Human eosinophil stimulation promoter lymphokine: Production by antigen stimulated lymphocytes and assay with a new electro-optical technique

A human lymphokine, eosinophil stimulation promoter (ESP), was shown to be produced by mononuclear leukocytes in response to non-helminthic antigens. Supernatants of tuberculin-stimulated cultures of mononuclear leukocytes from tuberculinskin test reactive humans contained non-dialyzable ESP activity; ESP could not be demonstrated in the supernatants of tuberculin stimulated cultures from skin test negative donors. Thus, human ESP, like murine ESP, is a soluble lymphokine product of sensitized mononuclear cells. ESP activity was also demonstrated in supernatants of mononuclear leukocyte cultures stimulated with streptococcal antigens. ESP selectively enhanced migration of eosinophils. ESP production could be blocked by puromycin, and its activity was diminished by prior incubation with eosinophil-rich granulocytes. Correlation of antigen stimulated ESP activity with skin test reactivity establishes ESP as another in vitro correlate of delayed hypersensitivity in man. A video method of data collection and analysis is described which greatly facilitates in vitro studies of ESP activity. The method is applicable to the study of other lymphokines which effect cellular migration.     

Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr ⁵¹Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.     

A new class of model glycolipids: synthesis, characterization, and interaction with lectins.

A method is described for preparing model glycolipids by linking aldobionic acids to an alkylamine through an amide bond. These compounds may be rapidly prepared in large quantities. The glycolipids precipitate specifically with lectins. Precipitation occurs at glycolipid concentrations just above their critical micelle concentration.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

BCG-induced macrophage suppression in mice: Suppression of specific and nonspecific antibody-medicated and cellular immunologic responses

The intravenous injection of killed BCG in an oil-in-saline emulsion (BCG-E) results in the development of intense chronic granulomatous inflammation in the lungs and spleen of C57B1/6 (B6) but not CBA mice. B6 mice injected intravenously with BCG-E exhibited marked suppression of antibody responsiveness and delayed hypersensitivity to sheep erythrocytes, as well as proliferation in response to PPD. In contrast, CBA mice similarly treated with BCG-E were not suppressed in any of these reactivities. The spleen is an important organ in this phenomenon since suppression was reversed by splenectomy and could be transferred to normal recipients with spleen cells from BCG-treated mice. Spleen cells responsible for suppression were adherent to plastic petri plates, removed with carbonyl iron, and were not eliminated with either anti-Thy-1 or anti-immunoglobulin serum + C. This study indicates that macrophages from BCG-inflamed spleen are capable of potent suppression of both antibody- and cellular-mediated immunologic reactivity.     

Lymphocyte-induced production of prostaglandin E2 by tumor cells in vitro: Requirements for direct contact and lymphocyte viability

The production of prostaglandin E₂ by tumor cell lines in response to exposure to purified lymphocytes has prompted the suggestion that this phenomenon may represent a defense mechanism whereby tumors may subvert an immune response mounted against them. To further characterize this phenomenon, cell lines derived from carcinogen-induced bladder tumors and embryo fibroblasts in Fischer rats were incubated with purified lymphocytes from peripheral blood, spleen, thymus, and lymph nodes from Fischer rats under a variety of conditions, and the amount of prostaglandin E₂ (PGE₂) production was determined by radioimmunoassay. Increased numbers of blood or splenic lymphocytes were associated with the induction of increased levels of PGE₂ production by the tumor cells. However, no prostaglandin was produced by the tumor cells after exposure to thymus or lymph node lymphocytes. Irradiation of lymphocytes prior to exposure to the tumor cells led to lower levels of PGE₂ production by the tumors, as did sonication of the lymphocyte preparations prior to addition to the tumor monolayers. Separation of lymphocytes from direct contact with the tumor cells resulted in less PGE₂ production by the tumor cell lines; however, when these lymphocytes were later layered onto fresh tumor cell monolayers, PGE₂ production occurred. Results in the present study suggest that direct contact between intact, viable, functionally active lymphocytes and tumor cells is necessary for tumor cell prostaglandin production to occur. Moreover, PGE₂ production only appears to occur in response to exposure to particular populations of lymphocytes, and this may correlate with the number of specific effector or attacker lymphocytes that are present. This specificity of response to effector cell challenge may be important in probing the defense mechanisms tumor cells may have to lymphocyte challenge, as well as in gauging the efficacy of a particular cellular immune response as it may be regulated both by cells involved in effecting this response as well as by the targets in lymphocyte/tumor cell interactions.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.     

Requirement of human chromosomes 19, 6 and possibly 3 for infection of hamster x human hybrid cells with baboon M7 type C virus.

The replication pattern of the endogenous baboon type C virus M7 was studied in 29 primary Chinese hamster × human hybrid clones generated with leukemic cells from two different patients with acute lymphoblastic or myeloblastic leukemia. There was no evidence of viral particulate RDDP or M7 antigen before viral infection. M7 virus replicated in human and some hybrid cells but not in Chinese hamster cells, indicating that M7 requires dominantly expressed human gene(s) for replication. Enzyme and cytogenetic analyses show that a gene(s) coded for by human chromosome 19 is necessary for M7 infection of these hybrids. Detailed cytogenetic correlations revealed, however, that the chromosome 19+/M7 + hybrid clones with intact chromosomes also had copies of chromosomes 3 and 6. Previously, Bevi, the putative integration site for M7 virus, has been assigned to human chromosome 6. Many clones with various combinations of chromosomes 3 and 6 lacked chromosome 19, however, and failed to replicate exogenously applied M7 virus, while tests of 27 secondary clones showed that M7 markers co-segregated with chromosome 19 markers. These findings all confirm the need for a chromosome 19-coded function in Chinese hamster × human hybrids. In addition, the yield of viral particulate RDDP produced into the culture fluid was 50–100 fold less per viral antigen-positive cell in the hybrids compared with human cells. Thus some form of regulation of viral components exists in the hybrid cells. When the virus replicating in hybrid cells was transferred back to human cells, this regulation was relaxed and the yield of RDDP per FA(+) cell greatly increased. We conclude that human chromosomes 6 and 19 code for functions involved in M7 virus metabolism, and we cannot exclude a function coded for by chromosome 3.     

Studies of antibody affinity in plasmacytoma-bearing mice: Evidence for a maturational defect of B lymphocytes

The antibody response of plasmacytoma-bearing mice (PC-mice) is severely reduced. In order to understand the nature of the effect of the tumor on the cells making antibody, quantitative and qualitative studies of the humoral response of PC-mice were undertaken. In these studies, the affinity of the antibody produced by tumor-bearing and normal mice was compared to determine whether the small amount of antibody produced by PC-mice is the product of a normal or an altered population of B cells. Antibody to TNP-Ficoll made by PC-mice 3 days after immunization was less heterogeneous and of an affinity lower than that of antibody made by normal mice. However, at 7 days, the antibody made by PC- and normal mice did not differ significantly. These data suggest that, prior to antigenic stimulation, the B cells of PC-mice are relatively immature, reflecting a possible retardation in the generation and turnover of B lymphocytes. The process of antigen-driven selection of high-affinity antibody-producing cells, however, appears to function normally in PC-mice. These studies, then, reveal a qualitative as well as quantitative defect in the primary humoral response of PC-mice which may reflect an abnormality in the development and differentiation of B cells in these mice.     

E-rosette receptors induced by phytohemagglutinin on human K cells expressing T-cell surface antigens.

Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.     

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.     

Calcium binding by troponin-C and homologs is correlated with the position and linear density of "beta-turn forming" residues.

The sequences of troponin-C, the light chains of myosin, the parvalbumins and modulator protein are known to contain one to four regions that are homologous with the sequence of the calcium binding region (the “EF-hand” configuration) of the carp parvalbumin of known three dimensional structure. Of the twenty-nine regions examined here, seventeen are known or inferred to bind calcium. Here we describe a correlation between the position and linear density of “β-turn forming” residues in these regions and their ability to bind calcium. The correlation provides a quantitative criterion for evaluating Kretsinger's proposal that “any protein which contains an EF-hand is calcium modulated”.     

Covalent binding of folic acid to dimethylglycine dehydrogenase

Dimethylglycine dehydrogenase (EC 1.5.99.2) carries out the oxidative demethylation of dimethylglycine to sarcosine in liver mitochondria. In vivo, the enzyme uses tightly bound tetrahydropteroyl pentaglutamate (H₄PteGlu₅) as an acceptor of the one-carbon group generated during the reaction. The purified enzyme can use, but does not require, H₄PteGluB and under these conditions formaldehyde is the one-carbon unit produced. It is reported that folic acid may be covalently linked to dimethylglycine dehydrogenase in a specific and saturable manner so that only 1 mole of folic acid is bound per mole of enzyme. Covalently bound folic acid blocks the subsequent binding of H₄PteGlu, and does not inhibit the rate of dimethylglycine dehydrogenase activity in vitro.     

Studies on the induction and expression of T-cell-mediated immunity. XIII. Membrane-associated antigens of cytotoxic T lymphocytes involved in cytotoxicity

An xenogeneic rat anti-mouse T-cell serum, designated RAT*, has been shown to block the cytolytic activity of cytotoxic T lymphocytes (CTL) at a postbinding step. RAT* serum or the IgG fraction was extensively absorbed with the target cell, P815, a DBA mastocytoma, and used with or without further absorption to immunoprecipitate specific molecules from radiolabeled membrane extracts of CTL derived from either in vivo-allosensitized mice or from cytotoxic clones maintained in in vitro cultures. Cell surface sialic acid residues were labeled by oxidation with sodium periodate (NaIO4) and reduction with tritiated sodium borohydride ([3H]NaBH4). Alternatively, cell surface proteins were labeled with 125I by lactoperoxidase-catalyzed iodination. Nonidet P-40 (NP-40)-solubilized radiolabeled membranes were then immunoprecipitated with RAT* serum and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Three membrane-associated molecules of 95,000, 140,000 and 180,000 Mr were found by such analysis. The sensitivity of these three molecules to trypsinization and their susceptibility to labeling with [3H]NaBH4 suggested that they are glycoproteins. Moreover, when RAT* serum or the IgG fraction was absorbed with various cell types, its ability to immunoprecipitate the three molecules correlated with its ability to block cytolysis. Adsorption of RAT* serum with CTL, but not with nonimmune thymocytes, significantly reduced the ability of RAT* serum to inhibit cytotoxicity and to immunoprecipitate the 95k, 140k, and 180k molecules. Thus, these findings suggest that one or more of these cell surface molecules of CTL may be involved in the cytolytic process.     

A general method for studying the secretion of macromolecules by single cells: II. A simplified procedure with enhanced sensitivity

A simple, sensitive precipitin-type single-cell secretion assay is described and applied to the study of immunoglobulin-secreting cells. Evidence is presented that its efficiency is comparable to that achieved with reverse hemolytic plaque techniques. Also described is a modification of the simplified procedure which possesses substantially increased sensitivity. Enhanced sensitivity is achieved through the use of monoclonal rheumatoid factors which preferentially react with rabbit or human immunoglobulins that are incorporated into immune complexes as a result of interaction with antigen. Addition of rheumatoid factor to the agarose-cell mixture leads to additional crosslinking of immune complexes that form around active cells, thereby increasing the probability of forming a detectable precipitate. The application of this procedure to the detection of cells producing T-cell products is also discussed.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Characterization and comparison of the major glycoprotein from three strains of Rous sarcoma virus.

The major glycoprotein g2 was purified from three strains of Rous sarcoma virus, representing subgroups A, B, and C. Carbohydrate analysis showed that glucosamine, mannose, galactose, fucose and sialic acid constitute 40% of the weight of the subgroup A glycoprotein and 15% of the subgroup B and C glycoproteins. The molar ratios of sugars were very similar and amino acid compositions were similar but not identical for the three glycoproteins. Glycosidase digestions of subgroup A and C glycoproteins suggested the presence of two types of oligosaccharide chains, the complex serum type, with terminal sequences sialic acidα-Galβ-GlcNAcβ- and the high mannose type with terminal α-linked mannosyl residues. After removal of 70% of the carbohydrate by glycosidases, subgroup A glycoprotein contained only glucosamine and mannose, in the molar ratio 2.0:1.3. The sequence of sugar release was consistent with oligosaccharide structures such as those which have been described for other glycoproteins. The plant lectins concanavalin A and wheat germ agglutinin were shown to interact strongly with the g2 glycoprotein from viruses of all three subgroups.     

Lipopolysaccharide (LPS) modulation of human monocyte accessory cell function in promoting T-cell colonies: Inability of LPS and IL-1 to abrogate the need for monocytes with high HLA-DR expression

The abilities of human monocytes differentially expressing HLA-DR and of lipopolysaccharide (LPS) to influence T-cell colony responses were investigated. Optimal T-cell colony responses stimulated by soluble Staph protein A were crucially dependent on monocytes. Also, monocyte facilitation of colony responses was markedly inhibited by 10 μg/ml LPS and the addition of indomethacin reversed this inhibition. In contrast the inhibition of T-cell colony responses with 100 μg/ml LPS was not reversed with indomethacin and preincubation experiments with high concentrations of LPS showed the inhibition could be mediated through T cells by mechanisms other than prostaglandins. The treatment of monocytes with a monoclonal anti-HLA-DR reagent + C reduced the frequencies of monocytes expressing high levels of HLA-DR ~ fivefold and the resulting monocytes which expressed low levels of HLA-DR also poorly functioned in the promotion of colony responses compared to controls. LPS in the presence of indomethacin improved the ability of monocytes expressing low levels of HLA-DR to promote colony responses. However, these monocytes consistently failed to augment colony responses to those levels observed with untreated monocytes and their failure was not secondary to deficient interleukin 1 release. These results indicate that although LPS can somewhat potentiate the accessory cell function of certain human monocytes, it cannot abrogate an additional requirement for those monocytes expressing high levels of HLA-DR.