SummaryExtracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyteinduced tumor destruction. We now provide evidence that a protein responsible for ATP synthesis in mitochondria may also play a physiologic role in major histocompatibility complex-independent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) beating structural and immunologic characteristics of the B subunit of H + transporting ATP synthase (E.C. 3.6.1.34, B-H +ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of three different human tumor cell lines studied. NH2-terminal amino acid sequence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the known sequence of human mitochondrial B-H § ATPase. Antibody directed against a 2l-iner peptide in the ATP binding region of B-H+ATPase (anti-B) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum reacts with a single species of protein. Anti-B reacted with the cell membranes of tumor ceils as determined by fluorescence-activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-labeled neoplastic cells (but not human erythrocytes and lymphocytes). Purified p51.5 bound to human lymphocytes and inhibited natural killer (NK) cell-mediated cytotoxicity. Furthermore, anti-B treatment of the K562 and A549 tumor cell lines inhibited NK (by >95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Soluble p51.5 upon binding to lymphocytes retained its reactivity to anti-B suggesting that the ATP binding domain and the lymphocyte-receptor binding domain reside in distinct regions of the ligand. These results suggest that B-H +ATPase or a nearly identical molecule is an important ligand in the effector phase (rather than the recognition phase) of a cytolytic pathway used by naive NK and LAK cells.

A growth factor that is mitogenic for vascular endothelial cells, with an ED50 of~'1 ng/ml, has been purified 170,000-fold to apparent homogeneity from tissue culture medium conditioned by a rat glioma-derived cell line. The pure protein is a 46-kDa dimer composed of two subunits of equivalent mass as established by comparison of migration in SDS/polyacrylamide gels with and without prior reduction.This glioma-derived growth factor is a glycoprotein and is not mitogenic for BALB/c 3T3 fibroblasts, properties that further distinguish it from other well-characterized vascular endothelial cell mitogens. In contrast to acidic and basic fibroblast growth factors and to platelet-derived endothelial cell growth factor, which have no secretory leader sequences and might only be released by leakage from damaged cells, the glycoprotein nature of this mitogen implies that it is processed through the glycosylating secretory pathway. This secretable growth factor could, therefore, be readily available in the extracellular space under normal physiological conditions in vivo to promote vascular endothelial cell proliferation associated with bloodvessel growth and maintenance. genes (1-7). All of these FGFs, with the exception of the int-2 and FGF-6 gene products, which have not been isolated, have been shown to be potent vascular endothelial cell mitogens in vitro. Furthermore, exogenously applied aFGF (8) and bFGF (9) induce angiogenesis in vivo. An angiogenic platelet-derived endothelial cell growth factor (PD-ECGF), a monomeric 45-kDa protein, has also been purified and structurally characterized. This protein, like aFGF and bFGF, lacks a secretory leader sequence and so is presumably released by lysis of platelets (10,11 We have identified and purified a vascular endothelial cell mitogen that is not only produced but also apparently secreted by cultured cells derived from a chemically induced rat glioma (13). This glioma-derived vascular endothelial cell growth factor (GD-VEGF) is structurally unrelated to the previously well-characterized mitogens for large-vessel endothelium. Pure GD-VEGF approaches the specific mitogenic activity of FGFs for cultured human umbilical vein endothelial (HUVE) cells. MATERIALS AND METHODSMitogenic Assays. HUVE cells were isolated and maintained as described (14). The cells were plated in gelatincoated 48-well tissue culture dishes (Costar) at 6000 cells per well in 400 ,ul of medium 199 (GIBCO) containing 15 mM Hepes buffer and supplemented with 20% heat-inactivated fetal bovine serum (Hazelton Research Products, Reston, VA) and antibiotics (penicillin G, 100 units/ml; streptomycin sulfate, 100lg/ml; amphotericin B, 0.25 ,ug/ml; GIBCO). Samples to be assayed were added at the time of cell plating and the tissue culture dishes were incubated at 37°C under 5% CO2. After a 12-hr incubation, 0.64 ,Ci of [methyl-3H]-thymidine (20 Ci/mmol; New England Nuclear; 1 Ci = 37 GBq) was added per well and the dishes were incubated for an additional 60 hr. The cells were then washed with Hanks' ...

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The ability of the vascular endothelium to elaborate cytokines in response to gram-positive sepsis has received limited attention. This study examined cytokine expression by human umbilical vein endothelial cells (EC) following infection with a gram-positive bacterial pathogen, Staphylococcus aureus. S. aureus infection of EC resulted in the production of interleukin-6 (IL-6) and IL-1␤. For IL-6, message was detected at 3 h after infection, protein was present at 24 h, and both message and protein persisted for 72 h. IL-1␤ message was detected at 12 h, IL-1␤ protein was detected at 24 h, and both persisted for 72 h. Message for colonystimulating factor 1 remained unaltered. UV-killed S. aureus also elicited IL-1␤ and IL-6 message and protein expression at 24 and 48 h. Twenty-one clinical isolates of S. aureus were tested, and all induced IL-6 release by 48 h. However, the laboratory strain 8325-4 did not induce cytokine expression at any time point and was internalized by EC 1,000-fold less than other strains were. Internalization of latex beads by EC did not induce IL-6 gene expression. Furthermore, cytochalasin D treatment of the EC prevented IL-1 and IL-6 induction by S. aureus but not by tumor necrosis factor alpha or lipopolysaccharide. These results indicate that S. aureus is a potent inducer of IL-1 and IL-6 in EC and that internalization of S. aureus by EC is necessary for their cytokine expression. Thus, our data suggest that the vascular endothelium may play an important role in the pathogenesis of septicemia caused by gram-positive organisms.

Differences in the ability of bacteria to adhere to normal valvular endothelium may account for the predominance of particular species as pathogens in acute endocarditis. An in vitro adherence assay was developed to simulate the host surface encountered in acute bacterial endocarditis by using confluent monolayers of human endothelial cells. Adherence of 32 gram-positive and -negative blood culture isolates to this surface was compared. All five Staphylococcus aureus strains tested were highly adherent to endothelial cells, as was one gram-negative strain (Serratia marcescens). The remaining gram-positive and -negative isolates, including four viridans streptococci, were relatively nonadherent. Transmission electron microscopy demonstrated attachment of Staphylococcus aureus and invagination of the underlying endothelial cell membrane at 1 h followed by engulfment of large numbers of bacteria after 3 h. The intracellular bacteria appeared to be contained within vacuoles. Preferential attachment of some strains of bacteria, in particular Staphylococcus aureus, to human endothelial cells occurred in vitro, suggesting that adherence is an important determinant of bacterial pathogenicity in acute endocarditis. Active uptake of bacteria by endothelial cells may help account for the virulence of Staphylococcus aureus in endovascular infections and for the ability of this organism to establish multiple metastatic foci of infection.