Identification of intercellular cell adhesion molecule 1 (ICAM-1) as a hypoglycosylation marker in congenital disorders of glycosylation cells

Many human inherited disorders cause protein N-glycosylation defects, but there are few cellular markers to test gene complementation for such defects. Plasma membrane glycoproteins are potential biomarkers because they may be reduced or even absent in plasma membranes of glycosylation-deficient cells. We combined stable isotope labeling by amino acids in cell culture (SILAC) with linear ion trap mass spectrometry (LTQ Orbitrap(TM)) to identify and quantify membrane proteins from wild-type CHO and glycosylation-deficient CHO (Lec9) cells. We identified 165 underrepresented proteins from 1447 unique quantified proteins, including 18 N-glycosylated plasma membrane proteins. Using various methods, we found that intercellular cell adhesion molecule 1 (ICAM-1) was reduced in Lec9 cells and in fibroblasts from 31 congenital disorder of glycosylation (CDG) patients compared with normal controls. Mannose supplementation of phosphomannose isomerase-deficient CDG-Ib (MPI-CDG) cells and complementation with PMM2 in PMM2-deficient CDG-Ia (PMM2-CDG) cells partially corrected hypoglycosylation based on increased ICAM-1 presence on the plasma membrane. These data indicate that ICAM-1 could be a useful hypoglycosylation biomarker to assess gene complementation of CDG-I patient cells and to monitor improved glycosylation in response to therapeutic drugs.     

A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1

Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions.     

Organic dust-induced activation, adhesion to substrate and expression of intercellular adhesion molecules in THP-1 monocytes

Inhalation of organic dust in a swine confinement building induces systemic reactions, increased bronchial responsiveness and intense airway inflammation in previously unexposed, healthy subjects. These effects are self-limiting, but chronic respiratory symptoms are frequently observed in swine confinement workers. The present study was aimed at investigating organic dust-induced activation of the monocytic leukemia cell line, THP-1. Unstimulated THP-1 cells proliferate in suspension but cultivation for several days in medium with complete dust or 0.22-mu-filtered suspension, caused a subset of the THP-1 cells to adhere to the substratum. As assessed by transmission light- and indirect immunofluorescence microscopy, dust-stimulated adherent THP-1 cells adopted macrophage-like morphology and expressed vimentin. Intercellular adhesion molecule (ICAM)-1 was expressed in all dust-activated adherent cells, but only in 1% of the unstimulated cells in suspension. Sialoadhesin, a macrophage marker, was detected in dust-stimulated adherent THP-1 cells but not in the parental monocytes. Serum factors were required for the dust-induced expression of sialoadhesin, but not for adhesion to substrate or expression of ICAM-1. In addition, morphology and expression of vascular endothelial growth factor (VEGF) of dust-stimulated adherent cells equalled that of PMA-differentiated THP-1 cells, but the PMA-differentiated cells exhibited weak sialoadhesin labelling. In conclusion, exposure to organic dust from a swine confinement building activated a subset of THP-1 monocytes inducing expression of intercellular adhesion molecules, which are important in inflammation. The sustained adhesion to substrate indicates that organic dust from a swine confinement building may contain agents that prevent deactivation and detachment of the cells.     

Molecular cloning of murine intercellular adhesion molecule (ICAM-1).

We have previously reported a murine lymphocyte surface antigen MALA-2 of approximately 95,000 Mr which is expressed mainly on activated lymphocytes. The rat monoclonal antibody YN1/1 that detects this antigen profoundly inhibits mixed lymphocyte response. We have now purified MALA-2 and determined its partial amino acid sequence. By using non-redundant synthetic oligonucleotides as probes, based on the amino acid sequence, we have isolated two full length cDNA clones encoding MALA-2. The two clones are identical except for the 5' end sequence. Expression of MALA-2 on transfected COS cells is only achieved with one of the two cDNA clones. The nucleotide sequence as well as the deduced amino acid sequence of MALA-2 display striking homology with those of the recently reported human intercellular adhesion molecule ICAM-1. All the unique features of the human ICAM-1, including its homology with the neural adhesion molecule NCAM, its internal repeat structure and the immunoglobulin-like structure, are found in MALA-2. Furthermore, purified MALA-2 crosslinked to a solid support binds Con A blasts that express LFA-1, the putative receptor for ICAM-1, and the binding can be blocked by YN1/1 antibody or antimurine LFA-1 antibody indicating a direct interaction of these molecules in cell adhesion. Therefore, we consider MALA-2 to be the murine homolog of human ICAM-1. Since ICAM-1 is known to be of primary importance in immune responses and inflammatory reactions, having a monoclonal antibody and a mouse model will provide the opportunity to study the functional role of ICAM-1 in vivo.     

A truncated recombinant intercellular adhesion molecule-1 inhibits adhesion of leukemic cell lines to upregulated endothelial cells

Summary Intercellular adhesion molecule-1, a member of the immunoglobulin supergene family, is the ligand for the integrin lymphocyte function associated antigen-1. Intercellular adhesion molecule-1 and lymphocyte function associated antigen-1 binding interactions mediate leukocyte adherence and migration. Previous work has shown that the adherence of lymphocyte function associated antigen-1 is directed to the first immunoglobulinlike domain of the endothelial cell surface protein intracellular adhesion molecule-1. We have constructed a truncated intercellular adhesion molecule-1 gene encoding the first 185 amino acids from the amino terminus and overexpressed it inEscherichia coli. The recombinant protein was purified from insoluble inclusion bodies and refolded into an active conformation by a denaturation/renaturation cycle. The identity of the protein was confirmed by microsequencing and by Western blot analysis using a polyclonal antibody to ICAM-1. We have demonstrated that this soluble region of the otherwise membrane-bound ligand is an inhibitor of Molt or HL-60 cell adhesion to cytokine-stimulated endothelial cells.     

Isolation of the murine intercellular adhesion molecule 1 (ICAM-1) gene. ICAM-1 enhances antigen-specific T cell activation

Cell adhesion molecules in the immune system are believed to play an important role in lymphocyte-target cell conjugate formation. One such molecule, intercellular adhesion molecule 1 (ICAM-1), is important in the function, aggregation, and adherence of leukocytes. Here, we report the isolation and characterization of the murine ICAM-1 gene. We report that the murine ICAM-1 gene is a member of the Ig gene superfamily, has limited homology to its human counterpart, and is expressed in cells of lymphocytic and myeloid lineages. Transfection of the ICAM-1 cDNA into MHC class II-transfected fibroblasts leads to enhancement of the Ag-specific T cell response when the transfectants are used as APC.     

Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses.     

细胞间粘附分子-1在血管内皮细胞冻融损伤中的作用

目的:探讨血管内皮细胞(VEC)表面细胞间粘附分子-1(ICAM-1)在VEC冻融损伤中的作用,以阐明冻融损伤的发病机制。方法:以大鼠主动脉VEC和大鼠外周血嗜中性粒细胞(PMN)为材料,使用WKL-Ⅴ型速率冷冻仪冷冻VEC然后在水浴中复温,制备VEC冻融模型。采用免疫组化法测定VEC冻融后4、12和24 h其表面ICAM-1的表达;将冻融VEC与正常PMN共同孵育后,以rose bengal染色法测定冻融VEC与PMN粘附,测定培养液中LDL活性确定VEC损伤程度。结果:冻融后4 h,VEC表面ICAM-1表达阳性率由冻融前的13.2%±3.6%增加至22.3%±4.4%,冻后12h达高峰(37.9%±2.5%)。冻融VEC与PMN共同孵育后,VEC-PMN粘附由对照组的0.204±0.025增加至0.363±0.022(P<0.01),培养液中LDH活性由对照组的104.64±20.14U/L增加至162.33±27.88U/L(P<0.01);ICAM-1Mab可部分阻断冻融VEC-PMN粘附(0.270±0.021,P<0.01),且使培养液中LDH活性降低至125.39±22.26U/L(P<0.05)。结论:冻融可诱发VEC表面ICAM-1的表达,进而增强VEC-PMN粘附而导致VEC损伤。     

In vitro analysis of the melanoma/endothelium interaction increasing the release of soluble intercellular adhesion molecule 1 by endothelial cells

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r ² = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1α (IL-1α) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1α/β neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1α secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines. Received: 15 October 1998 / Accepted: 17 February 1999     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

The clinical value of assaying maternal serum and amniotic fluid intercellular adhesion molecule 1 (ICAM-1) in cases of premature rupture of membranes

Intercellular adhesion molecule 1 (ICAM-1) expression and upregulation induced by pro-inflammatory cytokines may be of interest in defining human response to inflammation and infection. This study was initiated to determine the levels of ICAM-1 in sera and amniotic fluid of cases of premature rupture of membranes (PROM). Serum and amniotic fluid ICAM-1 levels were determined by ELISA in 33 cases of PROM and 10 cases of normal pregnancies of matched gestational age (controls). Both serum and amniotic fluid ICAM levels were significantly elevated in 76% and 85% of cases of PROM with mean fold increments of 3.13 and 3.95, respectively. This elevation was associated with intra-amniotic infection which was detected by microbiological culture and histopathological evidence of chorioamnionitis. Increased ICAM-1 in cases of PROM may be attributed to neutrophil activation and ICAM-1 expression on fetal membranes and mononuclear cells of amniotic fluid. These results demonstrate that determination of ICAM-1 may be a valuable biomarker for early detection of acute chorioamnionitis and the possibility of PROM.     

G241R and K469E polymorphisms of intercellular adhesion molecule 1 (ICAM-1) could predispose to Hashimoto thyroiditis

This study examined firstly the possible association of G241R and K469E single nucleotide polymorphisms (SNPs) of ICAM-1 gene with the occurrence of Hashimoto thyroiditis (HT). G241R and K469E SNPs in DNA from peripheral blood leukocytes of 190 HT and 247 healthy controls were investigated by real-time PCR combined with melting curve analysis using fluorescence-labeled hybridization probes. There was a significant increase of ICAM-1 241R allele frequency in patients with HT compared with healthy controls (P?=?0.04, OR?=?1.84, 95?% CI?=?1.00–3.37). Regarding ICAM-1 K469E polymorphism, patients homozygous for E allele had 1.73–fold increased risk for developing HT according to KK homozygotes (P?=?0.04, 95?% CI?=?1.00–3.01). The 469E allele frequency was higher in HT patients according to controls, however the difference was at borderline significance (P?=?0.05, OR?=?1.30, 95?% CI?=?1.00–1.70). No associations between polymorphisms and HT phenotypes were observed. We suggest that the G241R and K469E SNPs of ICAM-1 gene may be related to occurrence of HT. However, more studies with larger sample size including other loci of the ICAM-1 gene are necessary to support our findings before any definite statement can be made about the relationship between HT and ICAM-1 polymorphism.     

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate beta(2) integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of approximately 75 pm IL-8, corresponding to ligation of only approximately 10-100 receptors, was sufficient to activate approximately 20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.     

Cariporide inhibits high glucose-mediated adhesion of monocyte-endothelial cell and expression of intercellular adhesion molecule-1

The aim of this study was to examine whether cariporide, a new inhibitor of Na(+)/H(+) exchanger 1 (NHE-1), may inhibit high glucose-induced monocyte-endothelial cell adhesion and the expression of intercellular adhesion molecule-1 (ICAM-1). Cultured endothelial cells were incubated with normal glucose control (5.5 mM), cariporide control (5.5 mM glucose plus 10 microM cariporide), hyperosmolarity (5.5 mM glucose plus 16.5 mM mannitol), high glucose (HG, 22 mM), low-concentration cariporide (22 mM glucose plus 0.1 microM cariporide), medium-concentration cariporide (22 mM glucose plus 1 muM cariporide), and high-concentration cariporide (22 mM glucose plus 10 microM cariporide) for 24 h. Monocytes were isolated from peripheral human blood. Adhered monocytes were quantified by measuring their protein content. ICAM-1 expression and NHE-1 activity was determined with enzyme-linked immunosorbent assay (ELISA) and pH-sensitive fluorescent spectrophotometry. Exposure of endothelial cells to HG for 24 h caused an increase of adhesion of monocytes to endothelial cells and an increased expression of ICAM-1. However, these effects were reversed by treatment with cariporide (0.1, 1, 10 microM) in a concentration-dependent manner. Furthermore, cariporide (1 microM) was able to inhibit the activation of NHE-1 induced by HG in endothelial cells. These findings suggest that cariporide might inhibit HG-mediated monocyte-endothelial cell adhesion and expression of ICAM-1 by inhibiting the activation of NHE-1.     

细胞间粘附分子1的研究进展

细胞间粘附分子1(ICAM-1),又名CD54,是一种重要的细胞表面粘附分子,属免疫球蛋白超家族．它可与鼻病毒以及整合素家族成员结合,参与炎症,普通感冒,变态反应及移植排斥反应．文章就其细胞分布、表达调节、结构功能、基因工程以及临床应用进行了综述．     

Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3)

A cDNA encoding a putative bovine intercellular adhesion molecule (ICAM)-3, a ligand of the leukocyte integrin LFA-1 (CD11a/CD18), was sequenced and compared with human ICAM sequences. The 1635-bp bovine sequence codes for a protein of 544 amino acids (aa). This putative bovine ICAM-3 has five immunoglobulin (Ig)-like domains similar to human ICAM-1 and ICAM-3, and belongs to the Ig gene superfamily. The overall identities of the deduced aa sequence with those of human ICAM-3 and ICAM-1 are 61% and 58%, respectively. The predicted number and positions of Cys residues are all conserved between the bovine and human ICAM 3 aa sequences.     

Expression of intercellular adhesion molecule 1 (ICAM-1) on the human oviductal epithelium and mediation of lymphoid cell adherence

The epithelium of the human oviduct expresses the major histocompatibility complex (MHC) class II and shows endocytic properties towards luminal antigens. Therefore, the epithelial cells might behave as antigen-presenting cells, inducing a local immune response. The activation of antigen-specific T cells not only requires presentation of the peptide antigen by MHC class II, but also the presence of co-stimulatory molecules in the antigen-presenting cells. Therefore, the expression of the intercellular adhesion molecule 1 (ICAM-1) was examined in the epithelium of the human oviduct. Most oviducts showed epithelial ICAM-1 expression, as assessed by immunocytochemistry, western blot analysis and RT-PCR assay, and the expression was restricted to the luminal border of ciliated and secretory cells. Interferon gamma, interleukin 1 and lipopolysaccharide treatments increased the percentage of ICAM-1-positive cells in primary cultures, indicating that the expression of ICAM-1 in the oviduct might be upregulated in vivo by inflammatory cytokines or bacterial infections. Binding assays between allogenic phytohaemagglutinin-activated lymphocytes and epithelial monolayers expressing ICAM-1 demonstrated that this molecule stimulated lymphocyte adherence. The presence of ICAM-1, in addition to MHC class II, supports the putative role of the oviductal epithelium in antigen presentation. The exclusive apical distribution of ICAM-1 indicates that T-cell activation would occur in a polarized manner. Binding of lymphoid cells to the surface of the oviductal epithelium may help to retain these immune cells that are required for the clearance of pathogens.     

Small GTPase Rho signaling is involved in beta1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation

We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. Beta1 Integrin were highly expressed on osteoblasts. Engagement of beta1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on osteoblasts. Rho was activated by beta1 stimulation in osteoblasts. Beta1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of beta1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in beta1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.     

ICAM-1-mediated adhesion is a prerequisite for exosome-induced T cell suppression

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