Catherine Hession

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.

Isolation of the bovine and human genes for müllerian inhibiting substance and expression of the human gene in animal cells

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.

Lymphotoxin β, a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface

The lymphokine tumor necrosis factor (TNF) has a well-defined role as an inducer of inflammatory responses; however, the function of the structurally related molecule lymphotoxin (LT alpha) is unknown. LT alpha is present on the surface of activated T, B, and LAK cells as a complex with a 33 kd glycoprotein, and cloning of the cDNA encoding the associated protein, called lymphotoxin beta (LT beta), revealed it to be a type II membrane protein with significant homology to TNF, LT alpha, and the ligand for the CD40 receptor. The gene for LT beta was found next to the TNF-LT locus in the major histocompatibility complex (MHC), a region of the MHC with possible linkage to autoimmune disease. These observations raise the possibility that a surface LT alpha-LT beta complex may have a specific role in immune regulation distinct from the functions ascribed to TNF.

Two human 35 kd inhibitors of phospholipase A2 are related to substrates of pp60v-src and of the epidermal growth factor receptor/kinase

We have purified two 35 kd phospholipase A2 inhibitors from human placenta, which we refer to as lipocortin I and II. Both proteins exhibit similar biochemical properties and occur in placenta at about 0.2% of the total protein. By peptide mapping, sequence, and immunological analyses, we show that lipocortin I and the 35 kd substrate for the EGF-receptor/kinase from A431 cells are the same protein. By similar criteria, we determine that lipocortin II is the human analogue of pp36, a major substrate for pp60src, which has been characterized in chicken embryo fibroblasts and in bovine brush border preparations. The amino acid sequences of lipocortin I and II that we deduced from cDNA clones share 50% homology, indicating that they probably evolved from a common gene.

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.

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.

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.

Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxins alpha and beta.

The lymphotoxin (LT) protein complex is a heteromer of α (LT-α, also called tumor necrosis factor (TNF)-β) and β (LT-β) chains anchored to the membrane surface by the transmembrane domain of the LT-β portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-β receptor, which is generally present on nonlymphoid cells. The signaling function of this receptor-ligand pair is not precisely known but is believed to be involved in the development of the peripheral lymphoid organs. To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired. The LT-β molecule was engineered into a secreted form and co-expressed with LT-α using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-α3, LT-α2/β1, and LT-α1/β2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-α3-like components in the LT-α1/β2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-α3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors. Heteromeric LT complexes were prepared with this mutant LT-α form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors. A LT-α3 based cytotoxic activity was used to show that the LT-α1/β2 form cannot readily scramble into a mixture of forms following various treatments and storage periods. This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analysis of the specificity of the LT-β and TNF receptors for the various LT forms.

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.

Structure and Properties of a Secretable Phospholipase A2 from Human Platelets

Phospholipases A2 (PLA2S) constitute a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphoglycerides liberating free fatty acids and lysophospholipids (Dennis, 1983). Mammalian extracellular PLA2S are abundant in pancreatic secretions, but are also present in plasma, lymph and pulmonary alveolar secretions (Vadas and Pruzanski, 1986). Intracellular PLA2s are found in all tissues and cells (van den Bosch, 1980), where they are located either in the cytosolic compartment associated with the plasma membrane or stored within organelles of the vacuolar system, such as secretory granules and lysosomes. The granule-associated PLA2s are designed to effectively degrade phospholipids upon exocytosis thereby serving either a digestive (Verheij et al., 1981) or an anti-microbial function (Elsbach, 1980). In contrast, the cytosolic PLA2s play a key role in the metabolism of cellular lipids, including the biosynthesis of specifically tailored phospholipids and the degradation of peroxidized phospholipids thus protecting membranes from oxidation damage (Waite, 1987). Furthermore, such PLA2s are involved in the generation of rate-limiting precursors for various types of lipid mediators (Irvine, 1982; Snyder, 1985) that transmit stimulatory signals to other cells or function as intracellular messengers (O’Flaherty, 1982; Larsen and Henson, 1983). This amplification mechanism is an integral part of the inflammatory response of tissues to injury that normally leads to removal of the inciting agent and repair of injured site.