Biomedical subjects

R Lobb

Publications and source records attributed to R Lobb.

30 records · Page 2Linked to original sources

PubMed1992

Cloning of murine and rat vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.

Amino Acid Sequence↗

PubMed1992

Expression of vascular adhesion molecules in inflammatory bowel disease.

The expression of the vascular adhesion molecules ELAM-1 (endothelial leukocyte adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1) was evaluated in colonic mucosa of patients with inflammatory bowel disease and normal controls by immunocytochemistry. VCAM-1 was found to be constitutively expressed in lymphoid aggregates in normal colonic mucosa and was not significantly enhanced or altered in distribution in mucosa of patients with inflammatory bowel disease regardless of the activity of the inflammatory process. In contrast, ELAM-1 was not detected by these techniques in normal colonic mucosa (n = 11) or in colonic mucosa of patients with inflammatory bowel disease which was either uninvolved or quiescent (n = 30). However, high levels of ELAM-1 were consistently found on endothelial surfaces in association with active inflammation in affected areas of colonic mucosa in patients with either ulcerative colitis (n = 27) or Crohn's colitis (n = 9). In addition, ELAM-1 appeared to be present within neutrophils which had migrated into crypt abscesses in affected mucosa. Similar analysis was carried out in the cotton-top tamarin (CTT), a primate that experiences an idiopathic chronic diffuse colitis resembling human ulcerative colitis. Although anti-human VCAM-1 antibodies did not react with the CTT, anti-human ELAM-1 stained endothelial surfaces in mucosal biopsies from CTT with active colitis. No ELAM-1 was identified in mucosa of CTT in which colitis activity was quiescent. Thus ELAM-1 is expressed on colonic endothelial surfaces in association with inflammation and may play an important role in facilitating leukocyte migration into sites of active IBD involvement.

Cell Adhesion Molecules↗

PubMed1991

Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1.

Vascular cell adhesion molecule 1 (VCAM1) is a leukocyte adhesion molecule induced on human endothelium in vitro and in vivo by inflammatory stimuli. A truncated cDNA for VCAM1 was constructed, stably expressed in Chinese Hamster Ovary (CHO) cells, and the secreted recombinant soluble form of VCAM1 (rsVCAM1) purified to homogeneity by immunoaffinity chromatography. Immobilized rsVCAM1 is a functional adhesion protein, and selectively binds only VLA4-expressing cells, including human B and T lymphocytes, NK cells, and certain lymphoblastoid cell lines. T cell subset analyses indicate preferential binding of CD8+ memory cells. rsVCAM1 should prove valuable for the further study of the role of VCAM1 during inflammatory and immune responses in vivo.

Amino Acid Sequence↗

PubMed1991

Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription.

Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor alpha (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), we demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5' flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-kappa B and AP-1. Gel mobility shift assays demonstrate that TNF, IL-1, or LPS (but not IL-2, IL-4, IL-6, interferon gamma, histamine, or transforming growth factor beta) induces activation of NF-kappa B-like DNA binding activity in HUVEC. In contrast, neither TNF, IL-1, nor LPS activates proteins that bind to an AP-1 consensus sequence under these experimental conditions. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-kappa B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-kappa B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kappa B-like proteins but does inhibit ELAM-1 gene transcription. We conclude that PKC-independent activation of NF-kappa B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.

Base Sequence↗

PubMed1991

Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes.

The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.

Antibodies, Monoclonal↗

PubMed1991

Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils.

Two classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium. Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with ELAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18). Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3. The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractant." This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.

Antibodies, Monoclonal↗

PubMed1991

Human monocytes bind to two cytokine-induced adhesive ligands on cultured human endothelial cells: endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1) are adhesive proteins induced on endothelium by cytokines. We examined the contribution of these adhesive proteins to human peripheral blood monocyte adherence to endothelium using transfected Chinese hamster ovary (CHO) cells stably expressing these proteins and monoclonal antibodies (MoAbs) to ELAM-1, VCAM-1, or CD49d/CD29 (VLA-4), the leukocyte receptor for VCAM-1. Monocytes bound to CHO cells transfected with cDNA of ELAM-1 or VCAM-1. Binding to ELAM-1 was inhibited by MoAb to ELAM-1 and binding to VCAM-1 was inhibited by MoAb to VCAM-1 or the alpha-chain of very late activation antigen-4 (VLA-4) (CD49d). Additive inhibition of adherence to unstimulated human umbilical vein endothelium (HUVE) was observed when monocytes were pretreated with both MoAb to CD49d and MoAb to CD18, the common beta-chain of the leukocyte beta 2 integrin receptors. Adherence of monocytes to HUVE stimulated by recombinant human tumor necrosis factor-alpha was not reduced by MoAbs to CD18, CD49d, or ELAM-1 when used singly, but combinations of these MoAbs produced significant inhibition. We conclude that multiple receptor-ligand systems are involved in monocyte adherence to endothelium.

Animals↗

PubMed1991

Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1).

Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.

Amino Acid Sequence↗

PubMed1990

ELFT: a gene that directs the expression of an ELAM-1 ligand.

The LECCAMs are a family of cell adhesion molecules implicated in certain inflammatory processes. ELAM-1, a LECCAM found on the surface of activated endothelial cells, can mediate adhesion of neutrophils, monocytes, and certain cell lines to endothelial cells in vitro. No ligand for any LECCAM has yet been fully characterized. Here we report the cloning of a cDNA, ELFT (ELAM-1 ligand fucosyltransferase), that can confer ELAM-1 binding activity when transfected into nonbinding cell lines. ELFT encodes a 46 kd protein that has alpha(1,3)fucosyltransferase activity, suggesting that a fucosylated carbohydrate structure is an essential component of the ELAM-1 ligand. Furthermore, ELFT is expressed specifically in cell types that bind to ELAM-1, suggesting that this enzyme is an important regulator of inflammatory events in vivo.

Amino Acid Sequence↗

PubMed1990

Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine-activated cultured human endothelial cells.

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.

Animals↗

PubMed1989

Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes.

We have cloned a previously undescribed adhesion molecule, VCAM-1, which is induced by cytokines on human endothelial cells and binds lymphocytes. Using a novel method requiring neither monoclonal antibodies nor purified protein, VCAM-1-expressing clones were selected by adhesion to human lymphoid cell lines. VCAM-1 mRNA is present in endothelial cells at 2 hr after treatment with IL-1 or TNF-alpha and is maintained for at least 72 hr; leukocyte binding activity parallels mRNA induction. Cells transfected with VCAM-1 bind the human leukemia lines Jurkat, Ramos, Raji, HL60, and THP1, but not peripheral blood neutrophils. VCAM-1, which belongs to the immunoglobulin gene super-family, may be central to recruitment of mononuclear leukocytes into inflammatory sites in vivo.

Amino Acid Sequence↗

PubMed1986

Purification and characterization of heparin-binding endothelial cell growth factors.

Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo.

Amino Acids↗