Enhancement of antibody synthesis in vitro by mercaptoethanol

Click, Robert E.; Benck, Loretta; Alter, Barbara J.

doi:10.1016/0008-8749(72)90237-7

Cited by 153 publications

(69 citation statements)

References 8 publications

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“…Unattached cells were transferred to new flasks and incubated for a further hour under the same conditions . The unattached cells (NAPC) were washed once and resuspended (5 x 10 8 cells/ml) in EBSS with supplements as above and 5 x 10 -5 M 2-mercaptoethanol which helps to maintain functional lymphocyte activity (28) . Aliquots of the cells were then exposed to three or more selected antisera under conditions similar to the standard C-dependent cytotoxicity assay (see above) ; i.e .…”

Section: Methodsmentioning

confidence: 99%

Expression of T-cell differentiation antigens on effector cells in cell-mediated cytotoxicity in vitro. Evidence for functional heterogeneity related to the surface phenotype of T cells.

Shiku¹,

Kisielow²,

Bean³

et al. 1975

The Journal of Experimental Medicine

205

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The cell-mediated cytotoxicity (CMC) of nonadherent cells from the peritoneal cavity (NAPC) of alloimmunized mice can be measured by the [3H]proline microassay. The exhibition of thymus-derived (T) cell antigens on these killer cells was studied by incubating them with the relevant T-cell antisera and complement (C), under optimal conditions for lysis, before performance of the CMC assay. Under these conditions, the following T-cell antigens were demonstrable on the killer population in terms of percent reduction in CMC by the respective antisera: (a) Thy-1.1 (83%) and Thy-1.2 (100%), (b) MSLA (86%), (c) NTA-RA (a T-cell antigen recognized by naturally occurring autoantibody of NZB mice) (62%), (d) Ly1.1 )58%, (e) Ly-2.1 (11%; considered a marginal result) and Ly-2.2 (63%), and (f) Ly-3.2 (77%). The following were not demonstrable: (g) TL, and (h) Ly-1.2. (i) The antigen Ly-3.1 was not studied. Omission of C deprived all T-cell antisera tested of their capacity to suppress CMC, indicating that the cell components recognized by such antisera may perform no direct function in CMC. On the assumption that all Ly+ cells are Thy-1+, it is clear that the T-cell members of the immune NAPC population must be heterogenous. This follows from the fact that the proportions of T cells lysed by different Ly antisera did not correspond with ensuing degree of loss of CMC capacity. The extremes were represented by anti-Ly-1.2 (74% Thy-1+ cells lysed, but no reduction in CMC) and Ly-3.2 (54% Thy-1+ cells lysed, with 77% reduction in CMC). From this initial survey it appears that the C57BL/6 mice killer T-cell population active in CMC in vitro is relatively rich in surface antigens of the Ly-2/Ly-3 category and relatively poor in representation of the Ly-1 surface antigens. It remains to be seen whether this killer cell phenotype, poor in Ly-1 and rich in Ly-2/Ly-3, is characteristic of the mouse generally. From these results it appears that subsets of T cells with different immunological functions may exhibit qualitative or quantitative differences in surface antigens specified by different Ly loci; this will be easier to assess in the future when the results of experiments with the same Ly antisera but dealing with T-cell functions other than CMC become available.

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Section: Methodsmentioning

confidence: 99%

Expression of T-cell differentiation antigens on effector cells in cell-mediated cytotoxicity in vitro. Evidence for functional heterogeneity related to the surface phenotype of T cells.

Shiku¹,

Kisielow²,

Bean³

et al. 1975

The Journal of Experimental Medicine

205

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“…Broome and Jeng, in 1972, reported a requirement for sulfhydryl compounds in certain murine leukemia cells in vitro (2,3). Other reports describe enhancement by sulfhydryl compounds of blast transformation (4,5), of antibody formation (6)(7)(8)(9), and of viability (2,8) in lymphocyte cultures. This report describes a study of the sulfhydryl dependence of some hematopoietic cells and the inhibitory effect of vitamin B12 compounds on these cells.…”

mentioning

confidence: 98%

Sulfhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin B12 compounds.

Toohey¹

1975

Proc. Natl. Acad. Sci. U.S.A.

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Primary explants of P388, EL-4, and L1210 murine leukemia cells and of normal mouse bone marrow are shown to require sulfhydryl compounds for proliferation in vitro. Nine established cell lines show no stimulation by these compounds. Leukemia cells can lose the sulfhydryl dependence after various periods of adaptation to in vitro culture. Various sulfhydryl compounds have widely differing potencies in promoting in vitro proliferation of dependent cells. The effect appears to be specific for sulfhydryl groups in the reduced form. Vitamin B12 compounds inhibit the growth of sulfhydryl-requiring cells, apparently by catalyzing the oxidation of the sulfhydryl groups.Sulfhydryl compounds have received relatively little attention as essential nutrients for cells in vitro. In 1971, Park et al. (1) briefly described stimulation by cysteine and glutathione of plasmacytoma growth in vitro. Broome and Jeng, in 1972, reported a requirement for sulfhydryl compounds in certain murine leukemia cells in vitro (2, 3). Other reports describe enhancement by sulfhydryl compounds of blast transformation (4, 5), of antibody formation (6-9), and of viability (2,8) in lymphocyte cultures. This report describes a study of the sulfhydryl dependence of some hematopoietic cells and the inhibitory effect of vitamin B12 compounds on these cells. MATERIALS AND METHODSCell Culture. Eagle's minimum essential medium (MEM, ref. 10) was supplemented with 5-20% fetal calf serum (as indicated), 30 mg of glutamine/100 ml, 5 mg of asparagine/100 ml, 10 mg of serine/100 ml, and 11 mg of sodium pyruvate/100 ml. No antibiotics were added. In experiments involving a series of variable additives, a single homogeneous culture was used to pour all plates in the series. Variable additives were sterilized by filtration and placed in the plastic culture dishes before the culture was added.P388 murine leukemia cells (11) and L1210 murine leukemia cells (12) were obtained from peritoneal aspirates of DBA/2 mice which were inoculated 4-6 days previously with 106 cells. EI-4 murine leukemia cells (13) were obtained in a similar manner from C57 mice. Leukemia cells were cultured in 1.5-ml cultures in liquid medium in 35 X 10-mm Falcon plastic plates with an initial cell density of 1 X 105 cells per ml. After incubating at 370 in 5% CO2 for the indicated number of days (usually 3 days), the cells were suspended by swirling and diluted either 1 to 2 in trypan blue for counting in a hemocytometer or 1 to 800 in Isoton for counting in a Coulter counter.The For bone marrow culture, the medium was made at two times the normal concentration and mixed with an equal volume of 0.6% agar at 370 before adding fresh bone marrow cells obtained from the femurs of white Swiss Webster mice. Colony-stimulating factor was provided as an extract of gravid C3H or DBA/2 mouse uterus prepared by the method of Bradley et al. (21). Cultures of 1.5 ml were plated as a single layer in 35 X 10-mm Falcon plastic plates. Clones of more than 20 cells were counted as colonies on the seve...

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“…In fresh medium, at 5-50 ,M the thiol had the same growth-promoting-effectiveness (3,4), but in conditioned medium (concentrated to 1/8 volume) a change from 5 to 50 ,uM resulted in a decrease of inhibition from 75% to 27%. In more concentrated medium (145 volume) in which growth of L1210(V) was completely suppressed, thiol at 50 ,uM had no 1 (4). All the lines just described were found to condition medium so that the thymidine uptake of L1210(V) cells was inhibited (Table 3).…”

Section: Resultsmentioning

confidence: 99%

“…Specific thiols and their corresponding disulfides promote the survival and replication in vitro of mouse lymphoid cells, a property which is retained in numerous lymphoma lines (1)(2)(3)(4). Such substances, however, are either nonphysiological (particularly 2-mercaptoethanol which has been widely used in experimental systems) or are required at concentrations much greater than occur in vivo.…”

mentioning

confidence: 99%

Production of autoinhibitory factors by mouse lymphoma cells in vitro and its relationship to thiol dependence.

Tanapat¹,

Gaetjens²,

Broome³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Five different kinds of mouse lymphoma cells conditioned culture medium so that it became inhibitory to thiol-dependent cells but not to thiol-independent cells. Conditioning was rapid and appeared to be complete in 1-3 hr. The inhibitory effects could be reversed by concentrations of 2-mercaptoethanol higher than those usually required for growth promotion. Inhibitory factors were concentrated by ultrafiltration and chromatography on Sephadex G-10 which showed their apparent molecular weights to be 300-600. The factors were stable at 1000 and to proteases but were inactivated (after reduction) by iodoacetamide, indicating that they may be thiols or disulfides.Specific thiols and their corresponding disulfides promote the survival and replication in vitro of mouse lymphoid cells, a property which is retained in numerous lymphoma lines (1-4). Such substances, however, are either nonphysiological (particularly 2-mercaptoethanol which has been widely used in experimental systems) or are required at concentrations much greater than occur in vivo. On the other hand, thiol is not required by lymphoma cells when they are cocultivated with macrophages or other cells but, after separation, the supernatant medium does not continue to support growth (5, 6). The mechanism of these effects and their relationship to conditions in vio thus far have not been explained.We here present evidence that thiol-dependent lymphoma cells in vitro are subject to autoinhibition mediated through substances that they liberate into the medium. These have been partially characterized and appear to be thiols or disulfides. Growth promoting thiols (e.g., 2-mercaptoethanol) or cocultivated macrophages interrupt the autoinhibitory system, and these actions may therefore be significant for restoring cell proliferation. The experiments to be described were prompted by observations on the growth promoting effect of macrophages in cocultivation with lymphoma cells (6). Numerous attempts failed to show evidence that macrophages produced a stable conditioning of the medium, and an alternative explanation, that these cells might reverse an autoinhibition by L1210(V) cells, was therefore considered.In most of our experiments we used cells of the thiol-dependent lymphoma L1210(V), whose properties we compared with those of its thiol-independent variant L1210(A) (7). Three other cell lines were examined-EL4, RADA1, and ERLDand in these the same relationships between autoinhibition and thiol dependence were found. MATERIALS AND METHODSTumors. B6D2F1 mice were used to carry the following tumors in ascitic form: L1210(V), L1210(A), and EL4. ERLD (i) Supernatants were concentrated up to 20 times by using a UM05 or other ultrafiltration membrane (Amicon Co., Lexington, MA). Aliquots from these concentrated solutions were tested for their ability to support growth of the various cell lines in tissue culture.(ii) Supernatants were lyophilized to dryness and the residues were dissolved in water and chromatographed on Sephadex G-10.Measurement of Lymphoma Ce...

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Enhancement of antibody synthesis in vitro by mercaptoethanol

Cited by 153 publications

References 8 publications

Expression of T-cell differentiation antigens on effector cells in cell-mediated cytotoxicity in vitro. Evidence for functional heterogeneity related to the surface phenotype of T cells.

Expression of T-cell differentiation antigens on effector cells in cell-mediated cytotoxicity in vitro. Evidence for functional heterogeneity related to the surface phenotype of T cells.

Sulfhydryl dependence in primary explant hematopoietic cells. Inhibition of growth in vitro with vitamin B12 compounds.

Production of autoinhibitory factors by mouse lymphoma cells in vitro and its relationship to thiol dependence.

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