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.
Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of SabnoneUla typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the Gl stage of the cell cycle. Furthermore, [3lHluridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.In previous experiments designed to elucidate the immunoregulatory properties of endotoxin-associated protein (EP) and other polyclonal B-cell activators, we found that when lipopolysaccharide (LPS) was used in conjunction with these cell stimulators, activation of nonresponder C3H/HeJ splenic B lymphocytes was actually inhibited by the LPS (13). No evidence was found that the LPS stimulated suppressor T cells or macrophages of the C3HIIHeJ mouse, which would serve to shut off stimulated B-cell proliferation or polyclonal antibody synthesis by some means. Our working hypothesis, therefore, has been that the observed suppression was the result of a biochemical event(s) in C3H/HeJ
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