Vascular cell adhesion molecule-1 is involved in mediating hypoxia-induced sickle red blood cell adherence to endothelium: potential role in sickle cell disease

We investigated the effects of hypoxia on red blood cell (RBC)-endothelial cell (EC) adherence and the potential mechanism(s) involved in mediating this effect. We report that hypoxia significantly increased sickle RBC adherence to aortic EC when compared with the normoxia controls. However, hypoxia had no effect on the adherence of normal RBCs. In additional studies, we found that the least dense sickle RBCs containing CD36+ and VLA-4+ reticulocytes were involved in hypoxia-induced adherence. We next evaluated the effects of hypoxia on the expression of EC surface receptors involved in RBC adherence to macrovascular ECs, including vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and the vitronectin receptor (VnR). Hypoxia upregulated the expression of both VCAM-1 and ICAM-1, whereas no effect on VnR was noted. Potential involvement of VCAM-1 and ICAM-1 in mediating hypoxia-induced sickle RBC-EC adhesion was next investigated using monoclonal antibodies against these receptors. Whereas anti-VCAM-1 had no effect on basal adherence, it inhibited hypoxia-induced sickle RBC adherence in a concentration-dependent manner, with 50% to 75% inhibition noted at 10 to 60 micrograms/mL antibody (n = 6, P < .05 to P < .01). Anti-ICAM-1 (10 to 60 micrograms/mL, n = 8) had no effect on either basal or hypoxia-induced adherence. As noted in the bovine aortic ECs, hypoxia stimulated the adherence of sickle RBCs to human retinal capillary ECs, and this response appeared to be mediated via mechanisms similar to those observed with macro-endothelium, ie, via the adhesive receptor combination VCAM-1-VLA-4. Our studies show that hypoxia enhances sickle RBC adhesion to both macrovascular and human microvascular ECs via the adhesive receptor VCAM-1. Our findings are of interest because hypoxia is an integral part of the pathophysiology of the vaso-occlusive phenomenon in sickle cell anemia.     

Intercellular adhesion molecule-3 on endothelial cells. Expression in tumors but not in inflammatory responses

Intercellular adhesion molecule-3 (ICAM-3) was identified as the third counter-receptor for lymphocyte function-associated antigen-1. ICAM-3 is absent on endothelial cells in normal tissues but found on endothelial cells in lymphomas. Here, we examined ICAM-3 expression on vascular endothelial cells in lymphomas, nonlymphoid malignancies, benign tumors, and inflammatory diseases. We compared the expression of ICAM-3 on endothelial cells with the severity of inflammatory infiltrates and with the presence of E-selectin and VCAM-1. We found that ICAM-3 expression on endothelial cells was high on both benign and malignant tumors whereas it was low in inflammatory diseases. In contrast to E-selectin, ICAM-3 expression on endothelial cells was not correlated to the severity of inflammatory infiltrates. In hemangiomas, we showed by Northern blot analysis and immunocytochemistry that ICAM-3 expression was induced and that it was localized in immature areas that sustain the early stages of angiogenesis. Therefore, expression of ICAM-3 on blood vessels does not seem to play a role in the recruitment of leukocytes during inflammation but rather is correlated with angiogenesis and tumor development.     

Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood-brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB-MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S-PM) up-regulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on isolated HB-MVEC. In addition, HB-MVEC activated by S-PM secreted interleukin (IL)-6 and IL-8. The levels of ICAM-1, E-selectin, IL-6, and IL-8 expressed in S-PM-activated HB-MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM-1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (< or = 45%), but did not abolish the expression of cell adhesion molecules and IL-6 induced by S-PM, IL-8 production being insignificantly affected (< or = 10%). This suggests that membrane-associated TNF was partially involved in HB-MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB-MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.     

Circulating soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in patients with gastric cancer

The concentrations of the soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were investigated in 45 patients with gastric cancer before treatment and their correlation with clinical, histological and routine laboratory parameters was examined. Data were collected on tumour stage at presentation, presence and sites of metastatic disease, tumour pathology, survival and results of routine laboratory tests. Serum concentrations of ICAM-1 and VCAM-1 were significantly elevated in the patients with gastric cancer in comparison with the group of healthy subjects (P < 0.00001 and P < 0.0001 respectively). Increased serum concentrations of VCAM-1 were associated with locally advanced and metastatic disease whereas ICAM-1 was significantly elevated both in local and in advanced/metastatic disease. Soluble E-cadherin and E-selectin concentrations did not show any significant elevation in gastric cancer patients. Concentrations of soluble adhesion molecules showed significant correlation with each other (except E-selectin and VCAM-1) and with alkaline phosphatase. Soluble ICAM-1 and VCAM-1 were significantly associated with an elevated total white cell count. Patients with elevated VCAM-1 had significantly poorer survival in comparison with patients with normal serum levels (P = 0.0361).     

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.     

Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma

We investigated the effect of TNF alpha, IL-1alpha and IFN gamma on two neuroblastoma (NB) cell lines (SK-N-SH and SK-N-MC). These lines responded differentially to IL-1alpha, TNF alpha and IFN gamma for MCP-1 and IL-8 production and expression of the ICAM-1 and VCAM-1 adhesion molecules. None of the cytokines induced MCP-1 or IL-8 on SK-N-MC cells. Both chemokines were produced in response to IL-1alpha by SK-N-SH cells, while TNF alpha induced mainly MCP-1 production. Addition of IFN gamma decreased IL-8, but not MCP-1 production. These responses correlated with monocyte and neutrophil chemotactic activity in NB culture supernatants. This activity was neutralized by antibodies to IL-8 and MCP-1. The expression of ICAM-1 on SK-N-MC was up-regulated by TNF alpha or IFN gamma, while IL-1alpha also upregulated ICAM-1 on SK-N-SH cells. VCAM-1 expression on SK-N-SH was induced by IL-1alpha and TNF alpha and IFN gamma synergized with TNF alpha in this respect on both NB cell lines. These results suggest that mechanisms for chemokine production and VCAM-1 and ICAM-1 upregulation by inflammatory cytokines differ and IFN gamma, in conjunction with TNF alpha, stimulate neural cell responses (high MCP-1 and VCAM-1 and decreased IL-8) favouring mononuclear cell recruitment.     

Neutralization of TNF by the antibody cA2 reveals differential regulation of adhesion molecule expression on TNF-activated endothelial cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

Research on the mechanism of endothelin inflammatory effects on human mesangial cells

OBJECTIVE: To investigate the mechanism of endothelin (ET) inflammatory effects on human mesangial cells (HMC). METHODS: The following experiments were performed on cultured HMC after ET-1 stimulation: (1) the expression of tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) itself messenger ribonucleic acid (mRNA) was determined by Northern Blot analysis; (2) the TNF alpha concentration was tested with radioimmunoassay; the IL-1 activity was assayed by the enhancement of thymocyte proliferation in response to mitogen; the surface expression of ICAM-1 and VCAM-1 was measured with cell enzyme linked immunoadsorbent assay (ELISA) analysis. RESULTS: ET-1 (10(-7) mol/L) induced the following changes on HMC: (1) up-regulation of the expression of TNF alpha mRNA and protein; (2) up-regulation of the expression of ICAM-1 and VCAM-1 mRNA and protein; (3) up-regulation of the expression of ET-1 itself mRNA. However, the expression of IL-1 mRNA and protein was not changed. CONCLUSIONS: ET-1 can stimulate HMC to produce TNF alpha, ICAM-1 and VCAM-1, and thereby induce inflammatory effects. ET-1 can also stimulate HMC to up-regulate the expression of ET-1 itself, so as to amplify inflammatory effects. So, ET-1 is actually an inflammatory mediator and may play an important role in the pathogenesis of glomerulonephritis.     

Recombinant tumour necrosis factor-alpha and platelet-activating factor synergistically increase intercellular adhesion molecule-1 and E-selectin-dependent neutrophil adherence to endothelium in vitro

Neutrophil adhesion to microvascular endothelium at sites of acute inflammation is regulated by both chemotactic peptides and lipid-derived mediators. Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory peptide that up-regulates endothelial expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (E-selectin), while platelet-activating factor (PAF) is a potent lipid mediator that induces vascular changes via an unknown mechanism. Both have been shown to increase leucocyte-endothelial adhesion in various in vitro models of acute inflammation; however, the combined effects of recombinant TNF-alpha (rTNF-alpha) and PAF on neutrophil-endothelium adhesion have not been well described. In this study, we found rTNF-alpha at 0.5 ng/ml and PAF at 10 microM acted synergistically to increase neutrophil adherence to cultured umbilical vein endothelial cells 4 hr after stimulation. This increased neutrophil-endothelial adhesion was, in part, dependent on up-regulated expression of ICAM-1 and E-selectin since application of anti-ICAM1 and anti-E-selectin F(ab')2 fragments markedly diminished adhesion. Cultures stimulated with rTNF-alpha (0.5 ng/ml) or PAF (10 microM) alone did not show a significant increase in neutrophil adhesion, and neither ICAM-1 nor E-selectin expression was up-regulated as determined by flow cytometric analysis of endothelial cells. These results indicate that rTNF-alpha and PAF act synergistically to increase neutrophil-endothelial adhesion by stimulating endothelial expression of ICAM-1 and E-selectin and, thus, may play important roles in the onset and severity of acute inflammatory reactions.     

The Peyer's patch high endothelial receptor for lymphocytes, the mucosal vascular addressin, is induced on a murine endothelial cell line by tumor necrosis factor-alpha and IL-1

The specificity of lymphocyte homing from the blood into a tissue is determined in part by complementary pairs of adhesion receptors on lymphocytes and endothelial cells termed homing receptors and vascular addressins, respectively. The mucosal vascular addressin involved in lymphocyte homing to Peyer's patches is a 66-kDa glycoprotein, MAdCAM-1. Investigation of the regulation and molecular genetics of MAdCAM-1 have been hampered by the lack of a murine cell line expressing this adhesion molecule. We show herein using indirect immunofluorescence studies that MAdCAM-1 can be induced on a murine endothelial cell line, bEnd.3, by cytokines and LPS. Western blot analysis of MAdCAM-1 purified by affinity column chromatography from TNF-alpha-treated bEnd.3 cells demonstrates a 66-kDa protein that comigrates in SDS-PAGE with the MAdCAM-1 constitutively found on high endothelial venules in murine mesenteric lymph nodes. Comparison of MAdCAM-1 expression on the bEnd.3 cells was made to the expression of adhesion molecules ICAM-1 and VCAM-1. MAdCAM-1 and VCAM-1 are not constitutively expressed on the bEND.3 surface but can be induced in a concentration-dependent manner by LPS, TNF-alpha, and IL-1. ICAM-1 is constitutively expressed on the endothelioma surface and expression is increased by TNF-alpha, IL-1, LPS, and IFN-gamma. Surface expression of MAdCAM-1 peaks 12 to 18 h after exposure to TNF-alpha and remains elevated at 48 h, whereas expression of VCAM-1 peaks at 4 h and inducible ICAM-1 peaks between 4 and 18 h. Interestingly, IFN-gamma has differential effects on expression of these three adhesion receptors. IFN-gamma alone induces VCAM-1 and enhances ICAM-1 expression, but does not induce MAdCAM-1. Furthermore, although, preincubation of bEND.3 cells with IFN-gamma modestly increases the induction of ICAM-1 and VCAM-1 in response to TNF-alpha and IL-1, it dramatically reduces the TNF-alpha, IL-1, and LPS-induced expression of MAdCAM-1. MAdCAM-1 on bEnd.3 cells is functional as the murine T lymphoma TK1, known to bind MAdCAM-1, also binds to TNF-alpha-stimulated endothelioma but not to unstimulated cells. This binding is blocked by the antibodies against MAdCAM-1 and against the alpha 4-chain of its integrin receptor, alpha 4 beta 7, on TK1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of intercellular adhesion molecule-1 in mice with Pseudomonas-induced pyelonephritis

PURPOSE: To investigate the role of intercellular adhesion molecule-1 (ICAM-1) in the renal inflammatory process, we studied the time-course fluctuation of ICAM-1 expression on inflammatory lesions in mice with experimentally induced bacterial pyelonephritis and the effect of in vivo administration of an anti-ICAM-1 monoclonal antibody (mAb) on leukocytic migration. MATERIALS AND METHODS: Ascending pyelonephritis was induced by transurethral instillation of Pseudomonas aeruginosa, and the expression of ICAM-1 in the pyelonephritic lesions was studied by immunohistochemical methods. RESULTS: The expression of ICAM-1 on the pyelonephritic lesions closely paralleled the degree of infiltration of neutrophils and macrophages until 3 days after infection. At 7 days after infection, though the degree of infiltration of these cells was quite high, expression of ICAM-1 was reduced. Treatment with the anti-ICAM-1 mAb in mice with bacterial pyelonephritis resulted in suppression of influx of neutrophils and macrophages in the infected sites until 3 days after infection. However, at 7 days after infection inhibition of the influx of these cells was not seen. CONCLUSIONS: These results suggest that ICAM-1 expression is transient and plays a key role in the influx of neutrophils and macrophages associated with the early-phase response, and that in the late phase ICAM-1 independent adhesion molecules may be more predominant.     

Vascular adhesion molecules and immunogenicity in blood vessels used as coronary artery bypass grafts

OBJECTIVE: The performance of coronary bypass grafts can be affected by a variety of circulating cell types. The initial event in any biological effect of such cells is adherence to the vascular endothelium prior to migration into the perivascular space. We aimed to investigate the expression of molecules that regulate cell adhesion in blood vessels employed as bypass conduits. METHODS: Segments of human saphenous vein, internal mammary artery, gastroepiploic artery and inferior epigastric artery were stained using specific monoclonal antibodies against the endothelial workers EN-4, Pal-E, von Willebrand factor small (vWF), and the cell adhesion molecules platelet-endothelium cell adhesion molecule (PECAM), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, the leucocyte marker (CD45) and major histocompatibility complex (MHC) class I and II antigens, with visualisation by ABC immunoperoxidase method. RESULTS: All vessels had a strong expression of the endothelial specific antigens EN4, vWF, and PECAM as well as MHC class I. However, there was less expression of Pal-E, ICAM-1, E-Selectin and of the DR determinant of MHC class II. VCAM-1, DP and DQ determinants of MHC class II were expressed to a weaker extent. There were no marked differences in the expression of all the molecules examined between the four vessel types. CONCLUSION: Thus vessels used as bypass grafts are immunogenic and possess the potential to attract and interact with blood elements. Definition of the molecules responsible could offer opportunities for modulating the response to such interactions.