Augmentation by 2-mercaptoethanol of in vitro anti-TNP antibody production induced by butyrate plus IL-2 in murine splenic B cells

We previously reported that anti-trinitrophenyl (TNP) antibody production in murine splenic B cells stimulated with TNP-lipopolysaccharide in vitro was promoted by sodium butyrate (NaBu) in an IL-2-dependent manner. In the present study, we found that the effect of NaBu plus IL-2 was markedly augmented by 2-mercaptoethanol (2-ME), which showed a slight or null effect on the response of untreated, IL-2-treated or NaBu-treated B cells, as assessed by both anti-TNP plaque-forming cell assay and anti-TNP IgM ELISA. Other thiol compounds such as dithiothreitol, cysteamine and reduced glutathione (GSH) also had this activity. 2-ME enhanced the anti-TNP antibody production induced by other short-chain fatty acids with three to five carbon atoms plus IL-2. The proliferation of B cells was significantly inhibited by NaBu or NaBu plus IL-2, and the proliferation was completely restored by the simultaneous addition of 2-ME. These results demonstrate that 2-ME markedly enhanced anti-TNP antibody production in murine B cells induced by NaBu plus IL-2 and suggest that the effect of 2-ME is at least partly due to its blocking activity of the growth-inhibitory action of NaBu.     

Agents which block membrane lipid peroxidation enhance mouse spleen cell immune activities in vitro: relationship to the enhancing activity of 2-mercaptoethanol

A broad spectrum of agents known to block various steps in the lipid peroxidation process were tested for their ability to protect mouse spleen cells and thereby enhance their activities, in vitro, in either the primary antibody response or the lipopolysac-charide-stimulated proliferation response. Each agent (superoxide dismutase, butylated hydroxyanisole/butylated hydroxytoluene/n-propyl gallate, lucigenin, and α-tocopherol) was able to enhance the cellular response in both assay systems. The degree of enhancement of these immune functions was in proportion to the efficacy of each agent in blocking the overall process in lipid peroxidation. Previous work in this laboratory has shown that the enhancement of the primary antibody response by 2-mercaptoethanol (2-ME) is mediated by the enhanced availability of reduced glutathione in the culture medium. Suboptimal doses of each lipid antioxidant agent were able to enhance the antibody response in an additive manner with a suboptimal dose of 2-ME up to a maximum response equal to that achieved with an optimal dose of 2-ME alone. These data support the hypothesis that the enhancement of cellular responses in the presence of 2-ME is mediated by the lipid antioxidant activity of reduced glutathione.     

Restoration of impaired immune functions in aging animals. III. Effect of mercaptoethanol in enhancing the reduced primary antibody responsiveness in vivo.

The enhancing effect of 2-ME on the primary antibody forming capacity of young and old mice from 5 strains and hybrids was investigated by assessing the number of hemolytic antibody-forming spleen cells in response to sheep RBC stimulation. The following results were obtained: (1) the optimum dose of 2-ME is 4 micrograms per mouse; (2) the best time to administer 2-ME is just prior to, or at the same time as, antigen is given; (3) 2-ME can enhance response to suboptimum and optimum, but not supra-optimum doses of antigen; (4) 2-ME is effective in enhancing the primary antibody forming capacity of both young and old mice, with one exception, but the enhancement of old mice was greater than that of young mice (80% vs. 20%). The exception was old C57Bl mice, in which 2-ME was ineffective; (5) the level of primary antibody forming capacity of old mice can be restored to that of young mice by treating them with 3--4 weekly injections of 2-ME at a dose of 4 micrograms per injection.     

beta-Cyclodextrin as a substitute for fetal calf serum in the primary antibody response in vitro

Fetal calf serum (FCS) must be present at 10% in the culture medium for optimally eliciting the primary antibody response to sheep erythrocytes (SRBC) in murine lymphocytes. The response was no longer observed when FCS was reduced to less than 1%. However, we found that the addition of 250-500 micrograms/ml beta-cyclodextrin (beta-CD) to RPMI 1640 medium containing 1% FCS restored the immune response to a level comparable to that observed in 10% FCS-containing medium. beta-CD did not further augment the response in the presence of 10% FCS. The order of effectiveness of various cyclodextrin compounds tested was as follows: beta-CD (100) greater than alpha-CD(30) greater than gamma-CD(10) greater than heptakis (2,6-O-dimethyl)beta-CD (less than 1). The in vitro antibody response varied drastically depending on the lot of FCS added to the culture medium. The important observation was that even a deficient lot of FCS could elicit the antibody response as efficiently as a good lot if it was added to the culture medium at 1% in combination with beta-CD. beta-CD was also effective in inducing the primary antibody responses to both SRBC and dinitrophenylated Ficoll in serum-free RPMI 1640 medium containing bovine serum albumin, insulin and transferrin. In serum-free conditions, the responses were 40-50% of those in serum-containing medium. beta-CD did not increase the number of antibody-forming cells nonspecifically, nor did it show a significant mitogenic activity and cytotoxicity. These data suggest that beta-CD is a useful material as a serum substitute in inducing primary antibody response in vitro.     

The ability of horse serum to support an in vitro antibody response

It was found that 10% horse serum (HrS) could be used to support an in vitro antibody response to SRBC. It could replace the fetal calf serum (FCS) in both the culture medium and the nutritional feed. The response could be improved by the addition of 2-mercaptoethanol or by including both FCS (5%) and HrS (5%) in the culture medium.     

Restoration of impaired immune functions in aging animals. II. Effect of mercaptoethanol in enhancing the reduced primary antibody responsiveness in vitro.

The enhancing effect of 2-ME on the in vitro primary antibody forming capacity of young and old spleen cells from 5 different strains and hybrids was investigated by assessing the number of antibody-forming cells in response to sheep RBC stimulation. The following results were obtained: (1) the optimum concentration of 2-ME is 5 x 10(-5) M; (2) the best time to expose cultures to 2-ME is on day 0 together with the antigen, although days 3 and 4 were equally as effective with young but not old spleen cells; (3) 2-ME can enhance slightly the time of peak antibody response, but this appears to be strain dependent; (4) in response to antigenic stimulation over a 10,000-fold range in antidgen dose, antibody response by cultures exposed to it; (5) 2-ME is comparable to, or better than, those which had been exposed to it; (5) 2-ME is effective in enhancing antibody response because it probably promotes proliferation and transformation, as well as protects dividing cells which otherwise could not survive the culture conditions; (6) the relative number of viable cells detected on the third day of culture after antigen stimulation can be used in predicting with a reasonable accuracy (pie 2, 0.94) the magnitude of peak antibody response, which is detected normally about 2 days later; (7) 2-ME enhances antibody response of young spleen cells by about 30%, but is quite variable depending upon the genetic strain, varying from a low of --20% with random bred CV1 cells to a high of 100% with inbred C57B1 cells; (8) the enhancing effect of 2-ME on old spleen cells is much more impressive, being more that 10 times greater than on young spleen cells (i.e., 500 vs. 30%), although it too is quite variable, ranging from a low of 85% with C3H cells to a high of about 1100% with C57BL cells; (9) the enhancing effect of 2-ME on limiting numbers of spleen cells is most impressive, as judged by the relative magnitude of response by limiting (3 x 10(6)) and optimum numbers (10 x 10(6)) of young and old spleen cells and by the frequency of antibody response of cultures with limiting and optimum numbers of cells exposed to 2-ME was 23 and 3 times greater than that not exposed to 2-ME, respectively; and with old spleen cells, it was 30 and 12 times greater. In terms of the frequency of antibody responding cultures with limiting numbers of young spleen cells (3 x 10(6)), 77% responded in absence of 2-ME, whereas 100% of the cultures responded in the presence of 2-ME. The effect of 2-ME on cultures containing limiting numbers of old spleen cells was much more striking, for, in contrast to only 9% of the cultures responding in the absence of 2-ME, 78% responded in the presence of 2-ME.     

Effect of biological surfaces on neutrophil O2- production and its relationship to the CD11b/CD18 integrin-dependent adherence.

The production of O2- in response to LPS, PAF, FMLP, TNF and PMA by human neutrophils in suspension and residing on surfaces coated with fetal calf serum (FCS), fibronectin (FN), laminin (LM), collagen types I and IV (CI and CIV), fibrinogen (FBG) or fibrin (FBN) was studied. Of the agonists used, PAF and LPS failed to induce a response in any of the above conditions; FMLP and PMA stimulated neutrophils to produce similar amounts of O2- either in suspension or on biological surfaces; TNF induced O2- production only by cells residing on FN, FBG and FBN. These results indicate that production of oxygen-derived free radicals by neutrophils depends on the type of agonist and the nature of the surface they interact with. The relationship between the respiratory burst and adherence was studied by measuring O2- release and adherence of neutrophils residing on FN, LM, CIV, and FBG, in the absence and in the presence of the monoclonal antibody 60.3 that recognizes the common beta-chain of CD11/CD18 integrins. FMLP, PMA and TNF increased neutrophil adherence on all these surfaces except CIV. The monoclonal antibody markedly inhibited the FMLP and PMA-induced adherence but had no effect on the O2- release elicited by these two agonists. In contrast, the monoclonal antibody inhibited both the increased adherence and O2- release induced by TNF on FN and FBG. The TNF-induced increase in adherence to LM, that was not accompanied by an increase in O2- release, was also inhibited by the monoclonal antibody. We conclude that the respiratory burst of neutrophils residing on surfaces is not necessarily correlated with adherence.     

Restoration of impaired immune functions in aging animals. VI. Differential potentiating effect of 2-mercaptoethanol on young and old murine spleen cells

The differential effect of 2-mercaptoethanol (2-ME) on spleen and bone marrow cells of young and old mice was determined in vitro. Both the ability of spleen cells to proliferate and to generate Ig-secreting cells and the capacity of bone marrow cells to generate myeloid colonies were assessed. All three activities assessed in both young and old mice were enhanced by the presence of 2-ME, but a differential effect with respect to age was noted in only one. This was the polyclonal activating antibody response to bacterial lipopolysaccharide (LPS) in which 2-ME enhanced young spleen cells to a greater extent than old spleen cells, although their mitogenic responses to LPS were enhanced to the same extent. The ability of 2-ME to enhance old spleen B cells to proliferate but not differentiate in their response to LPS would suggest that aging alters certain subpopulations of spleen cells, some of which are sensitive and others insensitive to the potentiation effects of 2-ME. The enhancing action of 2-ME on the proliferative activity of LPS-stimulated young spleen cells was reduced drastically by decreasing the number of T cells by prior treatment of spleen cells with anti-T cell reagent. The proliferative activity was then brought back to normal pretreatment level by adding enriched T cells. Therefore it would appear that regulatory T cells are the target of the enhancing action of 2-ME. The failure of old spleen cells to respond vigorously to the polyclonal activating action of LPS and 2-ME individually and in combination would indicate that age-related alterations may be taking place in the B cells and/or the regulatory cells. Young-old spleen cell mixture study indicates that there are regulatory cells in old spleen cells which can inhibit B cell differentiation but not B cell proliferation.     

Effect of delayed addition of 2-mercaptoethanol on the generation of mouse cytotoxic T lymphocytes in mixed leukocyte cultures.

The generation of mouse cytolytic T lymphocytes (CTL) in primary or secondary mixed leukocyte cultures (MLC) is greatly enhanced by the addition of 2-mercaptoethanol (2-ME) to the culture medium. This enhancement can be equally demonstrated with either reduced or oxidized 2-ME. Addition of 2-ME to MLC as late as 3 days after the initiation of the culture results in a peak CTL response which is nearly as high as the one in cultures containing 2-ME throughout the 4 day-incubation period. Quantitative analysis of the CTL response in MLC supplemented with 2-ME on day 3 shows a 50-fold increase in lytic activity within 24 h, suggesting that cell differentiation, in addition to proliferation may be affected by this agent.     

Different optimal PHA concentrations for stimulation of Chinese hamster lymphocytes in cultures supplemented with foetal calf serum or horse serum

Chinese hamster lymphocyte cultures were supplemented with foetal calf serum (FCS) or horse serum (HS). Addition of 2-mercaptoethanol (2-ME) to cultures supplemented with these sera resulted in a marked increase in lymphocyte proliferation. The PHA concentrations necessary for optimal lymphocyte stimulation were much lower in cultures supplemented with HS than with FCS. Phytohaemagglutinin (PHA) concentrations optimal for stimulation of a given number of lymphocytes in cultures supplemented with FCS often gave strong inhibition of similar cultures supplemented with HS.     

Improved plaque production for short-term bone marrow and fetal liver cultures

The addition of 0.5% globulin-free (GF-BSA) or 0.5% delipidated BSA (D-BSA) to short-term murine bone marrow (BM) (cultures) increased the number of plaque-forming cells (PFC) responding to trinitrophenylated lipopolysaccharide (TNP-LPS) 2-5-fold (1.1 X 10(4)-2.7 X 10(4) PFC per 10 X 10(6) nucleated BM cells). Although it was necessary to continue to supplement these cultures with 5% fetal calf serum (FCS), the inclusion of the aforementioned BSA preparations provided enhanced PFC production for all lots of FCS tested. Similarly, these preparations of BSA made it feasible to also culture BM in autologous mouse sera (MS) or in medium without 2-mercaptoethanol (2-ME) if in the latter case the D-BSA was pretreated with 2-ME. Thus, the inclusion of GF-BSA or D-BSA in short term cultures of BM not only substantially increased the number of Ig-secreting B cells produced in response to TNP-LPS but seemed to eliminate the need to screen for supportive batches of FCS or MS. These preparations of BSA also facilitated hapten specific PFC responses of fetal liver cultures.     

Human rheumatoid factor-producing cell induction by 2-mercaptoethanol: immunomodulation by a simple thiol compound

Two-mercaptoethanol (2-ME), a simple 2 carbon thiol compound with a wide variety of in vitro and in vivo immunomodulating effects, was evaluated for its usefulness as a molecular probe of human antibody producing cell activation by adding 2-ME to cultures of peripheral blood mononuclear leukocytes from normal human volunteers. Culturing normal human leukocytes with 2-ME induced a significant number of cells producing rheumatoid factor as measured by a hemolytic plaque forming cell (PFC) assay. Dose response studies revealed 5 X 10(-5)M to be the optimum concentration of 2-ME for the induction of rheumatoid factor plaque forming cells (RF-PFC). This concentration of 2-ME also maximally induced PFC making antibodies to sheep red cells coupled to the trinitrophenyl (TNP) hapten demonstrating that 2-ME is a polyclonal inducer of human PFC. The addition of 5 X 10(-5) M 2-ME to cultures containing maximal concentrations of the polyclonal stimulators, pokeweed mitogen and human heat-aggregrated IgG, increased the number of RF-PFC detected in these cultures by approximately 50%, although both lower and higher concentrations of 2-ME suppressed the RF-PFC response. We conclude that 2-ME induces normal human leukocytes to produce rheumatoid factor as part of a polyclonal activation of antibody producing cells. 2-ME also has immunomodulating effects when added to other polyclonal stimulators of antibody producing cells.     

The effect of 2-mercaptoethanol on IgM and IgG antibody activity

The effect of reduction by 2-mercaptoethanol (2-ME) on antibody activity was studied in antisera with high anf low IgG concentrations.Sera obtained from B10.LP nu/nu mice during a primary response against a rat PVG/c skin graft contained only 2-ME sensitive antibodies. However, when analyzed on sucrose gradients, IgG as well as IgM antibody activity was present. After raising the low serum IgG concentration (0.3–0.7 mg/ml) of these sera by additional of normal mouse serum (5.1 mg IgG/ml), 2-ME resistant antibodies became detactable. Hyperimmune C57BL6 anti PVG/c lymphocyte serum with a high IgG concentration (20.2 mg/ml) and antibody activity predominatly located in the IgG class was not affected by 2-ME treatment.These data show that IgG antibodies, although less susceptible to reduction than IgM antibodies, are not resistant to this treatment. At high IgG concentrations the proportion of inactivated IgG with specific antibody will be negligible but a low IgG concentrations the use of this method leads to serious underestimation of IgG antibody activity.     

Lymphoproliferation Induced in Mouse Spleen Cells by Mycoplasma arthritidis Mitogen

Spleen cells of various mouse strains (e.g. BALB/c, C311, and CBA) reacted towards MAS (a mitogen derived from supernatants of cultured Mycoplasma arthritidis) with a marked lymphoproliferative response. This reactivity was T-cell-dependent. It was reduced by 90% after removal of macrophages by passage of the spleen cells through Sephadex G-10 columns. Addition of 2-mercaptoethanol (2-ME) to macrophage-depleted CBA spleen cells completely restored the response to MAS. Spleen cells of C57BL/6 and C57BL/10 mice were unreactive to MAS, even in the presence of macrophages, and this non-reactivity was controlled by the I-region of H-2. Other mouse strains that, similarly to C57BL/6, lack the expression of I-E on the cell surface (that is, mice of the haplotype H-2f, H-2q, and H-2s) were also non-responsive to MAS. However, the addition of 2-ME to spleen cells of non-responder mice resulted in high lymphoproliferative responses to MAS, which were as high as those of CBA spleen cells. The reaction of C57BL/6 spleen cells to MAS in the presence of 2-ME again was T-cell-dependent, as shown by data with spleen cells of homozygous nude mice and spleen cells treated by anti-thy-1 and C. A macrophage dependency of this response was also evident. When C57BL/6 spleen cells were vigorously freed of accessory cells by the use of nylon wool columns, the MAS response could no longer be restored by 2-ME.     

An assessment of the factors contributing to the killing of type 3 Streptococcus pneumoniae by human polymorphonuclear leukocytes in vitro

To characterize the factors that contribute to the killing of type 3 S. pneumoniae, human neutrophils were obtained from healthy donors and incubated with viable organisms. In contrast to prior observations with other pneumococcal serotypes, killing was not detected when 10(6) colony forming units (cfu) were incubated at 37 degrees C for 2-4 hours with 10(6) neutrophils in the presence of 20-80% fresh autologous serum; further, pneumococcidal activity was not found when preopsonized bacteria and primed neutrophils were employed in the standard assay. However, when the bacterium to cell ratio was reduced to 1:100 and 1:1000, microbicidal action was detected; a 10-fold reduction in the number of viable bacteria was observed when 2 x 10(3) cfu were incubated with 2 x 10(6) neutrophils and 80% autologous serum at 37 degrees C for 4 hours. To assess the effects of serum factors on killing, bactericidal assays were performed in the presence of normal human serum (NHS), heat-inactivated human serum (HIHS) and absorbed human serum (AHS); heating reduced and absorption eliminated the capacity of serum to support killing. Studies performed with mutanolysin, an enzyme that lyses type 3 pneumococci, demonstrated that the effects of HIHS and AHS on bactericidal activity were highly correlated with alterations in the ability of the sera to support phagocytosis. Studies of neutrophil activation revealed changes in the production of superoxide anion that correlated well with phagocytosis and killing; however, the results of assays of leukotriene B4 generation and degranulation (beta-glucuronidase and lactoferrin release) were more variable. In mixing experiments, the capacity of HIHS to support killing was normalized with NHS; however, the ability of AHS to promote killing was not restored with HIHS or NHS. Thus, these studies demonstrate the relatively limited capacity of human serum to support the killing of type 3 pneumococci, and they emphasize the importance of killing assays in assessing interactions between the bacterium and neutrophils.     

Fetal calf serum stimulates 'autoreactive' T-cell hybridomas

During attempts to generate Sendai virus-specific T-cell hybridomas, a number of autoreactive T-cell hybridomas were produced. These hybrids secrete IL-2 in response to class II-positive syngeneic cells in the absence of added Sendai virus. Class II molecules on the stimulator cells are involved since monoclonal anti-class II antibodies block the reaction, and mapping studies using recombinant mouse strains show the response is class II restricted. Hybridomas adapted to grow in serum-free medium do not respond to syngeneic cells in the absence of serum; however, addition of fetal calf serum (FCS) restores the response. The component of FCS responsible for the stimulation of the hybridomas has been partially purified and characterized, and its mode of action investigated. These findings suggest that the putative 'autoreactive' T-cell hybridomas reported by others may be specific for a component of the culture medium rather than being truly self-reactive.     

Immune response in Xenopus laevis and immunochemical properties of the serum antibodies

Antibody production in the African clawed toad (Xenopus laevis) against Salmonella flagella and the immunochemical properties of the antibodies were studied. The titre of the serum antibodies increased slowly and reached a maximum at 3 weeks after immunization and they were detectable even 20 weeks after antigenic stimulation. No protein components were electrophoretically demonstrated at the region corresponding to the IgG of mammalian antibodies. Two types of antibodies were found; the one produced early after immunization was heat-labile and the other produced later was heat-stable. The former was of low molecular weight (7S component) and the latter was macromolecular (19S component). Both antibodies moved electrophoretically to the cathode and were sensitive to 2-mercaptoethanol (2-ME) treatment, and their antigenicities were not identical. The low molecular weight antibody did not fix complement in the presence of antigen, in contrast to the macromolecular antibody. The low molecular weight antibody did not correspond to the IgG type of mammalian antibodies in the following properties; 2-ME sensitivity, heat-sensitivity, electrophoretic mobility and absence of complement-fixing ability.     

Augmentation by 2‐Mercaptoethanol of In Vitro Anti‐TNP Antibody Production Induced by Butyrate Plus IL‐2 in Murine Splenic B Cells

Abstract

We previously reported that anti‐trinitrophenyl (TNP) antibody production in murine splenic B cells stimulated with TNP‐lipopolysaccharide in vitro was promoted by sodium butyrate (NaBu) in an IL‐2‐dependent manner. In the present study, we found that the effect of NaBu plus IL‐2 was markedly augmented by 2‐mercaptoethanol (2‐ME), which showed a slight or null effect on the response of untreated, IL‐2‐treated or NaBu‐treated B cells, as assessed by both anti‐TNP plaque‐forming cell assay and anti‐TNP IgM ELISA. Other thiol compounds such as dithiothreitol, cysteamine and reduced glutathione (GSH) also had this activity. 2‐ME enhanced the anti‐TNP antibody production induced by other short‐chain fatty acids with three to five carbon atoms plus IL‐2. The proliferation of B cells was significantly inhibited by NaBu or NaBu plus IL‐2, and the proliferation was completely restored by the simultaneous addition of 2‐ME. These results demonstrate that 2‐ME markedly enhanced anti‐TNP antibody production in murine B cells induced by NaBu plus IL‐2 and suggest that the effect of 2‐ME is at least partly due to its blocking activity of the growth‐inhibitory action of NaBu.     

In vitro induction of specific immune responses in the absence of serum: requirement for nonspecific T or B cell mitogens

A mixed lymphocyte culture reaction (MLC) between histoincompatible mouse lymphocytes could be induced in the absence of serum in vitro. There was a steady increase of DNA synthesis for 7 days in the absence of serum, whereas peak activity occurred on day 4 in the presence of serum. Analogous results were obtained when the secondary responses to SRC were determined by stimulation of DNA synthesis. It was not possible to induce a primary humoral antibody response to SRC in the absence of serum. However, if the serum-free cultures were supplemented with optimal concentrations of T (concanavalin A) or B (lipopolysaccharide, LPS) cell mitogens, a primary specific antibody response to sheep red cells (SRC) could be induced and increased in magnitude for 4 days in culture. Also, a MLC reaction permitted the induction of primary antibody responses to SRC. In the presence of fetal calf serum (FCS), optimal concentrations of T cell mitogens, as well as a MLC reaction, suppressed the induction of a primary antibody response to SRC, whereas suboptimal doses of the mitogen stimulated it. It was possible to induce specific secondary IgM and IgG antibody responses to SRC in the absence of serum. In contrast to the findings with SRC, a primary – as well as secondary – antibody response could be induced to LPS, which is a relatively thymus-independent antigen. Taken together the results suggest that: a) FCS is necessary for the induction of primary antibody responses, because it contains B and possibly T cell mitogens. b) Primary antibody responses to SRC can be induced in the absence of FCS if nonspecific B cell activation is provided by the addition of either B cell mitogens or B cell activating factors, obtained by activating T cells by specific mitogens or by a MLC reaction. c) Secondary responses to T cell-dependent antigens are possible in the absence of FCS because of the large number of specifically activated T cells present in the culture system. d) Primary antibody responses to LPS are possible in the absence of FCS because LPS by itself is a B cell mitogen.