Modulation of SCE induction and cell proliferation by 2-mercaptoethanol in phytohemagglutinin-stimulated rat lymphocytes

2-Mercaptoethanol (2-ME) is used as a medium supplement to enhance the proliferation of lymphocytes culturedin vitro. In this study, we have examined the effects of 2-ME on cell growth and on SCE induction in cultures of unstimulated and phytohemagglutinin (PHA)-stimulated Fischer 344 rat lymphocytes. There were virtually no metaphases detected in cells cultured without PHA. In PHA-stimulated cultures, 2-ME decreased SCE-frequency but it enhanced SCE frequency in the presence of S to 12.5 µM bromodeoxyuridine (BRd U). Both mitotic and replication indices were increased in the PHA/2-ME system. The levels of incorporated exogenous thymidine, in the presence of 2-ME, were relatively low in unstimulated cells, suggesting that 2-ME is not mitogenic for T-cells. However, 2-ME enhanced PHA-induced response of T-cells as evidenced by increased levels of thymidine incorporation into cellular DNA. The growth promoting effects and the decrease in SCE frequency caused by 2-ME upon PHA stimulation indicate that 2-ME may alter the nature of interaction between PHA and cellular activating properties or the replicative processes.Abbreviations BRdU bromodeoxyuridine - FBS fetal bovine serum - SCE sister-chromatid exchanges - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - IL-2 interleukin-2 - 2-ME 2-mercaptoethanol - PBS phosphate buffered saline - PHA phytohemagglutinin - MI mitotic index - RI replication index - NADH nicotinamide adenine dinucleotide (reduced form)     

Selenium and immune cell functions. I. Effect on lymphocyte proliferation and production of interleukin 1 and interleukin 2

The dietary intake of selenium (Se) has been shown to influence the development and expression of various biologic processes. This study examined the immunologic competence of lymphocytes from C57BL/6J mice maintained for 8 weeks on Se-deficient (0.02 ppm Se), normal (0.20 ppm Se, as sodium selenite), or Se-supplemented (2.00 ppm Se) Torula yeast-based diets. The ability of the cells to recognize alloantigens, to proliferate in response to stimuli, and to produce interleukin 2 (IL-2) was determined. Se deficiency significantly inhibited the ability of the lymphocytes to proliferate in response to allogeneic stimulation in the mixed lymphocyte reaction or to mitogen stimulation by phytohemagglutinin, whereas Se supplementation significantly enhanced both responses. In contrast, the amounts of IL-2 and interleukin 1 (IL-1) produced by lymphocytes and macrophages, respectively, removed from Se-deficient or Se-supplemented animals did not differ significantly from the amounts of IL-2 and IL-1 produced by cells removed from animals maintained on the control diet. These results suggest that the mechanism(s) responsible for the observed effects of Se on lymphocyte proliferation are independent of the levels of IL-2 or IL-1.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

Immunoregulatory functions of paf-acether. I. Effect of paf-acether on CD4+ cell proliferation

Paf-acether or platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator of inflammation initially described as a potent platelet-aggregating compound. It is newly formed by a variety of cells including monocytes and is now recognized as a major mediator of cell-cell interactions. The present studies were undertaken to determine whether paf-acether could modulate T cell function. We found that addition of paf-acether to CD4+ cells cultured with phytohemagglutinin markedly inhibited the proliferative response in a dose-dependent manner. Maximal inhibition occurred when paf-acether was present during the first 24 hr of cell culture and the presence of paf-acether did not alter the kinetics of CD4+ cell proliferation. Importantly, the mechanism by which paf-acether inhibited the proliferative response was not related to inhibition of interleukin 2 (IL-2) secretion since the amount of IL-2 in cultures was not altered and addition of exogenous IL-2 failed to restore the CD4+ cell proliferative response. Further, as judged by indirect immunofluorescence, paf-acether did not inhibit IL-2 receptor expression. Taken together, these data indicate that paf-acether interferes with some processes leading to CD4+ cell proliferation. This new role for the chemically defined monokine paf-acether emphasizes the potential role of inflammatory lipid mediators in the regulation of T cell response.     

Effect of heat and 2-mercaptoethanol on intracytoplasmic membrane polypeptides of Rhodopseudomonas sphaeroides.

Solubilization at 75 degrees C of Rhodopseudomonas sphaeroides chromatophores in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME) resulted in the selective absence of reaction center B and C polypeptides from SDS-polyacrylamide gel electrophoresis profiles. A newly identified, chromatophore-specific polypeptide, with a mass of 35.2 kdaltons, was also missing under these conditions of chromatophore solubilization. Solubilization at 27 degrees C in the presence of SDS and beta-ME also resulted in the disappearance of these three polypeptides, but at much slower rates. Disappearance of either endogenous or exogenously supplied reaction center polypeptides B and C during SDS solubilization of whole chromatophores at either 27 or 75 degrees C was shown to be entirely dependent upon the presence of beta-ME. After chromatophore solubilization in the presence of beta-ME and subsequent SDS-polyacrylamide gel electrophoresis, exogenously added reaction centers B and C could be localized in a complex of no less than 100 to 200 kdaltons. However, the precise size of the complex was influenced by the stoichiometry of the reacting components. The disappearance of the 35.2-kdalton polypeptide was neither dependent upon the presence of beta-ME nor dependent upon the presence of any additional chromatophore polypeptides. The 35.2-kdalton polypeptide underwent a heat-induced oligomerization to yield several high-molecular-weight species.     

Modulation of T-cell function by type I interferon

Production of type I interferon (IFN-α/β) is a common cellular response to virus infection. IFN-α/β has a dual role in combating infection, triggering innate antiviral mechanisms and stimulating the generation of an adaptive immune response. This review focuses on the effects of IFN-α/β on one particular immune cell type, the T cell, and the impact of IFN-α/β-mediated signalling in T cells on the immune response. The critical role of T-cell responsiveness to IFN-α/β for the generation of productive T-cell responses after infections with certain viruses in vivo is discussed in the context of in vitro experiments investigating the mechanisms by which IFN-α/β modifies T-cell function. These studies reveal complex effects of IFN-α/β on T cells, with the consequences of exposure to IFN-α/β depending on the context of other signals received by the T cell.     

Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing

When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptoethanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven electrophoretically along the pH gradient, sweeping away, along its path, and focused carrier ampholytes.     

Human lymphocyte-eosinophil interactions. I. Modulation of phytohemagglutinin-induced lymphocyte proliferation by eosinophils

Do eosinophils modulate lymphocyte function? This question was studied by examining the effect of purified eosinophils (eos) on lectin-induced human lymphocyte proliferation. Intact resting or zymosan-stimulated eos or their extracts were cocultured with phytohemagglutinin-stimulated mononuclear cells in vitro and [3H]thymidine uptake was measured at 72 hr. Zymosan-stimulated eos consistently suppressed (up to 90%) the lectin-induced proliferative response by a noncytotoxic mechanism. Freeze-thaw extracts from zymosan-stimulated eos also significantly suppressed lymphocyte proliferation to a similar degree. The amount of suppression was directly proportional to the number of eos or the amount of extract added to the lymphocyte cultures. Intact resting eos and their extracts occasionally exhibited suppressive effects (up to 40%) on lymphocyte proliferation; this suppression, however, was always less than that of activated eos or their extracts. Eos pretreated with the protein synthesis inhibitor, pactamycin, exhibited significantly less suppressive activity, suggesting that a protein was responsible in part for the reduction in proliferation. The addition of superoxide dismutase or catalase to the eos-mononuclear cell cocultures did not reduce the amount of suppression observed, thus making it unlikely that active oxygen products were involved in the mechanism of suppression. Heating extracts from stimulated eos to 80 degrees C for 30 min resulted in partial loss of suppressive activity while extensive dialysis of the extracts had no effect. The studies reported here provide evidence that a nondialyzable and heat sensitive factor(s) produced by stimulated eos may exert feedback inhibition of lymphocyte function.     

In vitro interactions of murine peritoneal macrophages and sarcoma cells. I. Promotion of tumor cells proliferation by macrophages

An ascites subline (AA) of the murine sarcoma MC1M grows in vivo in the peritoneal cavity but dies in vitro when cultured on glass or collagen. The viability of AA cells in vitro is not influenced in cocultures with fibroblast cell line L929, and is diminished in cocultures supplemented with macrophage culture supernatant or in cocultures with non-adherent peritoneal cells. However, AA cells proliferate in vitro on glass or collagen when cocultured with syngeneic, semisyngeneic, and allogeneic peritoneal macrophages. This was demonstrated by tritiated thymidine incorporation assay, by AA cell number counting, and by measuring AA cell protein content. Proliferation also occurs when AA cells are separated from the macrophage monolayer by millipore filters.     

Effect of 2-mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells.

The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-mercaptoethanol for survival in tissue culture. The effect of 2-mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.     

Antigen-specific T-cell hybrids: I. Ovalbumin binding T-cell hybrid

Somatic cell hybrids were prepared between BW 5147 AKR T lymphoma cells and thymus-derived suppressor lymphocytes obtained from ovalbumin (OVA)-immunized, urea-denatured-ovalbumin (UD-OVA)-treated, suppressed BDF1 female mice. These T-cell hybrids express parental constitutive enzymes and differentiation antigens. Of the four hybrid lines developed, Hybrid 49A (Hyb 49A) has exhibited antigen-specific binding of OVA-coated syngeneic erythrocytes which is inhibited by preincubation in soluble OVA. This hybrid also interacts with self epitopes expressed on syngeneic erythrocytes. Antigen-specific rosette formation by Hyb 49A is not inhibited by preincubation in soluble keyhole limpet hemocyanin or normal mouse serum. This line and its agar-derived subclones have maintained their antigen-specific activity for 9 months and will provide extensive homogeneous quantities of T-cell products for molecular analysis.     

Effect of 2-mercaptoethanol on the individual periods of the mitotic cycle

Dynamics of the mitotic cycle of the KEPV cells being on different interphase stages at the start of a 20 hour 2-mercaptoethanol (0.001 M) treatment has been studied during the treatment and for 11 hours after washing out the agent. The KEPV cells affected by mercaptoethanol during the interphase (G1, S, G2) were shown to continue their passage through the cycle to enter mitosis, but part of the cells of the S period and of the first half of the G2 period were arrested in the interphase. In the presence of mercaptoethanol, mitotic cells reach the metaphase stage, and their further behaviour depends on the duration of the treatment. For the first 8 hours of treatment, a phase of "unstable block" exists for cells that were in S and G2 periods at the beginning of treatment, while other cells are transformed into K-metaphases. 8 hours later a phase of "stable block" occurs and all the normal metaphases are transformed into K-metaphases. After washing out the culture from mercaptoethanol the cells are ejected from the block in K-metaphase. The transformation from K-metaphase into the normal metaphase is realised in the course of this process. The cells which were in S and G2 periods at the beginning of the treatment are ejected from the block simultaneously after washing, while the cells of the G1 period--with a small delay. After washing out mercaptoethanol the cells that were in the interphase (G1, S, G2) at the beginning of the treatment are capable of producing both multipolar mitoses and mitoses without cytotomy.     

Whole-cell currents in macrophages: I. Human monocyte-derived macrophages

Summary We examined the variability of occurrence and frequency of voltage-dependent whole-cell currents in human peripheral blood monocyte-derived macrophages (HMDM) maintained in culture for up to three weeks. An increase in cell capacitance from an average value of 9 pF on the day of isolation to 117 pF at 14 days accompanied growth and differentiation in culture. The average resting potential was approximately –34 mV for cells beyond two days in culture. Cells exhibited a voltage-and time-dependent outward current upon membrane depolarization above approximately –30 mV, which appeared to be composed of a number of separate currents with variable expression from donor to donor. Three of these currents are carried by K⁺. The frequency of each outward current type was calculated for 974 cells obtained from 36 donors. The HMDMs in these studies exhibited two 4-aminopyridine (4-AP) sensitive, time-dependent outward currents (I _A andI _B ) that could be differentiated on the basis of the presence or absence of steady-state inactivation in the physiological potential range, time course of inactivation during maintained depolarization, as well as threshold of activation. The 4-AP-insensitive outward current activated at approximately 10 mV. One component of the 4-AP insensitive-outward current (I _C ) could be blocked by external TEA and by the exchange of internal Cs⁺ or Na⁺ for K⁺. The probability of observingI _B andI _C appeared to be donor dependent. Following total replacement of internal K⁺ with Cs⁺, two additional currents could be identified (i) a delayed component of outward current (I _D ) remained which could be blocked by low concentrations of external Zn²⁺ (4 m) and was insensitive to anion replacement in the external solution and (ii) a Cl^– current with a reversal potential which shifted in the presence of external anion replacement and which was irreversibly inhibited by the stilbene SITS. The activation of a prominent time-independent inward currents was often observed with increasing hyperpolarization. This inward current was blocked by external Ba²⁺ and corresponded to the inwardly rectifying K⁺ current. Neither inward nor outward current expression appeared dependent on whether cells were differentiated in adherent or suspension culture nor was there demonstrable differential current expression observed upon transition from suspension to adherent form.     

Effect of macrophages on proliferation of granulosa cells in the ovary in rats.

The effect of macrophages on proliferation of granulosa cells was examined in gonadotrophin-primed immature female rats. The mouse anti-rat macrophage monoclonal antibodies TRPM-2 and TRPM-3 were used and macrophages were observed in the granulosa layer and antrum of follicles and in corpora lutea and stroma around follicles. There was no difference in distribution between TRPM-2-positive cells and TRPM-3-positive cells. Macrophages with some cytoplasmic vacuoles of various sizes were also demonstrated in growing follicles. The average ratios of macrophages to granulosa cells in preantral, antral and mature follicles were 0.008, 0.007 and 0.002, respectively. Labelling with [3H]thymidine of granulosa cells cultured with peritoneal macrophages was significantly greater and the labelling index peaked to 25.0% when the ratio of macrophages to granulosa cells was 0.01, compared with the value of 14.2% when the granulosa cells were cultured alone. This ratio of macrophages to granulosa cells was similar to that in the preantral and antral follicles in vivo. These results suggest that macrophages participate in promoting proliferation of granulosa cells as local mediators in growing follicles.     

Inhibition of aflatoxin formation by 2-mercaptoethanol.

2-Mercaptoethanol inhibits growth of Aspergillus parasiticus NRRL 3240 and aflatoxin formation by the fungus. When added to the resuspended medium, 2-mercaptoethanol inhibited [1-14C]acetate incorporation into both aflatoxins and neutral lipids, thereby showing that it acts at an early stage of aflatoxin biosynthesis. The inhibition is probably due to its chelating action on zinc, which is essential for aflatoxin production. It is proposed that any chelating agent that selectively binds to zinc will inhibit aflatoxin formation.     

T-cell receptor expressed on an autoreactive T-cell clone, clone 4. I. Induction of various T-receptor functions by anti-T idiotypic antibodies

Three monoclonal antibodies (1G3, 2H11, and 3G12) specific for a syngeneic Ek-specific T-cell clone, clone 4, have been established. The antibodies specifically blocked not only the activation of the clone in response to the specific antigen Ek but also the activation by IL-2. Kinetic studies of the blocking activity revealed that the antibodies blocked activation not only through steric hindrance of the antigen-binding site of the receptor but also via inhibition of DNA synthesis. The antibodies induced unresponsiveness of the clone to the specific antigen Ek, but not to nonspecific activation by IL-2. The state of unresponsiveness induced by 1G3 continued for 14 days, the longest time so far examined. The recovery from the unresponsiveness (tolerance) was not observed unless the clone cells proliferated vigorously in response to IL-2. The idiotope recognised by 1G3 was different from that by 2H11 and/or 3G12. This might explain some functional differences elicited by the antibodies.     

Modulation of EL-4 mouse lymphoma cell proliferation by macrophages and tumor related factors

It is well known that macrophages play an important role in the control of tumor growth. This control may be the result of a direct action of macrophages or mediated by several biologically active products or factors elaborated by these and other cell populations. Our studies on the proliferation of a murine T-cell lymphoma (EL-4) showed that the treatment of the ascitic fluid (from the peritoneum of EL-4 bearing mice) with carbonyl iron resulted in a depletion of phagocytes concomitant with a significant increase of [3H] thymidine uptake by EL-4 cells. Further, the growth of EL-4 cells cultured in semisolid agar was significantly inhibited by an underlayer of large quantities of macrophages both from normal and EL-4 bearing mice as well as when cultured in the presence of PGE2. The underlayer of tumor macrophages P388 D1 resulted in an increase of the EL-4 cell growth. Also, conditioned media obtained from in vitro liquid cultures of EL-4 cells and L 1210 cells (B-lymphoma) produced a remarkable inhibition of the in vitro cloning capacity and [3H] thymidine uptake by EL-4 cells. These data support the hypothesis that different factors from normal and hemopoietic tumor cells may control the tumor growth and point out that self-produced factors may modulate the proliferation of tumor cells.