Gloria Chi-Rosso

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.

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.

Regulation of inflammation by collagen-binding integrins α1β1 and α2β1 in models of hypersensitivity and arthritis

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAbs against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

The alpha1beta1 integrin is expressed during neointima formation in rat arteries and mediates collagen matrix reorganization.

Remodeling of the extracellular matrix by activated mesenchymal cells (myofibroblasts) is a critical aspect of wound repair in all adult organs. Collagen-dependent gel contraction, a process requiring integrin function, is an established in vitro assay thought to mimic in vivo matrix remodeling. Numerous data have implicated the alpha2beta1 integrin in various cell types as the primary collagen receptor responsible for collagen gel contraction. However, evidence from the literature suggests that the major collagen binding integrin expressed on mesenchymally derived cells in situ is the alpha1beta1 integrin, not the alpha2beta1 integrin. In this report, we use a rat vascular injury model to illustrate that the alpha1beta1 integrin is the major collagen receptor expressed on vascular smooth muscle cells after injury. Using two smooth muscle cell lines, expressing either the alpha1beta1 integrin alone or both the alpha1beta1 and alpha2beta1 integrins, along with Chinese hamster ovary cells transfected with the alpha1 integrin, we demonstrate that alpha1beta1 supports not only collagen-dependent adhesion and migration, but also gel contraction. These data suggest that in vivo the alpha1beta1 integrin is a critical collagen receptor on mesenchymally derived cells potentially involved in matrix remodeling after injury.

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.

Fibronectin Type III Repeats Mediate RGD-independent Adhesion and Signaling through Activated β1 Integrins

Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of β1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and β1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated β1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.

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.

Epitope mapping of a function-blocking beta 1 integrin antibody by phage display.

Integrins are a major class of cell surface receptors involved in cell-cell and cell-matrix adhesion and communication. Ha2/11 is a function-blocking anti-rat beta 1 integrin hamster IgM that should be a useful reagent for understanding beta 1 integrin function. We demonstrate that Ha2/11 cross reacts with human, Xenopus, and Drosophila beta 1 integrins, and use phage display to map the epitope for Ha2/11 to residues within the sequence LRSGEPQTF which lies 18 amino acids proximal to the putative I domain in beta 1 integrins. Monoclonal antibody mapping experiments, mutational analyses, and direct binding assays have implicated integrin I domains in both cation and ligand binding. Our data therefore suggest that Ha2/11 blocks beta 1 integrin function by interfering with I domain-mediated ligand binding.