Abstract. mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immunohistochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/~/IFN, its distribution pattern at intercellular contact rims was severely altered, mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolicaUy and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K.Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7134 antigen is an endothelialspecific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.

The expression of PECAM, ICAM-1, VCAM-1, and E-selectin was studied in 64 samples of human coronary arteries taken from 15 explanted hearts obtained within 5 min of transplantation. Normal artery (n = 12), predominantly fibrous plaques (n = 23), and plaques containing extracellular lipid (n = 26) and three segments showing recanalization channels were studied. All endothelial cells strongly and equally expressed PECAM; positive staining was used to check that artefactual denudation of the endothelial surface had not occurred. PECAM was also present in some lipid-filled macrophages. Normal arteries showed no VCAM-1 staining but focal segments of the endothelium were positive for ICAM-1 and E-selectin. ICAM-1 was strongly and constantly expressed by the endothelium over all types of plaques and in macrophages. E-selectin expression was confined to endothelial cells and occurred on the surface in 35 per cent of fibrous and 22 per cent of lipid-containing plaques. VCAM-1 staining of surface endothelium occurred in 39 per cent of fibrous and 20 per cent of lipid-containing plaques. A population of spindle-shaped cells of macrophage type (positive for EMB11 antigen) expressed VCAM-1 in lipid-containing plaques. Adventitial vessels adjacent to plaques showed endothelial expression of ICAM-1 and E-selectin. VCAM-1 staining of adventitial vessel endothelium was associated with local lymphoid aggregation. In conclusion, the expression of cell adhesion molecules is an important element in the inflammatory component of atherosclerosis and contributes to both monocyte and lymphocyte activation and recruitment from adventitial vessels and the arterial lumen.

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The antigenic status of vascular endothelium from different sites of the normal adult and fetal human cardiovascular system was investigated. Tissues included aorta (n = 9), pulmonary artery (n = 8), coronary artery (n = 6), ventricle/atrium (n = >10), lymph node (n = 2), fetal whole heart (n = 3), and umbilical cord (n = 7). Frozen sections were studied using monoclonal antibodies recognizing endothelial markers (EN4, vWf; Pal-E, and 44G4), vascular adhesion molecules (ICAM-1, ELAM, VCAM, and PECAM), the monocyte/endothelial marker (OKM5), and major histocompatibility complex (AMHC)

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The pathology of multiple sclerosis (MS) is characterised by breakdown of the blood-brain barrier accompanied by infiltration of macrophages and T cells into the central nervous system (CNS). Myelin is degraded and engulfed by the macrophages, producing lesions of demyelination. Some or all of these mechanisms might involve proteinases, and here we have studied the cellular localisation and distribution of two matrix metalloproteinases (MMPs), MMP-7 (matrilysin) and MMP-9 (92-kDa gelatinase), in the normal human CNS and active demyelinating MS lesions. Cryostat sections of CNS samples were immunostained with antisera to MMP-7 and MMP-9. In addition, non-radioactive in situ hybridisation (ISH) was performed using a digoxygenin-labelled riboprobe to detect the expression of MMP-7. MMP-7 immunoreactivity was weakly detected in microglial-like cells in normal brain tissue sections, and was very strong in parenchymal macrophages in active demyelinating MS lesions. This pattern of expression was confirmed using ISH. MMP-7 immunoreactivity was not detected in macrophages in spleen or tonsil indicating that it is specifically induced in infiltrating macrophages in active demyelinating MS lesions. MMP-9 immunoreactivity was detected in a few small blood vessels in normal brain tissue sections, whereas many blood vessels stained positive in CNS tissue sections of active demyelinating MS lesions. The up-regulation of MMPs in MS may contribute to the pathology of the disease.

An essential property of vascular endothelial cells is the maintenance of a complete hemocompatible monolayer (1) . Preserving the integrity of this monolayer requires that cell-cell contacts are established and maintained during the normal cycles ofendothelial turnover. These contacts develop through adhesion molecules expressed on the cell surface, including ubiquitous integrin receptors for fibronectin, vitronectin, collagen, and laminin, and endothelial-specific molecules such as hec7 (2) .To define further endothelial cell adhesion molecules, we generated mAbs against proliferating human umbilical vein endothelial cells (HUVEC) (3) and used them to screen HUVEC cDNA libraries transiently expressed in COS cells (4). One of these mAbs, designated 9G11, recognized a 140-kD surface glycoprotein strongly expressed at cell-cell contacts. Clones encoding 9G11 epitopes were isolated, and sequence analysis revealed that this 9G11 endothelial antigen was identical to the previously identified leukocyte antigen CD31 (D. Simmons, unpublished observations). CD31 is a widely distributed, single-chain glycoprotein of mass 130-140 kD found on leukocytes (T and B cells, monocytes, granulocytes, platelets, 4070 ofbone marrow cells), endothelial, and smooth muscle cells (5) .Here, we report the complete sequence ofCD31/9G11 and show that it is a member of the Ig superfamily (6) . The extracellular domain of CD31 contains four contiguous C2-like Ig domains and is most closely related to the intercellular adhesion molecule carcinoembryonic antigen (CEA) (7, 8). Materials and Methods Brief Definitive ReportCell Culture, Generation of mAbs, and Immunofluorescence Staining. HUVEC were established from freshly isolated full-term umbilical cords and cultured using standard methods (3) . All cell lines were obtained from the ICRF cell bank and grown in DMEM/1070 FCS . mAbs against HUVEC were generated using a modification ofexisting methods (9) . Actively proliferating HUVEC were used as the immunogen . HUVEC were stained with mAb 9G11 or an anti-ICAM-1 mAb, RR1, followed by goat anti-mouse FITC conjugate, fixed in PBS/27o formaldehyde, and photographed on an Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY) with Ilford 400 ASA film.

The inducible adhesion molecules mediate important functions in the lymphoid tissues. We have investigated the expression of intercellular adhesion molecule 1 (ICAM-1), endothelial leucocyte adhesion molecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1), and platelet endothelial cell adhesion molecule (PECAM/CD31), using immunocytochemistry on cryostat sections of five lymph nodes from patients with Castleman's disease of the hyaline-vascular type. All five cases were characterized by marked hyperplasia of follicular dendritic reticulum cells, which were extensively present even in the mantle zone. Hyperplastic follicular dendritic reticulum cells showed marked expression of VCAM-1, and weak expression of ICAM-1. In two cases, several dysplastic giant cells with aberrant, polyploid nuclei showed aberrant expression of ELAM-1, an endothelium-restricted molecule. Dysplastic giant cells were positive with DRC-1 (an antibody to dendritic reticulum cells), VCAM-1 and occasionally ICAM-1, were negative for the endothelial cell markers factor VIII-related antigen and CD31 and were non-proliferating (Kl-67-). Cells positive for ICAM-1 or VCAM-1 were rare in the interfollicular areas. In all cases vascular hyperplasia was prominent, but endothelial cells were poorly activated in terms of expression of inducible adhesion molecules and of HLA-DR antigens. The possibility that dysplastic follicular dendritic reticulum cells have a pathogenetic role in Castleman's disease is discussed.