Increased cytolytic T lymphocyte activity induced by 2-mercaptoethanol in mixed leukocyte cultures: kinetics and possible mechanisms of action.

The 20- to 50-fold increase in cytolytic T lymphocyte (CTL) activity caused by the addition of 50 muM 2-mercaptoethanol (2-ME) at the onset of a one-way murine mixed leukocyte culture (MLC) between C57BL/6 and DBA/2 splenic lymphocytes appears to be unrelated to early events in the culture: if 2-ME was present for the first 24 hr of culture only, there was no increase on day 4, but if addition of 2-ME was delayed until the last 24 hr of culture, the CTL activity was almost as high as that of cultures that were exposed to 2-ME for the entire 4-day culture period. The increase of CTL activity caused by delayed addition of 2-ME ("2-ME rescue") was used to investigate the mechanism by which the thiol induces differentiation of CTL from precursor cells. 2-ME rescue was mimicked by two other thiols, dithiothreitol and cysteamine phosphate, but at higher concentrations. Because the latter compound has no free sulhydryl group until it diffuses into cells and is enzymatically dephosphorylated, we conclude that thiols may increase the differentiation of CTL from precursor cells by an intracellular process involving free sulphydryl groups rather than by interaction with membrane sulfhydryls or destruction of inhibitor cells or their products. Cell separation experiments indicated that 2-ME rescue was independent of the presence of B lymphocytes and of adherent cells (macrophages) and was restricted to a subpopulation of T lymphocytes that developed into large lymphoid precursor cells during the first 3 days in culture even without 2-ME. The development of this subpopulation required DNA synthesis between 24 nad 72 hr after the onset of MLC. When 2-ME was added to day-3 MLC, CTL activity increased slightly as early as 4 hr later, but the major increase occurred during the second half of the 24 hr "rescue"period. Because this increase was inhibited by cytosine arabinoside (ARA-C), it seems likely that DNA synthesis is associated with and may be required for the differentiation of large precursor lymphoid cells into CTL after the addition of 2-ME.     

Augmentation of proliferation and in vitro production of cytotoxic cells by 2-ME in the rat.

Low concentrations (10(-5) to 10(-8) M) of 2-mercaptoethanol (2-ME) greatly enhance the proliferation of allogeneic cells in the rat mixed lymphocyte culture (MLC). Studies were undertaken to determine the mode of action of 2-ME. MLC proliferation can occur in the absence of serum proteins (fetal calf serum, FCS) only if 2-ME is present; however, a synergistic effect is present with FCS plus 2-ME, with a 3-fold increase in 3HTdR incorporation with FCS concentrations as low as 0.1%. Kinetic studies show no shift in the peak of proliferation (92 hr) when comparing cultures with and without 2-ME; however, 2-ME-supplemented cultures have significant 3HTdR uptake at 24 hr, and the peak amount of uptake at 92 hr is two to four times higher. Delayed addition of 2-ME until 92 and 166 hr produces a further increase in 3HTdR uptake, indicating that the entire effect is not expressed at the time of allogeneic recognition. L-ascorbic acid, another reducing agent which lacks sulfhydryl groups, elicits a much lower effect on DNA synthesis than does 2-ME. The cytotoxicity of cells harvested from MLC supplemented with 2-ME is increased without loss of target specificity, whereas the same concentration of 2-ME has no direct effect upon the cytotoxicity assay except at higher concentrations where 2-ME suppresses cytotoxicity.     

2-Mercaptoethanol acts as a potentiating factor of interleukin-2-dependent lymphocyte proliferation

An interleukin-2 (IL-2) dependent cell line which could be grown continuously with crude concanavalin A-stimulated rat or mouse spleen cell culture supernatant could not survive for over 48 hr in the culture medium supplemented with partially purified or recombinant IL-2. The cell growth was restored by adding another factor obtained from the same crude culture supernatant. This potentiating activity which was physicochemically inseparable from serum albumin was also obtained from the culture medium containing 2-mercaptoethanol (2-ME) and incubated at 37 C for 24 hr without the spleen cells. By further experiments, it was demonstrated that 2-ME itself had the potentiating activity on the IL-2-dependent proliferation of this cell line and cysteine mediated the activity of 2-ME. The cells could not enter S-phase in the absence of 2-ME even in the presence of IL-2.     

Involvement and relative importance of at least two distinct mechanisms in the effects of 2-mercaptoethanol on murine lymphocytes in culture

2-Mercaptoethanol (2-ME) exerts several effects on murine lymphocytes in culture that might explain its ability to enhance survival and growth of these cells. The uptake of the essential amino acid cystine and consequently the maintenance of intracellular glutathione levels are enhanced by 2-ME. Furthermore, 2-ME (even in the disulfide form) causes lymphocytes to release thiols into the culture medium. These effects might protect the cells from oxidative damage. The additional cystine provided by treatment of lymphocyte cultures with 2-ME might also allow adequate protein synthesis to support survival and/or growth. This study was conducted to assess the relative importance of the antioxidant and protein synthesis effects of 2-ME. As expected, 2-ME increased cystine uptake at all concentrations that enhanced growth and survival, but four nonthiol antioxidants that enhanced growth and/or survival either did not substantially affect cystine uptake or decreased it and did not affect the release of cystine or its products. The results presented here demonstrate that antioxidant protection is necessary and sufficient for lymphocyte survival and that cystine uptake in untreated lymphocytes is sufficient to support the protein synthesis needed for survival and limited growth. However, we also noted that concentrations of 2-ME that stimulated maximal growth more than doubled protein synthesis as measured at 8 hr. Thus the portion of the effects of 2-ME not accounted for by antioxidant action could be accounted for by enhanced protein synthesis.     

2-Mercaptoethanol acts at the restricted stage of interleukin-2 dependent lymphocyte proliferation

An interleukin-2 (IL-2) dependent murine cell line (TN-9) which could be grown continuously with the crude culture supernatant of concanavaline A-stimulated rat or mouse spleen cells could not synthesize DNA in the culture medium supplemented with partially purified or recombinant IL-2. The cell growth was restored by adding another factor obtained from the same crude culture supernatant. This factor, physicochemically inseparable from serum albumin, was also obtained from the culture medium added with 2-mercaptoethanol (2-ME) and incubated at 37 degrees C for 24 hr without the cells. By the experiments using semi-synchronized cell population, it was demonstrated that 2-ME or 2-ME carrying protein acted at the restricted process(es) of cell proliferation which occurred between IL-2-acting stage and the initiation of DNA synthesis.     

Kinetics of long-term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin-activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen-stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H-thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.     

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes: III. Serum-bound and oxidized 2-mercaptoethanol are available for the augmentation

The fetal calf serum (FCS) that was incubated with 2-mercaptoethanol (2-ME) followed by the removal of free 2-ME could support the antibody response to sheep erythrocytes in vitro as effectively as native FCS plus 2-ME. The supporting activity of 2-ME-pulsed FCS was reversibly abrogated by the treatment with dithiothreitol followed by dialysis. In addition, iodoacetamidetreated FCS did not acquire the supportiveness by 2-ME pulsing. These observations suggest that the activity of 2-ME-pulsed FCS would be due to the mixed disulfide between 2-ME and FCS components. On the other hand, the disulfide form of 2-ME (2-ME_ox) could also augment the antibody response as effectively as fresh 2-ME (the reduced form). These derivatized forms of 2-ME as well as fresh 2-ME was found to stimulate the transport of [³⁵S]cystine into murine lymphocytes when the uptake was examined by the long-term experiments (24 hr). These stimulations were thought to be mediated by the formation of the mixed disulfide between 2-ME and cysteine because the lymphocytes promoted the reaction of [³⁵S]cystine with 2-ME_ox- or 2-ME-pulsed FCS to produce the mixed disulfide that had been shown to be taken up by the lymphocytes four to five times more rapidly than cystine. Therefore, it was suggested that 2-ME_ox and 2-ME-pulsed FCS could augment the antibody response in a similar fashion to 2-ME by stimulating the uptake of cystine, an essential amino acid.     

Specific binding of T lymphocytes to macrophages. I. Kinetics of binding.

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.     

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)     

Differentiation of the immunosuppressive and antiviral effects of interferon.

Virus-induced (virus-type) interferon suppression of the in vitro antibody response of mouse (C57B1/6) spleen cells to sheep red blood cells was blocked by 5 × 10^?5M 2-mercaptoethanol (2-ME). The blockade was not due to a direct effect on interferon since 2-ME was capable of blocking the suppression when added to cultures up to 48 hr after interferon. 2-ME blockade of virus-type interferon immunosuppression was not due to the immunoenhancing property of 2-ME. Similar protective effects of 2-ME were observed during immunosuppression by virus-type interferon inducers, but not T-cell mitogen inducers of interferon (immune interferon). The data suggest that the immunosuppressive properties of virus-type and immune interferon preparations involve different mechanisms. Virus-type interferon inhibited DNA synthesis in unstimulated spleen cell cultures and in 2-ME stimulated cultures, and the degree of inhibition of DNA synthesis appeared to be related to the immunosuppressive property of interferon in the absence or presence of 2-ME. 2-ME did not affect the antiviral properties of either virus-type or immune interferon in nonlymphoid cells. Further, the induction of virustype interferon in spleen cells was neither inhibited nor enhanced by 2-ME, while the induction of immune interferon was enhanced. This enhancement is consistent with 2-ME enhancement of the immunosuppressive effects of immune interferon inducers.There are two possibilities for 2-ME blockade of the immunosuppressive effect of virus-type interferon, while not affecting the antiviral property. Firstly, the immunosuppressive and antiviral properties of virus-type interferon may involve different mechanisms at the subcellular level. Secondly, the selectivity of the blockade by 2-ME could be due to the fact that spleen cells are the target cells in immunosuppression, while L cells are the target cells in inhibition of virus replication. Thus, virus-type interferon may suppress the immune response at the level of the macrophage and 2-ME may reverse this effect by replacing a blocked macrophage function.     

Function of 2-mercaptoethanol as a macrophage substitute in the primary immune response in vitro.

The mechanism by which 2-ME acts as a macrophage-substitute for the induction of a primary PFC response to SRC in vitro was studied in macrophage-depleted mouse spleen cell cultures. 2-ME could replace macrophages only in FCS-supplemented cultures. Evidence is presented that the function of 2-ME is independent of residual macrophages. Neither normal nor macrophage-depleted spleen cell cultures from congenitally athymic nude mice supplemented with 2-ME, with or without FCS, could give rise to a primary in vitro anti-SRC immune response. 2-ME, at an optimal concentration of 10(-5) M, induced DNA synthesis in normal and macrophage-depleted spleen cells in both FCS-containing and serum-free cultures. The peak response occurred on day 3. The stimulation was accompanied by a polyclonal B cell activation to antibody secretion which was much more pronounced in FCS-containing than in serum-free cultures. Spleen cells from nude mice showed a weaker DNA stimulation than did cells from normal mice in FCS-containing cultures, and nearly no response under serum-free conditions. T cells obtained by a nylon column adherence method from normal mouse spleen cells showed good DNA synthetic responses in FCS-containing, but no response in serum-free cultures. These results show that 2-ME has weak mitogenic activity for B cells, and in combination with FCS, strong mitogenic activity for T cells. Since the macrophage provides stimulation to the T cell in the primary anti-SRC PFC response in vitro, these results suggest that the direct mitogenic activity of 2-ME with FCS on T cells provides the functional substitution for macrophages.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G₁ period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G₁ period before cell fusion and differentiation during myogenesis.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. I. Kinetics

An in vitro culture method giving a high cell recovery rate of 25–38% has been developed to generate a relatively pure population of T cell progeny reacting to xenogeneic cell antigen. The method is basically a modification of the Ginsburg method and utilizes mosaic monolayers that are comprised of syngeneic Lewis and xenogeneic BALB/c fibroblasts. Thoracic duct lymphocytes of Lewis rats were cultured for 6 days on these monolayers and the resulting lymphocyte population, rich in blast cells, was collected and separated free of contaminating fibroblasts by an additional 6 hr incubation without monolayers. Cytotoxic activity of these harvested lymphocytes was assessed by a modification of the Hellström method in which embryonic target fibroblasts, Lewis or BALB/c, were plated at a critical density of 2200 cells/cm² of culture area. This, coupled with the use of a newly developed culture medium, allowed the demonstration of specific as well as nonspecific activity of up to 100% suppression at 48 hr of test incubation. The specific activity was clarified and determined critically by comparing the activity of these lymphocytes to that of lymphocytes grown on syngeneic monolayers, though rich in blast cells, reacting only to the horse serum in the medium. The nonspecific activity was not due to the deterioration of test cultures and needed the continued presence of fully active blast cells for complete suppression of the target cell growth in the later phase of test cultures. By changing the ratio of lymphocytes to target cells and following the kinetics of target cell destruction, it was shown that specific activity effects an active killing of target cells, whereas the nonspecific activity was primarily the suppression of target cell growth. The specific activity was effective with far less lymphocytes per target cell; was nearly complete by 12 hr of test cultures while the nonspecific activity was complete at 48 hr. Yet, the increase in the specific activity was nearly proportional to that of nonspecific activity and even the nonspecific activity seemed to kill sensitive Lewis target cells at its full intensity. These observations are in accordance with an interpretation of the mechanism of the specific target cell destruction as involving contact and recognition followed by the release of lymphotoxin into or onto target cells.     

Uptake of transferrin-bound zinc by human lymphocytes.

Uptake of transferrin-bound zinc was stimulated in phytohemagglutinin-treated human lymphocytes as compared to untreated lymphocytes. Stimulation of zinc uptake depended upon the concentration of phytohemagglutinin and was maximal for the first hour after addition of phytohemagglutinin to lymphocyte cultures. Thereafter, increased zinc uptake declined until approximately basal levels were reached 5 hr after addition of phytohemagglutinin. Stimulation of zinc uptake was insensitive to sulfhydryl reagents, but was decreased by KCN, actinomycin D, aurin tricarboxylic acid, and by lowering the incubation temperature. Two compounds, NaF and poly-l-ornithine, were found to markedly increase zinc uptake over that seen with only phytohemagglutinin. Additionally, compounds known to increase cellular levels of cyclic AMP, such as epinephrine, histamine, serotonin, glucagon, and prostaglandin E₁, as well as 8-bromo-cyclic AMP and dibutyryl-cyclic AMP, also increased uptake of transferrin-bound zinc by phytohemagglutinin-stimulated lymphocytes.     

Regulation of T cell growth factor production: arrest of TCGF production after 18 hours in normal lectin-stimulated mouse spleen cell cultures

The detailed kinetics of TCGF accumulation in Con A-stimulated spleen cell cultures shows a maximum at 24 hr, with a subsequent decrease in activity. This decrease is not due to the appearance of inhibitory substances "masking" TCGF activity. Pulse experiments show that the rate of TCGF production falls sharply after 18 hr and is completely arrested after 24 hr of Con A stimulation. The arrest in TCGF production is the result neither of culture depletion in medium components nor of limiting accessory cell function or inactivation of the lectin, and it thus seem to be the result of inactivation of TCGF-producing T cells. This regulation is not the result of a TCGF-mediated feedback mechanism but rather of lectin-induced suppressive cells that appear in culture after 24 hr and turn off de novo production of TCGF in fresh cultures.     

In vitro induction of polyclonal killer T cells with 2-mercaptoethanol and the essential role of macrophages in this process.

Killer T cells against allogeneic and syngeneic tumor cells were generated in vitro by the addition of 2-mercaptoethanol (2-ME) to the murine spleen cell culture in the absence of any antigenic stimulation. The maximum activity of T cell-mediated cytotoxicity (CMC) induced with 2-ME was observed on day 4 of culture and the induction of CMC was completely inhibited by the addition of inhibitor of DNA synthesis, hydroxyurea, or cytosin arabinoside. CMC induced with 2-ME was specifically inhibited by the addition of unlabeled target cells to the 51Cr-release assay system. These results indicated that killer T cells were generated in the presence of 2-ME as a result of nonspecific polyclonal activation of precursors into cytotoxic effector cells and that they recognized target cells with antigen-specific recognition receptors. Spleen cells deprived of adherent cells showed impaired induction of CMC with 2-ME. The addition of peritoneal exudate macrophages to splenic T cells restored this response. The result indicated that macrophages were essential for the induction of CMC with 2-ME. The possibility that the function of macrophages was mediated by soluble factor(s) released from macrophages was demonstrated by the separate culture of splenic T cells and macrophages in double-chambered, Marbrook-type vessels and by the addition of supernatants from macrophage cultures to splenic T cells. 2-ME and soluble factor(s) released from macrophages seemed to be required for the activation of precursors into killer cells.     

Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures

The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells.     

Withdrawal of 2-mercaptoethanol induces apoptosis in a B-cell line via fas upregulation

Mouse lymphoid cell cultures are dependent on reducing agents in their culture medium to allow proliferation and survival of the cells. In the case of the mouse CD5⁺-pre-B cell line SPGM-1, withdrawal of 2-mercaptoethanol (2-ME) resulted in rapid inhibition of proliferation and subsequent cell death by apoptosis. The pathways leading to cell death by withdrawal of 2-ME or by incubation with ionomycin, a known inducer of apoptosis, were compared. Both kinds of stimulation resulted in apoptosis of the whole population, but cell death occurred with different kinetics. Only apoptosis induced by ionomycin was inhibited by coincubation with the phorbol ester PMA, while apoptosis induced by withdrawal of 2-ME was not. Overexpression of the human bcl-2 proto-oncogene in these cells delayed the death process induced by either method. SPGM-1xbcl-2 cells accumulated in the G₀/G₁ and G₂/M cell cycle phases after removal of 2-ME from the medium, whereas treatment with ionomycin resulted in an arrest only in the G₀/G₁ transition. Interestingly, both stimuli induced the expression of the Fas receptor, but with different kinetics, while the Fas ligand (FasL) was expressed constitutively in SPGM-1 cells. These data demonstrate that withdrawal of 2-ME and incubation with ionomycin both induce rapid cell death by apoptosis, possibly mediated by an autocrine Fas/FasL loop. Although the initial pathways activated by the two forms of treatment must be different, they converge on a common level controlled by the anti-apoptotic gene product Bcl-2. J. Cell. Physiol. 177:68–75, 1998. © 1998 Wiley-Liss, Inc.