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Background: Kawasaki’s disease is a pediatric medium vessel vasculitis causing coronary arteritis and potentially coronary artery aneurysms. Higher systemic levels of Interleukin-6 (IL-6) in the first week of disease has been shown to be associated with a higher risk of coronary artery aneurysms (1, 2). Mice injected with Candida albicans water-soluble fraction (CAWS) develop coronary and aortic arteritis like that seen in Kawasaki’s disease. Our prior work has demonstrated that mast cell degranulation can inhibit lipopolysaccharide (LPS)-induced IL-6 gene expression in the aorta and systemic IL-6 production (3). Objectives: The objective of this study was to determine if mast cells play a role in the IL-6 homeostasis in an established mouse model of Kawasaki’s disease (CAWS model). Methods: 8-10-week-old male wild type controls (WT) and kitW-sh/W-sh mice (mast cell deficient, MCD) of C57Bl/6 background were randomly distributed into four groups (WT-PBS, WT-CAWS, MCD-PBS, MCD-CAWS). They were either injected intraperitoneally with PBS or CAWS (2 mg/mouse) daily for a period of 5 days. Eight animals from each group were sacrificed at either 7, 14 or 30 days after the last injection and blood, aorta and heart specimens were harvested. A pathologist (OT), blinded to treatment groups, examined aortic root and adjacent myocardium specimens and assigned a score for the degree of inflammation (0-6). Results: Seven MCD-CAWS mice died unexpectedly within 24 hours of the first CAWS injection. Autopsies reviewed marked liver sinusoidal dilation in all and patchy necrosis of the liver in 4. Similar, liver findings were seen in 75% of MCD-CAWS after 7 days, but not in other groups. MCD-CAWS mice had higher systemic IL-6 compared to WT-CAWS at both 7 days (208± 21 vs 152 ± 12 pg/ml; p Conclusion: Mast cell deficiency resulted in higher systemic levels of IL-6, TNFα and INFγ in the CAWS mouse model of Kawasaki’s disease. Similarly, mast cell deficiency resulted in more intense inflammation at the root of the aorta in this model. These results support the novel concept that mast cells play a protective role in reducing the initial systemic inflammatory response in Kawasaki’s disease. References: [1] Ueno Y, Takano N, Kanegane H, Yokoi T, Yachie A, Miyawaki T, et al. The acute phase nature of interleukin 6: studies in Kawasaki disease and other febrile illnesses. Clinical and experimental immunology. 1989;76(3):337-42. [2] Chow YM, Lin CY, Hwang B. Serum and urinary interleukin-6 (IL-6) levels as predicting factors of Kawasaki disease activity. Zhonghua Minguo xiao er ke yi xue hui za zhi [Journal] Zhonghua Minguo xiao er ke yi xue hui. 1993;34(2):77-83. [3] Springer J, Raveendran V, Smith D, Maz M, Dileepan K. OP0138 Novel Role of Mast Cells in the Regulation of Large Vessel Vasculitis. EULAR Paris, France: Ann Rheum Dis; 2014. p. 112-3. Acknowledgement: Acknowledgements: Joseph & Elizabeth Carey Endowment Fund and Lied Basic Science Research Development Award from University of Kansas Medical Center Disclosure of Interests: None declared

Mast cells play an important role in immunomodulation and in the maintenance of vascular integrity. Interleukin-6 (IL-6) is one of the key biomarkers and therapeutic target in systemic vasculitis. The objective of the current study is to describe the role of mast cells in arterial IL-6 homeostasis. Eight- to ten-week-old male C57BL/6 (wild-type) mice were injected with either (a) saline, (b) compound 48/80 (a systemic mast cell degranulating agent), (c) lipopolysaccharide (LPS), or (d) a combination of C48/80 and LPS. Twenty-four hours after the injections, mice were sacrificed and serum samples and aortic tissues were analyzed for determining inflammatory response and cytokine expression profile. The results revealed that induction of mast cell degranulation significantly lowers serum IL-6 levels and aortic expression of IL-6 in LPS-treated mice. Significantly higher aortic expression of toll-like receptor-2 (TLR-2) and TNF-α was seen in the LPS and LPS+C48/80 groups of mice compared to controls. Aortic expression of TLR-4 was significantly decreased in LPS+C48/80 compared to C48/80 alone. LPS+C48/80-treated mice presented with a 3-fold higher aortic expression of suppressor of cytokine signaling (SOCS-1) compared to saline-injected groups. The inhibition of LPS-induced increase in serum IL-6 levels by mast cell degranulation was not seen in H1R knockout mice which suggests that mast cell-derived histamine acting through H1R may participate in the regulatory process. To examine whether the mast cell-mediated downregulation of LPS-induced IL-6 production is transient or cumulative in nature, wild-type mice were injected serially over a period of 10 days (5 injections) and serum cytokine levels were quantified. We found no significant differences in serum IL-6 levels between any of the groups. While mice injected with C48/80 or LPS had higher IL-10 compared to vehicle-injected mice, there was no difference between C48/80- and LPS+C48/80-injected mice. In conclusion, in an in vivo setting, mast cells appear to partially and transiently regulate systemic IL-6 homeostasis. This effect may be regulated through increased systemic IL-10 and/or aortic overexpression of SOCS-1.

Mast cells are important cells of the immune system. Although, traditionally considered as key players in allergic and hypersensitivity reactions emerging evidence suggest that mast cells have many complex roles in vascular disease. These include regulation of vasodilation, angiogenesis, activation of matrix metalloproteinases, apoptosis of smooth muscle cells, and activation of the renin angiotensin system. Mast cells are also known to play an immunomodulatory role via modulation of regulatory T-cell (T-reg), macrophage and endothelial cell functions. This dual role of the mast cells is evident in myeloperoxidase anti-neutrophil cytoplasmic antibodies (MPO-ANCA)-mouse model of glomerulonephritis in which mast cell deficiency worsens glomerulonephritis, whereas inhibition of mast cell degranulation is effective in abrogating the development of glomerulonephritis. Our previous work demonstrated that mast cell degranulation inhibits lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) production in mice. This effect was not seen in histamine-1-receptor knockout (H1R-/-) mice suggesting a role for histamine in IL-6 homeostasis. In addition, mast cell degranulation-mediated decrease in IL-6 production was associated with an upregulation of suppressor of cytokine signaling-1 protein (SOCS-1) in the aorta. We propose that mast cells regulate large artery inflammation through T-cells, shifting a primarily Th1 and Th17 toward a Th2 response and leading to enhanced production IL-10, activation Treg cells and the inhibition of macrophage functions.

Background We have previously demonstrated that mast cell degranulation acutely downregulates lipopolysaccharide induced aortic expression and serum levels of IL-6 in vivo. This is accompanied by aortic upregulation of suppressor of cytokine signaling-1 (SOCS-1) gene expression1. This effect is not seen in histamine H1 receptor-knockout mice suggesting that mast cell-derived histamine is a key mediator involved in IL-6 homoeostasis2. Mice injected with Candida albican water-soluble-fraction (CAWS) have been shown to develop coronary and aortic vasculitis3. Our long-term objective is to determine the pathogenic mechanism of large vessel vasculitis (LVV). Objectives The aim of this pilot study was to replicate and develop a working mouse model to determine the regulatory role of mast cells in LVV. Methods Eight to ten weeks-old male C57Bl6/J mice were randomly distributed into two groups [CAWS, N=8; and Control, N=8] and were injected i.p. daily for 5 days with either CAWS in normal saline (2 mg/day/mouse) or normal saline alone (controls). All mice were sacrificed 30 days after the 5th injection. We examined serum levels of IL-6 and TNF-α, as well as aortic tissue expressions of IL-6, TNF-a, IL-10 and SOCS-1 mRNA. Heart and aortic sections were evaluated for inflammation and mast cells after staining with H & E and toluidine blue, respectively. Results Treatment of mice with CAWS for 5-consecutive days led to overexpression of IL-6, TNF-a and IL-10 genes in the aortic tissue with modest upregulation of SOCS-1. At the root of the aorta, all animals in the CAWS group had intense inflammatory infiltrates composed of mixed acute and chronic inflammatory cells. There is also evidence of vasculitis in the coronary arteries. In contrast, none of the control mice had any evidence of aortic inflammation or vasculitis. Serum IL-6 concentrations were below detectable levels in both controls and CAWS-treated mice whereas TNF-a levels were elevated in 3 out of 8 mice in the CAWS group. There were no signs or increased presence of intact or degranulating mast cells in the area of inflammation. Conclusions These results suggest that CAWS-induced LVV involves acute and chronic inflammatory response and vascular tissue expression of both pro- and anti-inflammatory cytokines and SOC-1. Detailed kinetic studies are warranted to determine the optimum windows of peak inflammatory response and the expression of these genes to understand the pathobiology of CAWS-induced LVV. References Springer J, Raveendan V, Smith D, et al. [abstract] Ann Rheum Dis 2014;73:112–113. Raveendran V, Springer J, Smith D, et al. [abstract]. Arthritis Rheumatol 2015;67(suppl 10). Miura N, Shingo Y, Adachi Y, et al. Immunopharmacol Immunotoxicol 2004;26(4):527–43. Acknowledgements Supported by Basic Science Development Award from Department of Medicine at the University of Kansas. Disclosure of Interest None declared

We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and apolipoprotein E (ApoE)−/− mice. Mice were fed a high-fat diet (HFD) for 3 mo, together with administration of either cetirizine (4 mg/kg body wt) or fexofenadine (40 mg/kg body wt) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight, and hepatic steatosis in wild-type mice but not in ApoE−/− mice. Lobular inflammation, acute inflammation, and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, whereas lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element-binding protein in wild-type mice, without altering the mRNA expression of sterol regulatory element-binding protein 1c, fatty acid synthase, or ApoB100, in either genotype. Fexofenadine increased both triglycerides and cholesterol ester, whereas cetirizine increased only cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild-type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 and bile salt export pump. These results suggest that H1-antihistamines increase the progression of fatty liver disease in wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE, and increase in hepatic bile acid levels.

Summary Histamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I(2) (PGI(2)), PGE(2) and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [(3)H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPS-induced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10- and 15-fold increases in PGI(2), PGE(2) and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI(2), PGE(2) and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.

STUDIES were carried out to evaluate the impact of a high-fat dietary regimen on aortic wall thickness, peripheral blood leukocyte profile, and plasma cholesterol and triglyceride levels in the mast cell-deficient Sl/Sldmouse. The results demonstrated that the mean aortic wall thickness of Sl/Sldmice was significantly higher than their normal littermates, and were increased in both genotypes after a 17-day high-fat regimen. In comparison with normal littermates, Sl/Sldgenotypes had elevated levels of plasma triglycerides with normal levels of plasma cholesterol, and the high-fat diet markedly lowered the triglyceride levels. Total peripheral blood leukocytes, the monocyte and granulocyte counts, and hemoglobin levels were significantly lower in Sl/Sldmice, although the number of lymphocytes, eosinophils and basophils were the same in both genotypes. Interestingly, the high-fat diet regimen elevated leukocyte counts and the number of monocytes and granulocytes in Sl/Sldmice.

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 functionsin vitro, and itsin vivoeffects 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 μM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 μM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-Cin 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.

Background:We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8.Aims:The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation.Methods:Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-κB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride.Results:Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-κB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide.Conclusions:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated protein kinase activation and nuclear factor-κB translocation.

Activation of histamine receptor 1 (H1R) reduces adiposity in mice, and chronic use of H1-antihistamines (H1-AH) increase weight gain in obese patients. Apolipoprotein E (ApoE) plays a major role in lipid transport. We examined the effects of chronic blockade of H1R by cetirizine or fexofenadine, two therapeutic H1-AH, on fatty liver disease, adiposity and weight gain in wild-type (WT) and ApoE-knockout (ApoE-/-) mice. Eight week old male WT or ApoE-/- mice were fed a high fat diet, and concurrently treated with equivalent therapeutic doses of either cetirizine or fexofenadine via drinking water for 3 months. H1-HA treatments increased weight gain, visceral adiposity, liver weight and macrovesicular hepatosteatosis in WT mice, but not in ApoE-/- mice. These changes were independent of food intake. Serum markers of liver injury tended to increase in H1-HA-treated WT mice in association with increased macrovesicular hepatosteatosis. Microvesicular hepatosteatosis and lobular inflammation were not affected b...

It has been hypothesized that hormonally regulated histamine production plays a role in preparation of the uterus for implantation. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production. The current study was designed to determine intrauterine expression of HDC mRNA expression during pregnancy in the mouse. High levels of HDC mRNA expression were observed in the preimplantation mouse uterus with peak expression occurring on day 4. High levels of HDC mRNA expression were also detected in the post-implantation uterus. In an effort to determine whether HDC mRNA is regulated by pro-inflammatory cytokines, the HDC mRNA pattern was compared to intrauterine expression of mRNA's for interleukin-1alpha (IL-1alpha), IL-1beta, macrophage chemotactic protein-1 (MCP-1) and RANTES (regulated on activation, normal T expressed and secreted) during the peri-implantation period. IL-1beta, MCP-1 and RANTES mRNA levels were increased in the uterus on days 1-2 and on days 4-5. Increased expression of IL-1alpha mRNA was observed on days 1-2 and days 5-7. There was no clear relationship between HDC mRNA expression and cytokine/chemokine mRNA expression. Progesterone-stimulated intrauterine expression of HDC mRNA. Intrauterine cytokine/chemokine mRNA was also hormonally regulated. This data allowed the possibility that one or more of these pro-inflammatory cytokines could be involved in regulating intrauterine HDC mRNA production. Recombinant IL-1alpha, IL-1beta, MCP-1 and RANTES all failed to induce HDC mRNA expression in the preimplantation uterus in a mouse pseudopregnancy model. At the same time, IL-1beta induced the expression of mRNA for each of the four cytokines/chemokines. Despite the fact that these were also produced in the uterus during pregnancy and were hormonally regulated, none of these cytokines induced intrauterine HDC mRNA expression. The data suggest that progesterone is involved in the regulation of HDC mRNA expression in the preimplantation uterus, but IL-1alpha/beta, MCP-1 and RANTES, which have been reported to regulate histamine synthesis during inflammatory processes, do not appear to play a role.

The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression.

Previous studies have shown that mast cell granules (MCG) inhibit numerous macrophage functions including tumour cytotoxicity, superoxide and nitric oxide (NO) production, and FCgamma2a receptor-mediated phagocytosis. In this study, the effect of MCG on macrophage TNF alpha and nitric oxide synthase (iNOS) mRNA expression, and the production and fate of TNF alpha were examined. Upon activation with LPS+IFN gamma, macrophages expressed both TNF alpha and iNOS mRNA and produced both TNF alpha and NO. Co-incubation of LPS+IFN gamma-activated macrophages with MCG resulted in dose-dependent inhibition of iNOS mRNA expression. TNF alpha production in the activated macrophages was decreased by MCG, which was associated with a reduction in TNF alpha mRNA expression. MCG were also capable of degrading both macrophage-generated and recombinant TNF alpha. The direct effect of MCG on TNF alpha was partially reversed by a mixture of protease inhibitors. These results demonstrate that MCG decrease the production of NO and TNF alpha by inhibiting macrophage iNOS and TNF alpha gene expression. Furthermore, MCG post-transcriptionally alter TNF alpha levels via proteolytic degradation.

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.

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 ( KitW-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−/−/KitW-sh/W-shcompared 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−/−/KitW-sh/W-shmice. The high-fat regimen did not induce atherosclerosis in either KitW-sh/W-shor 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−/−/KitW-sh/W-shmice, whereas no appreciable differences were noted in serum levels of triglycerides or very low density lipoprotein. ApoE−/−/KitW-sh/W-shmice 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−/−/KitW-sh/W-shmice compared with ApoE−/−mice. The assessment of systemic generation of thromboxane A2(TXA2) and prostaglandin I2(PGI2) revealed significant decrease in the production of PGI2in ApoE−/−/KitW-sh/W-shmice with no change in TXA2. The decrease in PGI2production 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−/−/KitW-sh/W-shmice. These results demonstrate the direct involvement of mast cells in the progression of atherosclerosis and hepatic steatosis.

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.

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.

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-kappaB 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-2.LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2.LPS complex. Finally we show that the MD-2.LPS 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-2.LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4(+)/MD-2(-) cells in the inflammatory microenvironment.

Although increased serum histamine levels and H1R expression in the plaque are seen in atherosclerosis, it is not known whether H1R activation is a causative factor in the development of the disease, or is a host defense response to atherogenic signals. In order to elucidate how pharmacological inhibition of histamine receptor 1 (H1R) signaling affects atherogenesis, we administered either cetirizine (1 and 4 mg/kg. b.w) or fexofenadine (10 and 40 mg/kg. b.w) to ApoE−/− mice maintained on a high fat diet for three months. Mice ingesting a low dose of cetirizine or fexofenadine had significantly higher plaque coverage in the aorta and cross-sectional lesion area at the aortic root. Surprisingly, the higher doses of cetirizine or fexofenadine did not enhance atherosclerotic lesion coverage over the controls. The low dose of fexofenadine, but not cetirizine, increased serum LDL cholesterol. Interestingly, the expression of iNOS and eNOS mRNA was increased in aortas of mice on high doses of cetirizine or fexofenadine. This may be a compensatory nitric oxide (NO)-mediated vasodilatory mechanism that accounts for the lack of increase in the progression of atherosclerosis. Although the administration of cetirizine did not alter blood pressure between the groups, there was a positive correlation between blood pressure and lesion/media ratio at the aortic root in mice receiving the low dose of cetirizine. However, this association was not observed in mice treated with the high dose of cetirizine or either doses of fexofenadine. The macrophages or T lymphocytes densities were not altered by low doses of H1-antihistamines, whereas, high doses decreased the number of macrophages but not T lymphocytes. The number of mast cells was decreased only in mice treated with low dose of fexofenadine. These results demonstrate that chronic ingestion of low therapeutic doses of cetirizine or fexofenadine enhance progression of atherosclerosis.

Polymorphonuclear leukocytes increase glomerular albumin permeability via hypohalous acid. Acute glomerulonephritis is characterized by the presence of neutrophils within glomeruli and the generation of reactive oxygen species (ROS) by activated polymorphonuclear leukocytes (PMNs). Hydrogen peroxide (H 2 O 2 ) and other ROS including hypohalous acids have been implicated in PMN mediated injury. To determine the role of specific ROS in PMN mediated glomerular injury, isolated rat glomeruli were incubated for 30 minutes at 37°C with H 2 O 2 , with H 2 O 2 and myeloperoxidase, or with activated PMNs. Scavengers of ROS were included in some experiments. PMNs were harvested from rat peritoneal cavity and activated with phorbol myristate acetate (PMA). Glomerular albumin permeability (P albumin ) was calculated from the volume response to an oncotic gradient. P albumin of glomeruli incubated with H 2 O 2 (10 -3 or 10 -1 M) was not increased, while P albumin after incubation with H 2 O 2 and MPO was markedly increased (0.94 ± 0.004). P albumin after incubation with PMA, or with non-activated PMNs was not different from that of control glomeruli. P albumin of the glomeruli incubated with activated PMNs increased (0.85 ± 0.01, P albumin was inhibited by superoxide dismutase, catalase, or taurine (P albumin = 0.035 ± 0.06, -0.39 ± 0.10, 0.028 ± 0.06, respectively) and ameliorated by sodium azide (P albumin = 0.21 ± 0.03). In contrast, dimethyl sulfoxide did not prevent the increase in P albumin (P albumin = 0.92 ± 0.01). Our results show that the hypohalous acid derived from that of H 2 O 2 -MPO-halide system is capable of increasing P albumin . We conclude that hypohalous acid may be the primary mediator of the immediate increase in glomerular protein permeability induced by PMNs.

It had been noted previously that the activity of mitochondrial dihydroorotate dehydrogenase was lower in crude tissue preparations containing cytosol than in isolated mitochondria. Closer examination reveals that the apparent lower enzyme activity is due to rapid conversion of newly-synthesized orotate to uridine-5-monophosphate by the cytosolic enzymes, orotate phosphoribosyltransferase and orotidylate decarboxylase.