Laurelee Osborn

VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.

Cytokine-activated human endothelial cells express vascular cell adhesion molecule-1 (VCAM-1), which binds lymphocytes. We now identify the integrin VLA-4 as a receptor for VCAM-1 because VLA-4 surface expression on K-562 cells (following transfection of the VLA alpha 4 subunit cDNA) resulted in specific cell adhesion to VCAM-1, and anti-VLA-4 antibodies completely inhibited VCAM-1-dependent cell-cell attachment. In addition, VLA-4 expression allowed K-562 cells to attach to the heparin II binding region (FN-40) of fibronectin. However, VLA-4/VCAM-1 and VLA-4/FN-40 interactions are readily distinguishable: only the former was inhibited by the anti-VLA-4 monoclonal antibody HP1/3, and only the latter was inhibited by soluble FN-40. The VCAM-1/VLA-4 ligand-receptor pair may play a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo.

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.

A blocking monoclonal antibody to endothelial-leukocyte adhesion molecule-1 (ELAM1)

ELAM1 is a leukocyte adhesion molecule induced on human umbilical vein endothelial cells (HUVECs) by inflammatory cytokines. Balb/C mice were immunized with COS cells transiently expressing cell-surface ELAM1 after transfection with ELAM1 cDNA. After fusion, ELAM1-specific monoclonal antibodies (Mabs) were identified by selective adhesion to ELAM1-expressing, but not control, CHO cells, and to cytokine-treated but not untreated HUVECs. One Mab, designated BB11, binds to and immunoprecipitates ELAM1 expressed on HUVECs, COS and CHO cells. BB11 blocks the interaction of ELAM1 with human PMN, the human myelomonocytic cell line HL60, and the human colon carcinoma line HT29.

Effect of Divalent Cations on the Affinity and Selectivity of α4 Integrins Towards the Integrin Ligands Vascular Cell Adhesion Molecule-1 and Mucosal Addressin Cell Adhesion Molecule-1: Ca 2+ Activation of Integrin α4β1 Confers a Distinct Ligand Specificity

A microtiter plate assay measuring the binding of cells expressing integrins f 4 g 1 or f 4 g 7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin g -chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn 2+ and Mg 2+ supported binding of either integrin to either ligand. Ca 2+ supported only the binding of f 4 g 1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC 50 ) ranged from 0.8-280 w M for Mn 2+ and 0.8-30 mM for Mg 2+, being thus 2-3 logs lower for Mn 2+ compared to Mg 2+ independent of ligand. EC 50 values for binding of f 4 g 1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while f 4 g 7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via f 4 g 1 was markedly lower with Mn 2+ versus Mg 2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn 2+ and Mg 2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated f 4 g 1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated f 4 g 7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca 2+ also binds to regulatory sites on both integrins. However, the Ca 2+ -activated state of f 4 g 1 is distinct from that achieved by Mn 2+ and Mg 2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.

Expression of Vascular Cell Adhesion Molecule-1 by Follicular Dendritic Cells

Follicular dendritic cells are the major supporting cell of the germinal center microenvironment. The major function of follicular dendritic cells is to present antigen to B cells in secondary lymphoid tissues. Through cell-cell interactions, FDCs are hypothesized to be central to the regulation of normal B cell growth and differentiation. The major receptor-ligand pair which mediates B cell-FDC adhesion is the beta 1 integrin VLA-4, present on B cells and VCAM-1 expressed on FDCs. Follicular non-Hodgkins lymphomas similarly employ this mechanism to bind to neoplastic germinal centers. The VCAM-1 molecule can exist as a 6 or 7 immunoglobulin domain form. The major form of VCAM-1 on activated endothelium is the 7 domain form. In this report we have determined by polymerase chain reaction of purified FDCs that they express predominantly mRNA for 7 domain VCAM-1. It is likely that the two forms of VCAM-1 are associated with distinct functions, therefore the expression of 7 domain VCAM-1 may be important in normal and neoplastic B cell-FDC interactions.