Tumor necrosis factor-alpha from bone marrow-derived cells is not essential for the expression of adhesion molecules in lipopolysaccharide-induced nasal inflammation

Both the role and source of tumor necrosis factor (TNF-alpha) in lipopolysaccharide (LPS)-induced nasal inflammation were investigated using TNF-alpha gene deficient (TNF-alpha -/-) mice and chimeric mice that are TNF-alpha gene deficient only in bone marrow-derived cells. In the present study, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in the nasal mucosa were significantly decreased following intranasal instillation of LPS in TNF-alpha gene deficient mice compared to those in wild type mice. In contrast, ICAM-1 and VCAM-1 mRNA expressions were not significantly decreased although TNF-alpha mRNA expression was dramatically decreased in TNF-alpha gene deficient bone marrow-transplanted-chimeric (TNF-alpha -/--->+/+) mice compared to those in wild type bone marrow-transplanted-control (TNF-alpha +/+-->+/+) mice. These results indicate that the elevation of TNF-alpha mRNA in the nasal mucosa is mainly originated from bone marrow-derived cells. However, even low expression of TNF-alpha at local inflammation sites is sufficient to induce the expression of adhesion molecules in acute LPS-induced experimental rhinitis.     

Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Effect of high glucose concentrations on expression of ELAM-1, VCAM-1 and ICAM-1 in HUVEC with and without cytokine activation

Diabetes mellitus is associated with an increased prevalence of endothelial dysfunction and development of atherosclerotic vascular diseases. We demonstrate here that hyperglycemia results in the expression of adhesion molecules on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) in a culture medium with 11.0 mM, 16.5 mM and 22.0 mM glucose concentrations induced the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). This effect was detectable after 24 h incubation of HUVEC with a high glucose concentration. The effect of high glucose concentration on TNF-alpha induced expression of ELAM-1, VCAM-1 and ICAM-1 was negligible, if at all. These results show that even a short-term exposure of endothelial cells (ECs) to high glucose concentration leads to their activation associated with increased expression of adhesion molecules such as ELAM-1, VCAM-1 and ICAM-1.     

MAdCAM mediates lymphocyte-endothelial cell adhesion in a murine model of chronic colitis

Previous studies have revealed that the expression of several endothelial cell adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)] is dramatically elevated in the chronically inflamed colonic vasculature of severe combined immunodeficient (SCID) mice reconstituted with congenic CD4+, CD45RB(high) T lymphocytes. The objective of this study was to define the contribution of different endothelial cell adhesion molecules to the lymphocyte-endothelial cell (L/E) adhesion observed in the colonic microvasculature in this experimental model of inflammatory bowel disease. Fluorescently labeled T lymphocytes, isolated from spleens of normal BALB/C mice, were injected intravenously into SCID mice that had been reconstituted with CD4+, CD45RB(high) T lymphocytes either before (3 wk after reconstitution) or after (7 wk postreconstitution) the onset of clinical signs of colitis (i.e., diarrhea, loss of body wt). Intravital fluorescence microscopy was used to quantify L/E adhesion in different-sized venules of the colonic submucosa during the development of colitis. L/E adhesion was noted in some segments of the vasculature in precolitic SCID mice (3 wk after reconstitution) but not in similar-sized vessels of control (wild type and SCID) mice. L/E adhesion was observed in a greater proportion of venules and occurred with greater intensity in the mucosa of colitic mice (7 wk postreconstitution). Pretreatment with a blocking monoclonal antibody against MAdCAM-1, but not ICAM-1 or VCAM-1, significantly and profoundly reduced L/E adhesion in colitic mice. Immunohistochemical staining also revealed the localization of T cells on colonic endothelial cells expressing MAdCAM-1. These findings indicate that MAdCAM-1 is largely responsible for recruiting T lymphocytes into inflamed colonic tissue.     

Alpha 4/beta 1 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.

Expression of vascular cell adhesion molecule-1 (VCAM-1) in synovial tissue was determined using the immunoperoxidase technique. Normal, rheumatoid arthritis (RA), and osteoarthritis (OA) synovia bound VCAM-1 antibodies in the intimal lining as well as blood vessels. The amount of VCAM-1 was significantly greater in the synovial lining of RA and OA tissues compared with normal synovium (p less than 0.002). There was also a trend toward greater levels of VCAM-1 staining in blood vessels of arthritic tissue (RA greater than OA greater than normal). Because VCAM-1 staining was especially intense in the synovial lining, VCAM-1 expression and regulation was studied on cultured fibroblast-like synoviocytes (FLS) derived from this region. Both VCAM-1 and intercellular adhesion molecule 1 were constitutively expressed on FLS. VCAM-1 expression was further increased by exposure to IL-1 beta, TNF-alpha, IL-4, and IFN-gamma. These cytokines (except for IL-4) also induced intercellular adhesion molecule 1 expression on FLS. ELAM was not detected on resting or cytokine-stimulated FLS. The specificity of VCAM-1 for FLS was demonstrated by the fact that only trace amounts were detected on normal and RA dermal fibroblasts. Cytokines induced intercellular adhesion molecule 1 display on dermal fibroblasts but had minimal effect on VCAM-1 expression. Finally, in adherence assays, Jurkat cell binding to resting FLS monolayers was inhibited by antibody to alpha 4/beta 1 integrin (VLA-4), CS-1 peptide from alternatively spliced fibronectin (which is another VLA-4 ligand), and, to a lesser extent, anti-VCAM-1 antibody. After cytokine stimulation of FLS, Jurkat-binding significantly increased, and this increase was blocked by anti-VCAM-1 antibody. Therefore, both CS-1 and VCAM-1 participate in VLA-4-mediated adherence to resting FLS in vitro, and VCAM-1 is responsible for the increase in Jurkat binding mediated by cytokines.     

Elimination of rheumatoid synovium in situ using a Fas ligand 'gene scalpel'

Surgical synovectomy to remove the inflammatory synovium can temporarily ameliorate rheumatoid inflammation and delay the progress of joint destruction. An efficient medically induced programmed cell death (apoptosis) in the rheumatoid synovium might play a role similar to synovectomy but without surgical tissue damage. Gene transfer of Fas ligand (FasL) has increased the frequency of apoptotic cells in mouse and rabbit arthritic synovium. In this study, we investigated whether repeated FasL gene transfer could remove human inflammatory synovial tissue in situ and function as a molecular synovectomy. Briefly, specimens of human synovium from joint replacement surgeries and synovectomies of rheumatoid arthritis (RA) patients were grafted subcutaneously into male C.B-17 severe combined immunodeficiency (SCID) mice. Injections of a recombinant FasL adenovirus (Ad-FasL) into the grafted synovial tissue at the dosage of 10(11) particles per mouse were performed every two weeks. Three days after the fifth virus injection, the mice were euthanized by CO2 inhalation and the human synovial tissues were collected, weighed and further examined. Compared to the control adenovirus-LacZ (Ad-LacZ) and phosphate buffered saline (PBS) injected RA synovium, the Ad-FasL injected RA synovium was dramatically reduced in size and weight (P < 0.005). The number of both synoviocytes & mononuclear cells was significantly reduced. Interestingly, an approximate 15-fold increased frequency of apoptotic cells was observed in RA synovium three days after Ad-FasL injection, compared with control tissues. In summary, our in vivo investigation of gene transfer to human synovium in SCID mice suggests that repeated intra-articular gene transfer of an apoptosis inducer, such as FasL, may function as a 'gene scalpel' for molecular synovectomy to arrest inflammatory synovium at an early stage of RA.     

The VLA-4/VCAM-1 pathway is involved in lymphocyte adhesion to endothelium in rheumatoid synovium.

Lymphocyte migration to inflammatory sites is an essential factor in the pathogenesis of chronic inflammation. An ensemble of adhesion receptors mediating lymphocyte-endothelial cell recognition and binding are thought to play a crucial role in this process. In the present study, we have explored the molecular basis of lymphocyte adhesion to endothelium in the synovial membrane of patients with rheumatoid arthritis. We established that the very late antigen-4 [VLA-4 (CD49d)] and the vascular cell adhesion molecule-1 (VCAM-1) are important mediators of binding to synovial endothelium of resting and, to a greater extent, of activated T lymphocytes, whereas the leukocyte-function associated antigen-1 [LFA-1 (CD11a/18)]/intercellular adhesion molecule-1 [ICAM-1 (CD54)] pathway is less important in this interaction. In contrast to its prominent role in lymphocyte interaction with endothelium in rheumatoid synovium, the VLA-4/VCAM-1 pathway does not significantly contribute to lymphocyte adhesion to peripheral lymph node high endothelial venule. Thus, the VLA-4/VCAM-1 pathway may be of primary importance in mediating lymphocyte adhesion to inflamed endothelium and in lymphocyte homing to rheumatoid synovium.     

Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells

Cell adhesion molecules expressed on endothelial cells in inflamed skin appear to be controlled by the actions of cytokines and reactive oxygen species. However, molecular mechanisms of the expression of adhesion molecules during skin inflammation are currently not well understood. To evaluate the role of antioxidants and nitric oxide in modulating inflammatory processes in the skin, we examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1 mM) and spermine NONOate (Sper-NO, 1 mM) on adhesion molecule expression and nuclear factor kappa B (NF-kappaB) activation induced by TNF-alpha (10 ng/ml) in cultured human dermal microvascular endothelial cells (HDMEC). Treatment of cells with TNF-alpha for 4 h significantly induced the surface expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1). Treatment with TNF-alpha for 8 h significantly induced the surface expression of E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1). The up-regulation of these adhesion molecules was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h. The mRNA expression of E-selectin, ICAM-1 and VCAM-1, and activation of NF-kappaB induced by TNF-alpha for 2 h were significantly decreased by the above two pretreatments. N-acetylcysteine (10 mM) and S-nitroso-N-acetylpenicillamine (1 mM) had no significant inhibitory effects on the cell surface and mRNA expression of these adhesion molecules stimulated by TNF-alpha. These findings indicate that both cell surface and mRNA expression of adhesion molecules in HDMEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly in part through blocking the activation of NF-kappaB. These results suggest a potential therapeutic approach using antioxidant agents or nitric oxide pathway modulators in the treatment of inflammatory skin diseases.     

The role of the cytoskeleton in cellular adhesion molecule expression in tumor necrosis factor-stimulated endothelial cells

Leukocyte infiltration is a hallmark of the atherosclerotic lesion. These cells are captured by cellular adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cell adhesion molecule (PECAM), and E-selectin, on endothelial cells (EC). We examined the role of the actin cytoskeleton in tumor necrosis factor-alpha (TNF-alpha)-induced translocation of CAMs to the cell surface. Human aortic EC were grown on 96-well plates and an ELISA was used to assess surface expression of the CAMs. TNF-alpha increased VCAM-1, ICAM-1, and E-selectin by 4 h but had no affect on the expression of PECAM. A functioning actin cytoskeleton was important for VCAM-1 and ICAM-1 expression as both cytochalasin D, an actin filament disruptor, and jasplakinolide, an actin filament stabilizer, attenuated the expression of these CAMs. These compounds were ineffective in altering E-selectin surface expression. Myosin light chains are phosphorylated in response to TNF-alpha and this appears to be regulated by Rho kinase instead of myosin light chain kinase. However, the Rho kinase inhibitor, Y27632, had no affect on TNF-alpha-induced CAM expression. ML-7, a myosin light chain kinase inhibitor, had a modest inhibitory effect on the translocation of VCAM-1 but not on ICAM-1 or E-selectin. These data suggest that the surface expression of VCAM-1 and ICAM-1 is dependent on cycling of the actin cytoskeleton. Nevertheless, modulation of actin filaments via myosin light chain phosphorylation is not necessary. The regulation of E-selectin surface expression differs from that of the other CAMs.     

Contrasting effects of interferon gamma and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor alpha.

Tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelial cells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-alpha-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from TNF-alpha-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-alpha-induced responses to those observed with endothelial cells of foetal origin. Additionally, we report here that TNF-alpha and interferon gamma (IFN-gamma) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-alpha-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-gamma plus TNF-alpha was markedly decreased when compared to the response induced by TNF-alpha alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

Neutralization of IL-18 attenuates lipopolysaccharide-induced myocardial dysfunction

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been implicated in cardiac dysfunction during endotoxemia. Because IL-18 is a proinflammatory cytokine known to mediate the production of TNF-alpha and IL-1beta and to induce the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), we hypothesized that neutralization of IL-18 would attenuate lipopolysaccharide (LPS)-induced cardiac dysfunction. Mice (C57BL/6) were injected with LPS (0.5 mg/kg ip) or vehicle (normal saline), and left ventricular developed pressure (LVDP) was determined by the Langendorff technique. LVDP was depressed by 38% at 6 h after LPS. LPS-induced myocardial dysfunction was associated with increased myocardial levels of TNF-alpha and IL-1beta as well as increased expression of ICAM-1/VCAM-1. Pretreatment with neutralizing anti-mouse IL-18 antibody attenuated LPS-induced myocardial dysfunction (by 92%) and was associated with reduced myocardial IL-1beta production (65% reduction) and ICAM-1/VCAM-1 expression (50% and 35% reduction, respectively). However, myocardial TNF-alpha levels were not influenced by neutralization of IL-18. In conclusion, neutralization of IL-18 protects against LPS-induced myocardial dysfunction. IL-18 may mediate endotoxemic myocardial dysfunction through induction of and/or synergy with IL-1beta, ICAM-1, and VCAM-1.     

Ultrastructural localization of adhesion molecules in the healthy

The functional expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and MAdCAM-1 in the choroid plexus is indicative of a role of this structure in the communication of the immune system with the central nervous system (CNS). In order to gain further insight into the possible functions of adhesion molecules expressed in the choroid plexus, we investigated the exact ultrastructural localization of VCAM-1, ICAM-1 and MAdCAM-1 on semithin and ultrathin cryosections of the choroid plexus of healthy mice and of mice suffering from experimental autoimmune encephalomyelitis (EAE). In the healthy choroid plexus VCAM-1 and ICAM-1, but not MAdCAM-1, could be detected on the apical surface of the choroid plexus epithelial cells. During EAE, immunoreactivity for VCAM-1 and ICAM-1 was dramatically increased. Additionally, apical expression of MAdCAM-1 was observed on individual choroid plexus epithelial cells during EAE. At the same time, VCAM-1, ICAM-1 or MAdCAM-1 were never present on the endothelial cells of the fenestrated capillaries within the choroid plexus. The polar expression of VCAM-1, ICAM-1 and MAdCAM-1 on the apical surface of choroid plexus epithelial cells, which form the blood-cerebrospinal fluid barrier, implies a previously unappreciated function of this barrier in the immunosurveillance of the CNS.     

TNF-alpha-dependent ICAM-1- and VCAM-1-mediated inflammatory responses are delayed in neonatal mice infected with Pneumocystis carinii

Neonatal mice have a delayed CD4-mediated inflammatory response to Pneumocystis carinii (PC) infection in the lungs that corresponds to a delayed TNF-alpha response and a delayed clearance of the organisms compared with adult mice. Since TNF-alpha is known to drive the up-regulation of adhesion molecules, we examined the expression and function of adhesion molecules in the lungs of neonatal mice. The expression of both ICAM-1 and VCAM-1 was significantly lower in the lungs of PC-infected neonatal mice compared with adults. Additionally, migration of neonatal T cells across endothelial cells expressing VCAM-1 and monocyte chemotactic protein-1 was aberrant compared with that in adult T cells, although alpha(4)beta(1) integrin-mediated adhesion of neonatal lymphocytes was comparable to that of adult lymphocytes. Treatment of neonatal mice with exogenous TNF-alpha resulted in increased expression of ICAM-1 and VCAM-1 as well as increased expression of chemokines, resulting in infiltration of CD4(+) cells into the lungs. Treatment with exogenous TNF-alpha resulted in a trend (although not statistically significant) toward a reduction of PC organisms from the lungs. These data indicate that neonatal lung endothelial cells do not up-regulate ICAM-1 and VCAM-1 in response to PC infection, probably due to depressed TNF-alpha production. Additionally, neonatal T cells are defective in the ability to migrate across endothelial cells.     

Characterization of a human synovial cell antigen: VCAM-1 and inflammatory arthritis

The contribution of synovial cells to the pathogenesis of rheumatoid arthritis (RA) is only partly understood. Monoclonal antibody (mAb) 1D5 is one of very few mAb ever raised against RA synovial cells in order to study the biology of these cells. Studies on the expression pattern and structural features of the 1D5 Ag suggest that 1D5 recognizes human vascular cell adhesion molecule-1 (VCAM-1), which is an intercellular adhesion molecule. Vascular cell adhesion molecule-1 may be involved in a number of crucial intercellular interactions in RA.     

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, alpha-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.     

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture

Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.