[Show abstract][Hide abstract] ABSTRACT: Obese patients display an exaggerated morbidity during sepsis. Since consumption of a western-style diet (WD) is a major factor for obesity in the United States, the purpose of the present study was to examine the influence of chronic WD consumption on hepatic inflammation in mice made septic via cecal ligation and puncture (CLP). Feeding mice diets high in fat has been shown to enhance evidence of TLR signaling and this pathway also mediates the hepatic response to invading bacteria. Therefore, we hypothesized that the combined effects of sepsis and feeding WD on TRL-4 signaling would exacerbate hepatic inflammation. Male C57BL/6 mice were fed purified control diet (CD) or WD that was enriched in butter fat (34.4% of calories) for 3 weeks prior to CLP. Intravital microscopy was used to evaluate leukocyte adhesion in the hepatic microcirculation. To demonstrate the direct effect of saturated fatty acid on hepatocytes, C3A human hepatocytes were cultured in medium containing 100 μM palmitic acid (PA). Quantitative real-time PCR was used to assess mRNA expression of tumor necrosis factor-alpha (TNF-α, monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), toll-like receptor-4 (TLR-4) and interleukin-8 (IL-8).
Feeding WD increased firm adhesion of leukocytes in the sinusoids and terminal hepatic venules by 8-fold six hours after CLP; the increase in platelet adhesion was similar to the response observed with leukocytes. Adhesion was accompanied by enhanced expression of TNF-α, MCP-1 and ICAM-1. Messenger RNA expression of TLR-4 was also exacerbated in the WD+CLP group. Exposure of C3A cells to PA up-regulated IL-8 and TLR-4 expression. In addition, PA stimulated the static adhesion of U937 monocytes to C3A cells, a phenomenon blocked by inclusion of an anti-TLR-4/MD2 antibody in the culture medium.
These findings indicate a link between obesity-enhanced susceptibility to sepsis and consumption of a western-style diet.
[Show abstract][Hide abstract] ABSTRACT: Sepsis remains a leading cause of death in critically ill patients. Because endotoxemia is viewed as a key mediator of sepsis-induced inflammation, administration of bacterial endotoxin (LPS) is often used to simulate sepsis in experimental animals. This study tests the hypothesis that LPS is a critical determinant of the hepatic microvascular dysfunction in mice made septic by cecal ligation and puncture (CLP).
Intravital videomicroscopy was used to quantify sinusoidal perfusion, and platelet and leukocyte adhesion in terminal hepatic venules (THV) and sinusoids in LPS-sensitive and LPS-insensitive mice subjected to CLP or LPS (i.p.). mRNA expression of TLR-2, TLR-4, MyD-88, and Ly-96 was also assessed.
While LPS-sensitive mice responded to both CLP and LPS challenges with elevated leukocyte and platelet adhesion in THV and sinusoids, and a reduced sinusoidal perfusion density, LPS-insensitive mice exhibited comparable blood cell adhesion and sinusoidal malperfusion following CLP, but not LPS. Hepatic mRNA of MyD-88 and TLR-2 was elevated in the CLP and LPS groups. Endotoxin was not detectable in the blood of LPS-sensitive mice after CLP, but was elevated after LPS administration.
These findings do not support a major role for LPS in the hepatic microvascular disturbances associated with polymicrobial sepsis.
Journal of Hepatology 01/2008; 47(6):799-806. DOI:10.1016/j.jhep.2007.07.021 · 10.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cerebral endothelial cells in the rat, pig, and, most recently, human have been shown to express several types of receptors specific for glutamate. High levels of glutamate disrupt the cerebral endothelial barrier via activation of N-methyl-d-aspartate (NMDA) receptors. We have previously suggested that this glutamate-induced barrier dysfunction was oxidant dependent. Here, we provide evidence that human cerebral endothelial cells respond to glutamate by generating an intracellular oxidant stress via NMDA receptor activation. Cerebral endothelial cells loaded with the oxidant-sensitive probe dihydrorhodamine were used to measure intracellular reactive oxygen species (ROS) formation in response to glutamate receptor agonists, antagonists, and second message blockers. Glutamate (1 mM) significantly increased ROS formation compared with sham controls (30 min). This ROS response was significantly reduced by 1) MK-801, a noncompetitive NMDA receptor antagonist; 2) 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate, an intracellular Ca(2+) antagonist; 3) LaCl(3), an extracellular Ca(2+) channel blocker; 4) diphenyleiodonium, a heme-ferryl-containing protein inhibitor; 5) itraconazole, a cytochrome P-450 3A4 inhibitor; and 6) cyclosporine A, which prevents mitochondrial membrane pore transition required for mitochondrial-dependent ROS generation. Our results suggest that the cerebral endothelial barrier dysfunction seen in response to glutamate is Ca(2+) dependent and may require several intracellular signaling events mediated by oxidants derived from reduced nicotinamide adenine dinucleotide oxidase, cytochrome P-450, and the mitochondria.
[Show abstract][Hide abstract] ABSTRACT: Mesothelial cell monolayers cover the serous cavities and internal organs, and provide a protective low-friction interface between apposed organs and tissues. The mesothelium also regulates inflammation, fluid and cell exchange, and tissue repair in these compartments and possibly tumor metastasis. In the present study, a stable pleural mesothelial cell line (MIM) was isolated and characterized, and the expression of several lymphatic specific markers by these cells examined.
MIM were isolated from mice stably expressing a temperature-sensitive SV40 large T antigen ('Immortomouse', strain: H-2K(b)-tsA58). These cells were compared with lymphatic endothelial cells (LEC) derived from the mesenteric adventitia of the Immortomouse. MIM and LEC expression of lymphatic-specific markers (Flt-4, LYVE-1, and Prox-1) was examined, and the tight junction protein (ZO-1) was studied by immunofluorescence and immunoblotting in these cells.
LYVE-1, Prox-1, and Flt-4 were detected in both MIM and LEC, with Prox-1 and LYVE-1 more strongly expressed on LEC than MIM. Conversely, Flt-4 was more densely expressed on MIM than on LEC. Spatially, ZO-1 was prominent at MIM junctions, but was less well organized in LEC.
MIM and LEC share several characteristic markers usually associated with lymphatic endothelium. MIM might be useful for studying the biology and pathology of mesothelial cells in vitro and help in the development of therapies for mesothelial-related diseases, such as mesothelioma and pleural effusion.
Lymphatic Research and Biology 02/2005; 3(3):117-25. DOI:10.1089/lrb.2005.3.117 · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lymphatic system regulates interstitial fluid and protein balance and modulates immune responses by regulating leukocyte and antigen traffic to lymph nodes. The present article describes a stable mouse lymphatic endothelial cell line from mesenteric adventitial tissue (SV-LEC) which is distinct from blood aortic (AEC) and venous (VEC) endothelial cells, based on expression of several lymphatic markers (e.g., Prox-1, LYVE-1, Flt-4). SV-LEC also expresses MAdCAM-1 in response to TNF-alpha, an effect seen in VEC, but not AEC.
Lymphatic endothelial cells (SV-LEC) were isolated from mesenteric adventitia from mice expressing temperature-sensitive SV40 large T ('Immortomouse', H-2K(b)tsA58) selected with hypoxia culture in D-valine-substituted MEM supplemented with VEGFC in a low oxygen atmosphere (0% O2, 5% CO2, and 95% N2) with 5 mM thioglycolate. Expression of lymphatic-specific markers (Flt-4, LYVE-1, Prox-1) and the tight junction proteins (ZO-1) were examined by RT-PCR, immunoblotting, and fluorescent microscopy. MAdCAM-1 (a high endothelial venular marker) expression was also examined in response to TNF-alpha IL-1beta and IFN-gamma.
Message for Flt-4 and LYVE-1 was detected on SV-LEC. Immunoblotting for LYVE-1 and Prox-1 showed strong expression on SV-LEC and VEC, but not AEC. Occludin expression was seen in all cell types, junctional ZO-1 was detected at SV-LEC and VEC junctions, not AEC.
SV-LEC expresses several lymphatic endothelial markers, some of which are shared with VEC, but not AEC, and may represent a useful system for modeling lymphatic function in vitro.
Lymphatic Research and Biology 02/2005; 3(3):105-15. DOI:10.1089/lrb.2005.3.105 · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to assess the roles of NK cells, B cells and/or intraepithelial lymphocytes (IEL) in suppressing the development of colitis in nude mice reconstituted with CD4(+)CD45RB(high) T cells. BALB/c nude mice were lethally irradiated and reconstituted with bone marrow from different immunodeficient mice to generate athymic chimeras devoid of one or more lymphocyte populations. Transfer of CD4(+)C45RB(high) T cells into chimeric recipients devoid of B cells, T cells and IEL produced severe colitis within 6-8 weeks, whereas transfer of these same T cells into B cell- and T cell-deficient or T cell-deficient chimeras produced little to no gut inflammation. In addition, we found that nude mice depleted of NK cells or RAG-1(-/-) mice reconstituted with IEL failed to develop colitis following transfer of CD45RB(high) T cells. Severe colitis could, however, be induced in nude mice by transfer of activated/T(h)1 CD4(+)CD45RB(low) T cells. Taken together, our data suggest that IEL, but not B cells or NK cells, play an important role in suppressing the development of chronic colitis in this model. In addition, our data demonstrate that suppression of disease may be due to polarization of naive CD4(+) cells toward a non-pathogenic and/or regulatory phenotype.
International Immunology 02/2004; 16(1):77-89. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.
[Show abstract][Hide abstract] ABSTRACT: Abstract
L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1) cerebral endothelial barrier and 2) cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells) have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR) expression via immunohistochemistry and murine neuroepithelial cell line (V1) were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.
[Show abstract][Hide abstract] ABSTRACT: Enhanced expression of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is associated with the onset and progression of inflammatory bowel disease. The clinical significance of elevated MAdCAM-1 expression is supported by studies showing that immunoneutralization of MAdCAM-1, or its ligands reduce inflammation and mucosal damage in models of colitis. Interleukin-10 (IL-10) is an endogenous anti-inflammatory and immunomodulatory cytokine that has been shown to prevent inflammation and injury in several animal studies, however clinical IL-10 treatment remains insufficient because of difficulties in the route of IL-10 administration and its biological half-life. Here, we examined the ability of introducing an IL-10 expression vector into endothelial cultures to reduce responses to a proinflammatory cytokine, TNF-alpha
A human IL-10 expression vector was transfected into high endothelial venular ('HEV') cells (SVEC4-10); we then examined TNF-alpha induced lymphocyte adhesion to lymphatic endothelial cells and TNF-alpha induced expression of MAdCAM-1 and compared these responses to control monolayers.
Transfection of the IL-10 vector into endothelial cultures significantly reduced TNF-alpha induced, MAdCAM-1 dependent lymphocyte adhesion (compared to non-transfected cells). IL-10 transfected endothelial cells expressed less than half (46 +/- 6.6%) of the MAdCAM-1 induced by TNF-alpha (set as 100%) in non-transfected (control) cells.
Our results suggest that gene therapy of the gut microvasculature with IL-10 vectors may be useful in the clinical treatment of IBD.