Cyclosporin A and FK506 inhibit activation-induced cell death in the murine WEHI-231 B cell line.

The WEHI-231 B lymphoma cell line expresses the phenotype of immature B cells. Cross-linking of surface IgM induces programmed cell death (PCD) with typical features of apoptosis demonstrated by the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Activation of protein kinase C (PKC) by phorbol esters was reported to protect WEHI-231 cells against apoptosis induced by ligation of antigen receptors. It was therefore hypothesized that PCD could result from a defect in PKC response with an imbalance in the phosphoinositide pathway in favor of Ca2+ mobilization. In support of this hypothesis, we show here that apoptosis can be readily triggered by the calcium ionophore ionomycin. Furthermore, pretreatment of cells with cyclosporin A or FK506 which inhibit selectively the phosphoprotein calcineurin, a calcium-and calmodulin-dependent serine/threonine phosphatase, protects WEHI-231 cells against apoptosis induced by ionomycin or ligation of surface IgM. Unlike phorbol esters, cyclosporin A did not impair the rise of intracellular Ca2+ induced by cross-linking of antigen receptors. Altogether, the data indicate that the phosphorylation status of yet undefined key cellular substrates controls the cellular response to calcium-dependent apoptotic signals in this B cell lymphoma.

Requirement for tyrosine phosphorylation in lipopolysaccharide-induced murine B-cell proliferation.

Bacterial lipopolysaccharide (LPS) induces a strong B-cell proliferative response with subsequent differentiation, through a complex signal transduction pathway. This process is known to be mediated through protein kinase C (PKC) translocation without Ca2+ mobilization. Here, we show that B-cell proliferative responses induced by five different LPS preparations, as well as by F(ab')2 anti-IgM antibodies, are inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A. In contrast, B-cell proliferation induced by the combination of phorbol 12-myristate 13-acetate (PMA) plus ionomycin was not influenced by treatment with either herbimycin A or genistein. These data indicate that tyrosine phosphorylation is required to initiate B-cell proliferation by LPS.

Induction of mouse IgA B cell differentiation by phorbol ester in the absence of proliferation.

Previously, we have demonstrated that protein kinase C-activating phorbol esters selectively induce IgA synthesis by mouse B cells whether stimulated or not by lipopolysaccharide (LPS). In this study, we investigated in detail whether this maturation into IgA-secreting cells is preceded by cell proliferation. T-depleted spleen B cell suspensions were fractionated by Percoll gradient into a dense fraction that contained a majority of small resting B cells, and a hypodense fraction relatively enriched in large B cells. Phorbol 12-myristate, 13-acetate (PMA) induced IgA synthesis in both types of cells, though to a greater extent in large B cells. The phorbol ester accelerated and increased DNA synthesis in LPS-stimulated B cells but did not induce any DNA synthesis in small or large B cells, while at the same time decreasing the percentage of cells in the S, G2/M phases of the cell cycle. Inhibition of proliferation by colcemid, a cell cycle blocker, did not prevent PMA-induced IgA synthesis. The number of IgA secreting cells determined by the enzyme-linked immunospot reached a maximum at 24 h, by which time more than 50% of IgA B cells showed morphological features characteristic of plasma cells. No increase in percentage of large or blastic IgA cells was observed. Collectively the data indicate that PMA induces the terminal differentiation of mIgA+ B lymphocytes into IgA plasma cells without any DNA synthesis and cell proliferation.

Selective induction of high levels of IgA synthesis in Peyer's patch B cells by protein kinase C-activating phorbol esters.

To understand mechanisms of signal transduction involved in the regulation of isotype differentiation of B lymphocytes, we investigated effects of activation of protein kinase C (PKC) by phorbol esters and elevation of intracellular free calcium (Ca2+) by the calcium ionophore ionomycin (Ion) on Ig secretion by mouse Peyer's patch (PP) and spleen B cells. Results show that Ion suppressed production of IgM, IgG, and IgA by LPS-stimulated B cells whereas PKC-activating phorbol esters also inhibited LPS-induced IgM and IgG secretion, but induced a substantial IgA synthesis, as well as alpha-chain mRNA transcription, in B cells whether stimulated or not by LPS. Phorbol esters enhanced IgA response by directly activating PKC, inasmuch as the other phorbol ester, 4 alpha-phorbol 12,13-didecanoate, which is inactive with respect to PKC, had no effect on B cell differentiation. The increase in IgA secretion occurred in whole PP B cells, but not in the membrane IgA- B cell subset, suggesting that PKC activation does not promote the switching rate of IgM+ cells to IgA+ cells. Results from double staining studies of mIgA using FITC-labeled anti-IgA antibodies and DNA content using the DNA-binding propidium iodide showed that enhanced IgA response was not caused by IgA B cell clonal expansion. PMA induced low level of IL-6 production by highly purified PP B cells. However, addition of anti-mouse IL-6 antibody did not prevent PMA-enhanced IgA secretion, suggesting that IL-6 was not responsible for IgA induction by PMA. Collectively, the present data demonstrate that PKC activation and Ca2+ mobilization, which synergistically trigger cell proliferation, have differential effects on B cell isotype differentiation. Elevation of intracellular Ca2+ suppresses Ig production, but activation of PKC selectively enhances IgA secretion by directly promoting terminal differentiation of IgA-committed PP B cells into IgA-secreting plasma cells.

Role for protein kinase C activation in IgA B cell terminal differentiation.

In the present study, we demonstrate that the PKC-activating phorbol ester PMA selectively induced IgA synthesis by PP B cells. PKC activation triggered neither B cell proliferation nor the switching rate of IgA- to IgA+ cells. Together with the fact that the rate of IgA secretion by the myeloma cell line MOPC 315 was not altered by PMA, the data demonstrate that activation of PKC enhances IgA secretion by promoting terminal differentiation of IgA-committed B cells into IgA-secreting plasma cells.

In vitro proliferative responses of spleen lymphocytes from rats bearing progressive or regressive tumors induced by cell variants of a syngeneic colon carcinoma.

From one colonic carcinoma chemically induced in the rat, 2 sublines of tumor cells have been cloned, one (PROb) inducing progressive tumors, the other (REGb) generating tumors that regress a few weeks after s.c. injection into syngeneic hosts. Our study was aimed at comparing cellular immunity between animals bearing PROb or REGb tumors. Spleen cells were first tested for in vitro proliferation in response to mitomycin-treated PROb or REGb cells. Only spleen cells from rats injected with REGb cells proliferated significantly when mixed with PROb or REGb cells. The proliferative response induced by REGb cells was considerably higher than the response to PROb cells. When spleen cells from rats bearing REGb tumors were cultured with a mixture of REGb and PROb cells at various PROb/REGb cell ratios, PROb cells significantly suppressed the strong proliferative response generated by the same number of REGb cells alone. REGb-immune spleen cells, after in vitro stimulation by PROb or REGb cells, were not cytotoxic for either cell variant. REGb-immune spleen cells did not differ in their content of T lymphocytes expressing CD4 or CD8 markers when they were stimulated by PROb or REGb cells in vitro, but REGb cells induced a larger number of activated lymphocytes expressing the IL-2 receptor. Our results indicate that, compared to REGb cells, PROb cells are poorer stimulators of proliferation of tumor-immune spleen cells, and that they are able to suppress the proliferative response induced by REGb cells.

Comparative study of biological properties of proteins synthetised in vitro by murine decidua and deciduoma.

Supernatants from short-term in vitro cultures of murine decidual tissue obtained from the uteri of pregnant mice on day 8 postcoïtum (decidua, in the presence of paternal antigens) or from pseudopregnant mice (deciduoma, without any histocompatibility antigens were assessed for their regulatory activity; both statuses (physiological and experimental) can only be developed under hormonal conditions. All these supernatants possess no complement inhibitory activity, but they markedly impair the generation of cytotoxic T cells (CTL) and suppress anti-sheep red blood cell antibody response. These effects seem essentially located in a single AcA 22 fraction of 60 KD that inhibits CTL, while the plaque-forming cell response is strongly stimulated. Electrophoretic and gel filtration profiles of supernatants from cultures of decidua and deciduoma seem similar. The results suggest that the factors synthetised by decidua or deciduoma are the same and are not induced by exposure to foreign histocompatibility antigens of the fetus. According to these results, it seems likely that decidualization represents a localized general mechanism that can be induced, under a particular hormonal background, by different stimuli (trauma) rather than being a specific response to a particular signal (blastocyst).

Specific and non-specific immunosuppressive factors in salamander pregnancy serum.

In vitro experiments employing pregnant Salamandra salamandra have previously shown a maternal specific cytotoxic cellular reaction against larval cells and an inhibition of this reaction by maternal serum. Two serum fractions, 19S and 13S, have been implicated in this inhibition. In the present work, the site of action and the identity of the factors responsible for this activity have been determined. Pre-incubation experiments indicate that the 19S fraction from pregnancy serum specifically protects embryonic epithelial cells derived from the same pregnant female. The 13S fraction non-specifically inhibits cytotoxic activity of maternal cells. Both factors implicated in these reactions are respectively an IgM and an alpha 2-macroglobulin. The question of whether the alpha 2M is responsible for immunosuppression is discussed, with particular reference to its vitellogenin analogue and also in view of the fact that the presence of this alpha 2M is not always linked with the immunosuppressive properties (except during pregnancy).

Immunoregulatory activity of supernatants from short-term cultures of mouse decidual tissue.

Supernatants from short-term in-vitro cultures of decidual tissue, obtained from the uteri of pregnant mice from Days 4 to 13 post coitum (Day 1 = day of mating), were assessed for immunoregulatory activity by their addition to a mixed lymphocyte reaction (MLR), an in-vitro analogue of the afferent arm of the immune response. All culture supernatants tested possessed inhibitory activity in the MLR, although the extent of inhibition was affected by seeding density, length of culture, and the day of pregnancy from which decidual tissue was obtained. Inhibitory activity produced by decidual cultures increased from Day 4 to reach a maximum on Day 8, and then declined to Day 11. Two morphologically distinct cell types were present in all decidual cultures; flat dendritic cells, considered to represent decidual cells, and small round cells, but whether immunoregulatory factors are associated with both is uncertain. The results suggest that decidual tissue could fulfil a role in the local partial blockade of the afferent arm of the maternal immune response during pregnancy.

Partial characterization of immunosuppressive factors from short-term cultures of murine decidual tissue.

Previous in vitro experiments provided evidence for the immunosuppressive properties of supernatants from short-term cultures of decidual tissue obtained from the uterus of the pregnant mouse. The inhibitory activity of supernatants, previously detected in a mixed lymphocyte reaction (MLR), has now been demonstrated in an in vitro thymocyte proliferation assay. Fractionation of these supernatants on Sephadex G-15 and Sephacryl S-300 revealed that inhibition of the thymocyte proliferation assay was associated only with a low molecular weight fraction (less than 1,500), whilst MLR inhibitory activity was associated with three different molecular weight fractions (less than 1,500, 60,000 and 1,000,000). These results are discussed in relation to the different biological factors previously isolated in decidua or deciduoma of rats and humans.

[Inhibition of an immune hemolysis reaction in higher vertebrates by a serum fraction (fraction P) from the pregnant Salamandra salamandra L]. / Inhibition d'une réaction d'hémolyse immune chez les vertébrés supérieurs par une fraction sérique (fraction P) de femelle gestante chez Salamandra salamandra L.

Serum "fraction P" from Salamandra pregnant female (containing essentially an alpha 2-macroglobulin) possesses non specific cellular immunosuppressive properties [2]. This fraction has been tested in an immune hemolysis reaction: hemolytic Rabbit IgG + guinea-pig complement + Sheep erythrocytes. This fraction, after incubation with guinea-pig complement, inhibits immune lysis.

[Analogies between "fraction P" with immunosuppressive properties in pregnant females and a protein fraction induced by estradiol treatment in the amphibian Salamandra salamandra L]. / Analogies entre la "fraction P" à propriétés immunosuppressives chez les femelles gestantes, et une fraction protéique induite par un traitement à l'oestradiol, chez l'Amphibien Urodèle, Salamandra salamandra L.

A "fraction P" analogue possessing immunosuppressive properties during pregnancy in the Salamandra, can be induced by treating males and females with oestradiol. Both fractions possess the same physiochemical characteristics, and equally react against a Rabbit immune serum anti "fraction P" of pregnant Salamandra. But the proteic fraction induced by hormonal treatment does immunosuppressive properties.

[Separation and study of two serum factors involved in the inhibition of the maternal cytotoxic reaction against embryonic cells in pregnant salamandra (author's transl)]. / Séparation et étude de deux facteurs sériques responsables de l'inhibition de la réaction cytotoxique maternelle à l'égard des cellules embryonnaires, chez la salamandre gestante.

In vitro experiments in pregnant salamanders show a maternal specific cytotoxic reaction against larvae cells and an inhibition of this reaction by maternal serum. The last one is double: non-specific inhibition of maternal cellular reaction and specific protection of embryonic cells. Two fractions have been isolated from maternal serum, with biological properties in cell cultures: the first one is a protein (migrating as alpha2-macroglobulin does) which is implied in non-specific immunosuppression of maternal cells; the second fraction contains an IgM and specifically protects embryonic cells against maternal cytotoxicity.

[In vivo and in vitro study of the effect of pregnant female serum graft rejection in Salamandra salamandra Lin]. / Etude in vivo et in vitro de l'activité du sérum de femelle gestante sur la réaction de rejet de greffe chez Salamandra salamandra Lin.

Serum from pregnant female Salamandra salamandra inhibits the cytotoxic reaction from the mother towards its larvae. Such a serum accelerates the allograft rejection reaction. In vitro studies show that a serum from pregnant female inhibits the cytotoxic reaction of host spleen cells towards epithelial cells of the donor of the graft.

Antagonistic maternal immune reactions (rejection and facilitation) to the embryo in the urodele amphibian Salamandra salamandra lin.

In vitro assays have been employed to demonstrate that pregnant salamanders mount an immune reaction against their embryos. Maternal spleen cells kill up to 85% of dissociated embryonic epidermal cells during a 48 h incubation period. The degree of killing depends upon the ratio of maternal to embryonic cells and on the number of embryos borne by the mother. The cytotoxicity shows considerable specificity for the embryos of a given mother although a weak degree of killing can occur with embryos from other mothers, presumably due to some form of cross-reactivity. The effect is inhibited by the addition of maternal serum to the cultures. The degree of protection is also a function of the number of embryos borne by the mother. Pre-incubation experiments indicate that the maternal serum has a protective action on the embryonic cells which is largely specific for the female's own embryos (and suggested to be antibody in nature) and an inhibitory action on the maternal spleen cells which occurs also with spleen cells of other females (and suggested to be either an immune complex or a nonimmunological substances). An increase in beta protein peaks is seen following electrophoresis of sera from pregnant (and also allografted) salamanders. These findings indicate that the pregnant salamander mounts a double immune reaction against her embryos, an aggressive (rejection) reaction and a protective (facilitation) reaction.

[The maternal protection reaction against a mother's rejection of her own larva in Salamandra salamandra L]. / Etude de la réaction de protection maternelle contre une réaction de rejet de la mère à l'égard de ses larves chez salamandra salamandra L.

In vitro experiments have shown that maternal spleen cells from Salamandra salamandra are cytotoxic to cells from their embryos. This reaction can be inhibited by maternal serum. In this paper, we show that maternal serum protection acts through two effects: by inactivating spleen cells and by protecting embryonic cells. The more numerous the embryos are in a female, the stronger the protection is. The effect of the maternal serum does not appear to be individual specific.

[Demonstration and in vitro study of a complex reaction (rejection and facilitation) of the mother towards the embryo in the urodele amphibian Salamandra salamandra L]. / Mise en évidence et étude in vitro d'une réaction immunitaire complexe (rejet et facilitation) de la mére envers l'embryon chez l'Amphibien Urodèle Salamandra salamandra L

Spleen cells from pregnant (but not from non-pregnant) Salamandra salamandra are cytotoxic in vitro for dissociated epidermal cells from their own embryos. Maternal serum inhibits this cytotoxicity. Analogies and differences are stressed in Salamandra salamandra between allograft rejection mechanisms and immune reactions towards embryos. They may contribute to an explanation of the delayed expulsion of embryos.