Mast cells are immune cells of the myeloid lineage and are present in connective tissues throughout the body. The activation and degranulation of mast cells significantly modulates many aspects of physiological and pathological conditions in various settings. With respect to normal physiological functions, mast cells are known to regulate vasodilation, vascular homeostasis, innate and adaptive immune responses, angiogenesis, and venom detoxification. On the other hand, mast cells have also been implicated in the pathophysiology of many diseases, including allergy, asthma, anaphylaxis, gastrointestinal disorders, many types of malignancies, and cardiovascular diseases. This review summarizes the current understanding of the role of mast cells in many pathophysiological conditions.

SUMMARYHistamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-jB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.

MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2⅐LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2⅐LPS complex. Finally we show that the MD-2⅐LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2⅐LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4 ؉ /MD-2 ؊ cells in the inflammatory microenvironment. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, stimulates an exceptionally potent innate immune response in mammals that can result in septic shock and death (1). The LPS response is mediated by four proteins (2): LPS binding protein extracts single molecules from LPS micelles and transfers them to CD14, a glycosylphosphatidylinositol-anchored cell surface receptor that also exists as a serum protein. In turn, the CD14⅐LPS complex activates two proteins that comprise the essential signaling complex of the LPS response, Toll-like receptor 4 (TLR4) and MD-2 (3-6). TLR4 is a type I integral membrane glycoprotein and is one of 10 TLR paralogs that activate NF-B, mitogen-activated protein kinases, and other transducers of inflammatory signals in response to pathogen-specific structural motifs. MD-2, a small cysteine-rich glycoprotein, binds to the ectodomain of TLR4 (7) in the endoplasmic reticulum and then transits to the cell surface in an active TLR4⅐MD-2 complex. However, MD-2 is also secreted into the medium as a soluble, active protein (sMD-2) by primary cells such as immature dendritic cells (iDC), and by MD-2-transfected cell lines (8). The activity of sMD-2 was shown by its ability to bind to TLR4 and confer LPS responsiveness to cells that express TLR4 but lack MD-2 (8, 9). Forward genetic and gene deletion studies have demonstrated that both MD-2 and TLR4 are required for normal responsiveness to LPS in vitro and in vivo (3)(4)(5)10).Analyses of species specificity differences for various forms of LPS provide strong evidence that LPS interacts directly with the TLR4⅐MD-2 complex (11-16). However, the molecular events leading to LPS binding and TLR4 activation are only partially understood. Photoaffinity labeling (17) and binding (9,18,19) studies have shown that LPS binds directly to MD-2 and TLR4, and that binding of LPS to TLR4...

Human endothelial cells respond to extracellular proteases, endotoxin (lipopolysaccharide, LPS), and inflammatory cytokines. Endothelial cells express several protease-activated receptors (PAR), including the thrombin-activated receptors PAR-1 and PAR-3 and a thrombin-independent, protease-activated receptor, PAR-2. To examine the potential cooperation between PAR and inflammatory stimuli, we investigated the effects of the PAR-1 agonist peptide Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and PAR-2 agonist peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro with SFLLRN or SLIGKV in the presence and absence of LPS or tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) levels in the culture supernatants were assayed. Both SFLLRN and SLIGKV induced detectable levels of IL-6 production in a dose-dependent fashion, with the PAR-1 receptor agonist being more potent. In the presence of all stimulatory concentrations of LPS or TNF-alpha tested, both peptides were found to further enhance IL-6 production. The effects of SFLLRN and SLIGKV were specific, as related peptides with identical amino acid compositions, but lacking in consensus sequences, were biologically inactive either alone or in the presence of LPS. Both the direct and the amplifying effects of PAR agonist peptides on IL-6 production were pertussis toxin sensitive and caused an increase in the intracellular levels of calcium, implicating G-proteins and calcium mobilization in these pathways. Furthermore, the amplifying effect of LPS or TNF-alpha on PAR-mediated cytokine production was associated with corresponding increases in nuclear NF-kappaB proteins. The results demonstrate significant potentiation of PAR-induced signaling by LPS and TNF-alpha and indicate the potential cooperation of proteases and inflammatory stimuli in amplifying vascular inflammation.

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