Mechanisms of Endotoxin Tolerance in Human Intestinal Microvascular Endothelial Cells

Abstract

Lipopolysaccharide (endotoxin) tolerance is well described in monocytes and macrophages, but is less well characterized in endothelial cells. Because intestinal microvascular endothelial cells exhibit a strong immune response to LPS challenge and play a critical regulatory role in gut inflammation, we sought to characterize the activation response of these cells to repeated LPS exposure. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were stimulated with LPS over 6–60 h and activation was assessed using U937 leukocyte adhesion, expression of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, manganese superoxide dismutase, HLA-DR, and CD86. Effect of repeat LPS stimulation on HIMEC NF-κB and mitogen-activated protein kinase (MAPK) activation, generation of superoxide anion, and Toll-like receptor 4 expression was characterized. LPS pretreatment of HIMEC for 24–48 h significantly decreased leukocyte adhesion after subsequent LPS stimulation. LPS pretreatment inhibited expression of E-selectin, VCAM-1, IL-6, and CD86, while ICAM-1, IL-8, and HLA-DR were not altered. Manganese superoxide dismutase expression increased with repeated LPS stimulation, with a reduction in intracellular superoxide. NF-κB activation was transiently inhibited by LPS pretreatment for 6 h, but not at later time points. In contrast, p44/42 MAPK, p38 MAPK, and c-Jun N-terminal kinase activation demonstrated inhibition by LPS pretreatment 24 or 48 h prior. Toll-like receptor 4 expression on HIMEC was not altered by LPS. HIMEC exhibit endotoxin tolerance after repeat LPS exposure in vitro, characterized by diminished activation and intracellular superoxide anion concentration, and reduced leukocyte adhesion. HIMEC possess specific mechanisms of immunoregulatory hyporesponsiveness to repeated LPS exposure.

Human Intestinal Microvascular Endothelial Cells Express Toll-Like Receptor 5: A Binding Partner for Bacterial Flagellin

“…Interestingly, tolerance toward the TLR5 stimulus has been demonstrated in human monocytes and THP-1 cells, and this tolerance mechanism was shown to be independent of the TLR5 surface expression levels (28). In homology, tolerance toward bacterial LPS, the stimulus for TLR4, was similarly shown for HIMEC in vitro (12), further suggesting that human microvascular endothelial cells, a "nonimmune" cell population, possess features characteristic of immune cells involved in the pathogenesis of intestinal inflammatory disease. Defining mechanisms of endotoxin tolerance in various gut cell populations is presently an area of intense investigation in mucosal immunity and inflammatory bowel disease research.…”

“…Infection of intestinal epithelial cells with Salmonella thereby leads to an active transport of flagellin to the subepithelial compartment and subsequent binding to basolaterally expressed TLR5 in vitro, resulting in phosphorylation and nuclear translocation of the transcription factor NF-B in intestinal epithelial cells (9,11). Recent work has demonstrated that gut-specific endothelial cells that exist in proximity to the enteric flora express TLR4, which plays an integral role in LPS-mediated activation and tolerance (12). In light of the notion that intestinal Salmonella infection elicits endothelial adhesion molecule expression and that bacterial flagellin was found to be present in the mucosal layer, we hypothesized that human intestinal microvascular endothelial cells (HIMEC), representing the barrier between circulating immune cells and the intestinal mucosa, functionally express TLR5 and are readily activated by its ligand, translocated bacterial flagellin.…”

Immune Regulation by Microvascular Endothelial Cells: Directing Innate and Adaptive Immunity, Coagulation, and Inflammation

“…Neutrophil accumulation appears to depend on TLR4 expression by ECs rather than leukocytes as sequestration of neutrophils in the lung is deeply impaired in endothelial TLR4 Ϫ/Ϫ mice (31). The latter observation contrasts with the significant decrease of leukocyte binding caused by LPS in human intestinal microvascular ECs, perhaps reflecting a tolerance of ECs to high levels of endotoxin to which they constantly exposed in the gut microenvironment (32). Finally, LPS can directly initiate angiogenesis through TNFR-associated factor 6-dependent signaling pathways (33).…”

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

“…1 A, iii and iv). We next purified mouse intestinal microvascular endothelial cells (MIMECs) according to the technique of Ogawa et al (10) from wild-type, TLR4 −/− , and TLR4 Δendoth mice, along with peritoneal macrophages, and evaluated the expression of TLR4 on single cell suspensions by RT-PCR and flow cytometry. As shown in Fig.…”