signals which influence the activation state of the endothelium, and consequently, the continued influx of inflammatory cells.

Induction of tissue factor expression in human monocyte/endothelium cocultures.

Induction of tissue factor (TF) expression on monocytes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 x 10(4)/well) and endothelial cells (2 x 10(4)/well) produced 35.3 +/- 8.5 mU of PCA/well, representing a 5-fold increase over the combined PCA of each cell type cultured alone (7.1 +/- 1.5 mU, n = 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN-gamma. When endothelium was prestimulated with 500 U/ml IFN-gamma there was 142 +/- 11% increase over unstimulated cocultures (n = 5, P < 0.01). TF induction was inhibited by 32 +/- 6% in the presence of anti-ICAM-1 mAb (n = 5, P < 0.01). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.