The inhibitory effect of alacepril, an angiotensin-converting enzyme inhibitor, on endothelial inflammatory response induced by oxysterol and TNF-alpha

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-alpha. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-alpha-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-alpha. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-alpha.     

The inhibitory effect of alacepril,an angiotensin-converting enzyme inhibitor,on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.     

ICAM-1 and CD11b/CD18 expression during acute pancreatitis induced by bile-pancreatic duct obstruction: effect of N-acetylcysteine

Different molecules are involved in the recruitment of leukocytes during inflammation. The aim was to investigate (i) the contribution of acinar cells to the overall production of ICAM-1 and (ii) the kinetics of leukocyte CD11b/CD18 expression during acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) to evaluate the contribution of both molecules to leukocyte homing. The role of reactive oxygen species (ROS) as mediators in the expression of ICAM-1 and CD11b/CD18 was examined by using N-acetylcysteine (NAC) as an antioxidant treatment. By mechanisms resistant to NAC treatment, acinar cells were able to produce ICAM-1 at first onset of AP; other cell sources contribute to maintaining increased ICAM-1 plasma levels during AP. By contrast, CD11b/CD18 was overexpressed in leukocytes in the course of AP by oxidant-dependent mechanisms. Since NAC treatment reduced neutrophil infiltration in the pancreas, we conclude that CD11b/CD18 over-expression is required for leukocyte recruitment; however, other adhesion molecules in addition to ICAM-1 seem to contribute to leukocyte homing during BPDO-induced AP.     

The enhanced monocyte adhesiveness after UVB exposure requires ROS and NF-kappaB signaling in human keratinocyte

The infiltration of both monocyte and activated T cells in the skin is one of critical steps in the development of UVB-induced inflammation. Upregulation of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) on the surface of keratinocytes plays an important role in this process. In this study, we examined the molecular mechanism responsible for UVB-induced expression of ICAM-1 and subsequent monocyte adhesion by keratinocyte. We observed that (1) UVB induced protein and mRNA expression of ICAM-1 in a dose- and time-dependent manner in human keratinocyte cell HaCaT; (2) UVB induced the translocation of NF-kappaB and inhibition of NF-kappaB by NF-kappaB inhibitors suppressed UVB-induced mRNA and protein expression of ICAM-1; (3) UVB increased the intracellular level of reactive oxygen species (ROS) by HaCaT cells; (4) UVB-induced increase of intracellular ROS level was suppressed by pretreatment with diphenyl iodonium (DPI) and N-acetyl cysteine (NAC); and (5) inhibition of UVB-induced ROS production by DPI or NAC suppressed UVB-mediated translocation of NF-kappaB, expression of ICAM-1 and subsequent monocyte adhesion in HaCaT cells. These results suggest that UVB-induced ROS is involved in the translocation of NF-kappaB which is responsible for expression of ICAM-1 and subsequent increased monocyte adhesion in human keratinocyte.     

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.     

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.     

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

Abstract

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, α-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.     

A systemic lupus erythematosus-associated R77H substitution in the CD11b chain of the Mac-1 integrin compromises leukocyte adhesion and phagocytosis

The Mac-1 integrin is expressed mainly on myeloid cells and binds several ligands, including members of the ICAM family and the complement factor iC3b. It is involved in essential immunological processes, such as leukocyte extravasation and phagocytosis. In addition, Mac-1 has been described to negatively regulate immune cell signaling. Recently, a single nucleotide polymorphism conferring an amino acid change in the Mac-1 integrin extracellular domain, R77H, was shown to be associated with systemic lupus erythematosus. Here, we demonstrate that the R77H-substituted Mac-1 can be expressed on the cell surface in transfected cells and can undergo conformational changes in response to integrin activation. The affinity of the integrin for ICAMs is only partially reduced, but cell adhesion to ICAM-1 and ICAM-2 is severely compromised, and Jβ2.7 cells expressing R77H substituted integrins are deficient in adhesion to ICAM-1 under shear flow conditions. Importantly, cell adhesion to the complement factor iC3b is also diminished, and COS cells expressing R77H-substituted integrins display reduced iC3b-dependent phagocytosis. In addition, U937 cells expressing R77H-CD11b display increased IL-6 production as compared with WT-CD11b-expressing cells. These results suggest that the R77H substitution results in the deficiency of the mutated integrin to mediate cell adhesion to ligands such as ICAMs and iC3b. These deficiencies may ultimately lead to detrimental effects on the immune system and contribute to the development of systemic lupus erythematosus.     

Adhesion-dependent interactions between eosinophils and cholinergic nerves

Eosinophils adhere to airway cholinergic nerves and influence nerve cell function by releasing granule proteins onto inhibitory neuronal M(2) muscarinic receptors. This study investigated the mechanism of eosinophil degranulation by cholinergic nerves. Eosinophils were cocultured with IMR32 cholinergic nerve cells, and eosinophil peroxidase (EPO) or leukotriene C(4) (LTC(4)) release was measured. Coculture of eosinophils with nerves significantly increased EPO and LTC(4) release compared with eosinophils alone. IMR32 cells, like parasympathetic nerves, express the adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (ICAM-1). Inhibition of these adhesion molecules alone or in combination significantly inhibited eosinophil degranulation. IMR32 cells also significantly augmented the eosinophil degranulation produced by formyl-Met-Leu-Phe. Eosinophil adhesion to IMR32 cells resulted in an ICAM-1-mediated production of reactive oxygen species via a neuronal NADPH oxidase, inhibition of which significantly inhibited eosinophil degranulation. Additionally, eosinophil adhesion increased the release of ACh from IMR32 cells. These neuroinflammatory cell interactions may be relevant in a variety of inflammatory and neurological conditions.     

Distinct ICAM-1 forms and expression pathways in synovial microvascular endothelial cells.

Human synovial endothelial cell (HSE) intracellular adhesion molecule-1 (ICAM-1) is upregulated maximally by synergy of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Such synergy is not as pronounced in human umbilical vein endothelium (HUVE). ICAM surface staining and ELISA detection reflected similar levels on HUVE and HSE cells, yet mRNA levels were much higher in HSE cells in response to TNF alpha/IFN gamma. To correlate protein and mRNA levels of ICAM-1, both cell types were permeabilized and stained with a monoclonal antibody against ICAM-1. HSE cells displayed a distinct vesicular cytoplasmic staining for ICAM while HUVE cells were devoid of such stained vesicles upon staining with the antibody. ICAM-1 immunostaining of HSE cytoplasmic vesicles appeared enhanced in cells treated with TNF alpha/IFN gamma and monensin, an endosomal processing inhibitor. Monensin inhibited HSE cell surface expression of ICAM-1 routinely up to 70%, while HUVE cell expression was unaffected. In addition, monensin also inhibited soluble ICAM-1 release from HSE cells while not effecting HUVE cells. Immunoprecipitation of ICAM-1 followed by gel electrophoresis indicated that HUVE and HSE cell ICAMs are expressed in cell-specific forms. These results define distinct forms and distinct secretory pathways for ICAM-1 in HSE cells and HUVE cells that indicate functional differences between these human endothelia.     

Mast cells induce upregulation of P-selectin and intercellular adhesion molecule 1 on carotid endothelial cells in a new in vitro model of mast cell to endothelial cell communication

It is suggested that mast cells contribute to cell recruitment in inflammation through the upregulation of endothelial adhesion molecules. P-selectin and intercellular adhesion molecule(ICAM)-1 are two key adhesion molecules that have been associated indirectly with mast cell activity. The canine C2 mastocytoma cell line and primary cultures of canine carotid endothelial cells were used to establish a new in vitro model to help study the interaction between mast cells and endothelial cells. Carotid endothelial cells were incubated with mast cell mediators to uncover their effect on endothelial ICAM-1 and P-selectin expression. To assess the relative contributions of tumour necrosis factor (TNF)-alpha and histamine to such effect, an H1 antihistamine and a TNF-alpha blocking antibody were used. Prior to activation by mast cell mediators, P-selectin was expressed only within the cytoplasm, and ICAM-1 was constitutively expressed on the surface of the canine carotid endothelial cells. Both adhesion molecules were enhanced significantly and strongly upon mast cell activation at various time points. Unstored TNF-alpha was fully responsible for ICAM-1 upregulation. P-selectin was up-regulated by both preformed and newly synthesized mast cell mediators, but neither histamine nor TNF-alpha accounted for such an effect. Therefore,a new model is proposed in which the pro-inflammatory effect of mast cells on endothelial cells can be studied in vitro.In this model, it has been demonstrated that only TNF-alpha accounts for the overexpression of ICAM-1 induced by mast cells, and that mast cells up-regulate P-selectin expression through a histamine-independent mechanism.     

Interleukin-1α Released from Epithelial Cells after Adenovirus Type 37 Infection Activates Intercellular Adhesion Molecule 1 Expression on Human Vascular Endothelial Cells

A key event in virus-induced inflammation (leukocyte extravasation through the endothelium) is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. In order to identify triggers of inflammation in adenovirus infection, we inoculated respiratory and ocular epithelial cells with adenovirus type 37 (Ad37), a human pathogen associated with keratoconjunctivitis as well as urogenital and respiratory infections. Fluids from virus-infected epithelial cells activated ICAM-1 (and to a lesser extent, VCAM-1) expression on cultured human umbilical vein endothelial cells. Blocking studies with anticytokine antibodies implicated interleukin-1alpha (IL-1alpha) as the epithelial cell-derived factor which activated endothelial cell ICAM-1 expression. The results thus identify epithelial cell-derived IL-1alpha as a potentially important activator of endothelial cells in Ad37-induced inflammation.     

Family with Sequence Similarity 5, Member C (FAM5C) Increases Leukocyte Adhesion Molecules in Vascular Endothelial Cells: Implication in Vascular Inflammation

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microcopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity.     

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.     

Extracts from cigarette smoke induce DNA damage and cell adhesion molecule expression through different pathways

Cigarette smoke is a major risk factor for human diseases, such as lung cancer and atherosclerosis. The present study was undertaken to investigate the effect of non-fractionated water-soluble cigarette smoke extract (NFWS CSE) on DNA damage and cellular adhesion molecule expression in human umbilical vein endothelial cells (HUVECs). DNA damage and the surface expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin were determined by the use of the comet assay and flow cytometry, respectively. NFWS CSE-induced DNA damage in a dose-dependent manner during a 2 h exposure. Pretreatment with ascorbic acid or -tocopherol completely inhibited the NFWS CSE-induced DNA damage. NFWS CSE exposure also up-regulated the surface expression of ICAM-1 and E-selectin in HUVECs. Pretreatment with ascorbic acid or -tocopherol had no effect on NFWS CSE-induced E-selectin and ICAM-1 expression. In contrast, the non-antioxidant metal chelator 1,10-phenanthroline partially suppressed the surface expression of ICAM-1 and E-selectin. These results suggest that NFWS CSE exposure induces both DNA damage and the surface expression of adhesion molecules in HUVECs. However, the molecular mechanism of these effects may be through different pathways: reactive oxygen species are involved in NFWS CSE-induced DNA damage but have little relation to NFWS CSE-induced E-selectin and ICAM-1 expression.     

Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types

The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H₂O₂ production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H₂O₂ levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC. phenotype; reactive oxygen species; inflammation; shear stress     

Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34(+) cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34(+) cells in response to stromal cell-derived factor-1alpha. Stromal cell-derived factor-1alpha-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34(+) cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.