The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens.

Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunological activity and that bacterial DNA can induce the in vitro proliferation of normal murine B cells. To delineate structural features of DNA associated with mitogenic activity, the response of murine lymphocytes to various natural and synthetic polynucleotides was determined. Both ss and dsDNA from two different bacterial strains were equally effective in inducing proliferation. This response was independent of adenosine methylation, since DNA from dam- Escherichia coli stimulated proliferation. Among the synthetic polymers tested, only the duplexes poly(dG).poly(dC), and poly(dG.dC) were mitogenic, while polymers containing dA, dT, or dI alone or in combination with dG and dC were inactive. The mitogenic activity of poly(dG.dC) was eliminated, however, upon substitution of rG for dG or 5medC for dC. The mitogenic activity did not require high molecular weight DNA since active polymers ranged in size from approximately 260 to 800 base pairs. In addition, E. coli DNA fragments of 50-300 and 125-600 bases were mitogenic. Together, these data suggest that the mitogenic activity of DNA is dependent on sequence-specific determinants that can be presented by synthetic DNA duplexes as well as bacterial ss and dsDNA.

Effects of 2-mercaptoethanol and buthionine sulfoximine on cystine metabolism by and proliferation of mitogen-stimulated human and mouse lymphocytes.

Cysteine is an essential amino acid for lymphocytes and its anabolic products are intimately involved in lymphocyte activation. The purpose of this study was to assess the uptake and subsequent utilization of cyst(e)ine by mitogen-stimulated human peripheral blood mononuclear cells (PBMC), to evaluate the effect of an exogenous thiol, 2-mercaptoethanol (2ME), on these processes, and to compare human and mouse lymphocyte reactivities. Unlike mouse lymphocytes, the proliferation of human T-cells was inhibited by addition of 2ME although 2ME enhanced cystine uptake. Optimal responses to T-cell mitogens (Con A and PHA) were obtained with a cystine concentration of greater than or equal to 25 and 200 microM for human and mouse cells, respectively, and 2ME enhanced DNA synthesis of Con A-stimulated mouse cells regardless of the cystine dose; however, 2ME enhanced the response of human cells only in the presence of suboptimal doses of cystine. To assess whether 2ME's inability to enhance human PBMC responses was related to their glutathione (GSH) content, the human PBMC were pretreated with buthionine sulfoximine (BSO, an inhibitor of GSH synthesis). Even when the initial intracellular GSH concentration was lowered to below that of mouse lymphocytes, 2ME still inhibited proliferation. In contrast, addition of 2ME to human PBMC maintained in the presence of BSO enhanced the proliferative response suggesting that a critical level of thiols is needed for proliferation. The ability of 2ME to enhance proliferative responses in cystine deficient medium supports this contention. Consistent with thiol involvement in activation, Con A increased [35S]cystine uptake 2-fold within 4 h of incubation and enhanced subsequent conversion of cystine into cysteine and GSH. Interestingly, BSO treatment only slightly inhibited Con A-induced protein synthesis (5%), but it significantly suppressed conversion of cystine into cysteine or GSH (80-95%) and blocked DNA synthesis (90%). Overall, the results indicate that various differential thiol characteristics must exist between human and mouse lymphocytes and that a reducing equivalent is necessary for DNA synthesis but not lymphocyte activation.

Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA.

Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunologic activity and that bacterial DNA, unlike mammalian DNA, can induce significant antibody responses in mice. To explore further the immunologic activities of bacterial DNA, its ability to stimulate in vitro proliferation of murine lymphocytes was tested. The stimulation of lymphocytes with highly purified ssDNA from Escherichia coli resulted in a dose-dependent response that was maximal at 48 h. Several lines of evidence indicate that DNA, rather than endotoxin contamination, induced this response: 1) LPS at doses equivalent to those detected in the DNA preparation caused significantly less proliferation than the DNA; 2) the response to DNA was insensitive to polymyxin B; 3) pretreatment of DNA with DNase completely abrogated the response; and 4) DNA induced the proliferation of cells from endotoxin-resistant C3H/HeJ mice. Furthermore, although DNA from three different bacterial species induced proliferation, mammalian DNA from three species were nonmitogenic. Depletion of T cells from lymphocytes did not reduce proliferation, suggesting that bacterial DNA directly triggered B cell proliferation. These studies provide further evidence that DNA are not uniform in their immunologic activities likely because of their content of nonconserved structural determinants.

Cell cycle progression of glutathione-depleted human peripheral blood mononuclear cells is inhibited at S phase.

Glutathione (GSH) the most abundant nonprotein thiol, is involved in the maintenance of the cellular redox state. In this capacity it may influence lymphocyte responsiveness to various stimuli. We have investigated the requirement of GSH during the activation and proliferation of PBMC. The intracellular GSH content of PBMC was altered by continuous culture or pretreatment with buthionine-S,R-sulfoximine (BSO), a specific and irreversible inhibitor of GSH synthesis. Initial experiments demonstrated that the addition of BSO at the initiation of culture, or shortly thereafter (6 hr), inhibited DNA synthesis and produced a simultaneous decrease in intracellular GSH. It was necessary that the BSO be present in the culture for at least 24 hr prior to the initiation of DNA synthesis for maximal inhibition. Cell cycle analysis revealed that BSO did not affect the entry and progression of PBMC through G1 of the cell cycle, however, entry into S-phase was inhibited in a dose-dependent fashion. These results were further substantiated by the inability of BSO to inhibit IL-2 production and expression of the IL-2R. In addition the timely expression of the transferrin receptor by BSO-treated cells indicated that the block occurred at the G1/S transition. The influence of GSH on early activation events was determined by BSO pretreatments. Lowering the intracellular GSH level of PBMC to less than 10% of the initial content prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. In the course of these studies we also observed a modest (17%) albeit consistent increase during activation in the total thiol levels of GSH-depleted PBMC. These thiols may have a key role in the activation process. These data support our hypothesis that GSH is required for lymphocyte proliferation and that additional thiols are involved during the activation process.

Modification of a direct enzyme-linked immunosorbent assay for the detection of immunoglobulin G and M antibodies to pneumococcal capsular polysaccharide.

In contrast to the usual indirect enzyme-linked immunosorbent assay (ELISA) method for detection of antibody responses, a modified direct ELISA technique was used to measure immunoglobulin G (IgG) and IgM responses to pneumococcal capsular types 1, 3, 9N, and 23F in humans. Individual capsular polysaccharides were covalently bound to poly-L-lysine before adsorption to the solid phase. The coupling reaction was enhanced by maintenance of a constant pH of 8.2 after the addition of all reactants. The evaluation of four diluents (phosphate-buffered saline [PBS]-Tween; PBS-Tween plus 10% fetal calf serum; PBS-Tween plus 10% bovine serum albumin; and PBS-Tween plus 20% normal goat serum) showed that the sensitivity and specificity of the assay was increased with normal goat serum (10-fold). Serum samples from 10 subjects immunized with polyvalent pneumococcal vaccine were tested by direct ELISA and by radioimmunoassay. At 4 weeks postimmunization, the ELISA method showed that IgG was the predominant antibody and that IgM responses were lower or had diminished. Isotype shifts during this period would have been undetected by the radioimmunoassay method. The changes in antibody response measured by ELISA were comparable to the radioimmunoassay results. The direct ELISA method for the detection of antipneumococcal capsular antibody has been found to be a sensitive and reproducible assay for the detection of IgG and IgM antibodies.