Kottarappat N. Dileepan

Interleukin-6 Production by Endothelial Cells via Stimulation of Protease-Activated Receptors Is Amplified by Endotoxin and Tumor Necrosis Factor-α

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.

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

Histamine 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‐κB 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.

Mast Cell: A Multi-Functional Master Cell

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.

Histamine Directly and Synergistically with Lipopolysaccharide Stimulates Cyclooxygenase-2 Expression and Prostaglandin I2 and E2 Production in Human Coronary Artery Endothelial Cells

Although histamine plays an essential role in inflammation, its influence on cyclooxygenases (COX) and prostanoid homeostasis is not well understood. In this study, we investigated the effects of histamine on the expression of COX-1 and COX-2 and determined their contribution to the production of PGE2, prostacyclin (PGI2), and thromboxane A2 in human coronary artery endothelial cells (HCAEC). Incubation of HCAEC monolayers with histamine resulted in marked increases in the expression of COX-2 and production of PGI2 and PGE2 with no significant change in the expression of COX-1. Histamine-induced increases in PGI2 and PGE2 production were due to increased expression and function of COX-2 because gene silencing by small interfering RNA or inhibition of the catalytic activity by a COX-2 inhibitor blocked prostanoid production. The effects of histamine on COX-2 expression and prostanoid production were mediated through H1 receptors. In addition to the direct effect, histamine was found to amplify LPS-stimulated COX-2 expression and PGE2 and PGI2 production. In contrast, histamine did not stimulate thromboxane A2 production in resting or LPS-activated HCAEC. Histamine-induced increases in the production of PGE2 and PGI2 were associated with increased expression of mRNA encoding PGE2 and PGI2 synthases. The physiological role of histamine on the regulation of COX-2 expression in the vasculature is indicated by the findings that the expression of COX-2 mRNA, but not COX-1 mRNA, was markedly reduced in the aortic tissues of histidine decarboxylase null mice. Thus, histamine plays an important role in the regulation of COX-2 expression and prostanoid homeostasis in vascular endothelium.

Mast cell granules inhibit macrophage-mediated lysis of mastocytoma cells (P815) and nitric oxide production

The effects of mast cell granules (MCGs) on macrophage‐mediated lysis of P815 mastocytoma cells and nitric oxide (NO) production were studied. Murine peritoneal macrophages exhibited tumor cell killing and NO production only when activated with lipopolysaccha‐ ride (LPS) or interferon‐7 (IFN‐γ). Coincubation of macrophages with MCGs during LPS activation dose‐depen‐ dently inhibited macrophage‐mediated tumor cell lysis. The MCG effect was not due to inactivation or removal of LPS by MCG. The inhibitory effect was also not due to histamine or serotonin present in the MCGs. The granules were not toxic to macrophages or P815 mastocytoma cells. The effect of MCGs on macrophage‐mediated tumor cell killing was evident whether MCGs were added before or after a 4‐h exposure of macrophages to LPS. However, the inhibitory effect was not seen if MCGs were added after macrophages had been exposed to LPS for 24 h. To assess whether MCGs could inhibit a non‐LPS trigger, MCGs were tested on macrophages activated with IFN‐γ. In these experiments, MCGs dose‐dependently inhibited macrophage‐mediated tumor cell killing induced by IFN‐ γ, LPS, or IFN‐γ plus LPS. Furthermore, in parallel experiments, MCGs significantly inhibited macrophage NO production induced by LPS, IFN‐γ, or IFN‐γ plus LPS. Pretreatment of MCGs with diisopropylfluorophosphate, a serine protease inhibitor, only partially abrogated the effects of MCGs. The results demonstrate that MCGs inhibit both LPS‐ and IFN‐γ‐induced macrophage killing of P815 cells and the inhibition is associated with the decrease of NO production.

Endotoxin and Mast Cell Granule Proteases Synergistically Activate Human Coronary Artery Endothelial Cells to Generate Interleukin-6 and Interleukin-8

Mast cells (MC) are strategically located along blood vessels and, on activation, exocytose granules that contain many vasoactive mediators. Endothelial cell (EC) activation, which includes the production of such cytokines as interleukin-6 (IL-6) and IL-8, is a key event in vascular inflammation. In this study, the effects of purified MC granules (MCG) on the production of IL-6 and IL-8 by human coronary artery EC (HCAEC) were examined. HCAEC were cocultured with MCG in the presence or absence of lipopolysaccharide (LPS), and IL-6 and IL-8 levels in the culture medium were assayed by ELISA. Unactivated HCAEC produced only low levels of IL-6 or IL-8, and the addition of MCG alone resulted in little or no increase in production of these cytokines. LPS-activated HCAEC produced significant amounts of IL-6 and IL-8 in a dose-dependent and time-dependent fashion, which was amplified 2-3-fold by MCG at EC/MC ratios of 16:1-2:1. Scanning electron microscopy revealed direct communication between MCG and HCAEC. The enhancement of IL-6 and IL-8 production by MCG was abrogated when MCG were pretreated with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). These results demonstrate that MCG interaction with HCAEC causes amplification of endotoxin-stimulated cytokine production via serine proteases present in MCG. The synergistic activation of EC by endotoxin and MCG proteases emphasizes the role of MC in amplifying vascular inflammation.

Mast cell deficiency attenuates progression of atherosclerosis and hepatic steatosis in apolipoprotein E-null mice

Mast cells are important cells of the immune system and are recognized as participants in the pathogenesis of atherosclerosis. In this study, we evaluated the role of mast cells on the progression of atherosclerosis and hepatic steatosis using the apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)/mast cell-deficient (Kit(W-sh/W-sh)) mouse models maintained on a high-fat diet. The en face analyses of aortas showed a marked reduction in plaque coverage in ApoE(-/-)/Kit(W-sh/W-sh) compared with ApoE(-/-) after a 6-mo regimen with no significant change noted after 3 mo. Quantification of intima/media thickness on hematoxylin and eosin-stained histological cross sections of the aortic arch revealed no significant difference between ApoE(-/-) and ApoE(-/-)/Kit(W-sh/W-sh) mice. The high-fat regimen did not induce atherosclerosis in either Kit(W-sh/W-sh) or wild-type mice. Mast cells with indications of degranulation were seen only in the aortic walls and heart of ApoE(-/-) mice. Compared with ApoE(-/-) mice, the serum levels of total cholesterol, low-density lipoprotein and high-density lipoprotein were decreased by 50% in ApoE(-/-)/Kit(W-sh/W-sh) mice, whereas no appreciable differences were noted in serum levels of triglycerides or very low density lipoprotein. ApoE(-/-)/Kit(W-sh/W-sh) mice developed significantly less hepatic steatosis than ApoE(-/-) mice after the 3-mo regimen. The analysis of Th1/Th2/Th17 cytokine profile in the sera revealed significant reduction of interleukin (IL)-6 and IL-10 in ApoE(-/-)/Kit(W-sh/W-sh) mice compared with ApoE(-/-) mice. The assessment of systemic generation of thromboxane A(2) (TXA(2)) and prostaglandin I(2) (PGI(2)) revealed significant decrease in the production of PGI(2) in ApoE(-/-)/Kit(W-sh/W-sh) mice with no change in TXA(2). The decrease in PGI(2) production was found to be associated with reduced levels of cyclooxygenase-2 mRNA in the aortic tissues. A significant reduction in T-lymphocytes and macrophages was noted in the atheromas of the ApoE(-/-)/Kit(W-sh/W-sh) mice. These results demonstrate the direct involvement of mast cells in the progression of atherosclerosis and hepatic steatosis.

Poloxamer 407-induced atherosclerosis in mice appears to be due to lipid derangements and not due to its direct effects on endothelial cells and macrophages.

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functions in vitro, and its in vivo effects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 microM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 microM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-C in vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation.

Constitutive Expression of Functionally Active Protease-Activated Receptors 1 and 2 in Human Conjunctival Epithelial Cells

Protease-activated receptors (PARs) are G-protein-coupled receptors which initiate inflammatory responses when activated by specific serine proteases. This study was conducted to examine whether human conjunctival epithelial cells (HCECs) express functionally active PAR1 and PAR2 using Chang conjunctival epithelial cells as in vitro model. We performed RT-PCR and immunofluorescence analyses to determine the expression of PAR1 and PAR2, and monitored the production of IL-6 after activating HCECs with PAR1 activating agents (thrombin or TFLLRN) or PAR2 activating agents (tryptase, trypsin, or SLIGKV). The results show that HCECs constitutively express PAR1 and PAR2 mRNA and proteins, and produce significant amounts of IL-6 when incubated with specific PAR-activating enzymes or agonist peptides. Thrombin- and tryptase-induced HCEC activation was blocked by PAR1 and PAR2 neutralizing antibodies, respectively, and by specific enzyme inhibitors. The constitutive expression of PAR1 and PAR2, and their activation by thrombin and tryptase, respectively, may have important implications in ocular inflammation.

Mast cell granules potentiate endotoxin-induced interleukin-6 production by endothelial cells.

Mast cells are constituent cells of vascular tissue and their numbers are increased in atherosclerotic vessels. To gain insight into the role of mast cells in vascular inflammation, the effect of mast cell granules (MCG) on endothelial cell production of interleukin‐6 (IL‐6) was examined. Human umbilical vein endothelial cells (HUVEC) were cultured with lipopolysaccharide (LPS) in the presence or absence of rat peritoneal MCG and IL‐6 production was assayed by enzymelinked immunosorbent assay. The interaction of MCG with HUVEC in culture was examined by electron microscopy (EM). The EM studies revealed that MCG are internalized by HUVEC and appear intact even after 24 h in culture. Unactivated HUVEC produced little or no IL‐6 either in the presence or absence of MCG. Treatment of HUVEC with LPS stimulated IL‐6 production in a dose‐ and time‐dependent fashion. Addition of MCG to LPS‐activated HUVEC resulted in the potentiation of IL‐6 production at all LPS doses. MCG‐induced enhancement of IL‐6 production was evident even at a mast cell‐to‐endothelial cell ratio of 1:32. The enhancement of IL‐6 production by MCG was also seen when tumor necrosis factor α was used as an activator. Although potentiation was evident when MCG were added 6 h before or after LPS stimulation, the maximum effect was noted when MCG and LPS were added simultaneously. MCG‐mediated enhancement of IL‐6 production was abrogated by pretreating MCG with protease inhibitors. Although MCG proteases potentiate IL‐6 production by HUVEC, they do not degrade secreted IL‐6. These results demonstrate that MCG interact with endothelial cells and modulate the production of an important inflammatory cytokine. J. Leukoc. Biol. 62: 210–216; 1997.