Activation of Bad trafficking is involved in the BCR-mediated apoptosis of immature B cells.

The activity of Bad, a pro-apoptotic protein, is regulated by reversible phosphorylation. Moreover, sequestration of Bad within subcellular compartments may be a new mechanism of apoptosis regulation. In this study, we report that Bad interacts with 14-3-3 protein in WEHI-231 immature B cells. This association is disrupted following BCR stimulation in correlation with Bad translocation to mitochondria and apoptosis. Confocal microscopy was further used to examine the co-localization of Bad with lipid rafts in WEHI-231 and murineex vivoB cells. Bad was found colocalized to lipid rafts in freshly isolated mature B lymphocytes, in contrast to immature cells. Finally, co-immunoprecipitation experiments performed on WEHI-231 B cells revealed that PP1alpha interacts with Bcl-2 and Bad, and dissociation of the complex was found correlated with appearance of apoptosis. Bcl-2 seemed to be required to assemble the complex which may regulate Bad phosphorylation status and consequently cell survival. Collectively, present data outline the role of Bad trafficking in the BCR-mediated apoptosis and suggest that differences in intracellular Bad trafficking may be involved in the differential outcome of BCR signaling.

Changes in bad phosphorylation are correlated with BCR-induced apoptosis of WEHI-231 immature B cells.

Signaling through the B cell antigen receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not completely known. Using the murine B lymphoma cell line WEHI-231 as a model system, we investigated the role of Bad phosphorylation, a pro-apoptotic member of the Bcl-2 family, in anti-IgM mediated apoptosis. For apoptotic analysis we focused in particular on the mitochondrial potential (deltapsi(m)) collapse which has been reported as a rate-limiting step in the BCR-induced cell death of immature B lymphocytes. Bad phosphorylation at serine 112, 136 and 155 was found in WEHI-231 cell control cultures and its hypophosphorylation on the three sites correlated with the appearance of apoptosis when cross-linking surface IgM. Furthermore, treatment of cells with specific PK inhibitors known to be involved in serine phosphorylation of Bad (LY294002 for PI3K and H-89 for PKA) mimiced or enhanced BCR-induced cell death. These results strongly suggest that regulation of Bad phosphorylation plays an active role in mediating anti-IgM-induced apoptosis of immature B cells.

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes.

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.

Optimization of cell permeabilization for multiparametric flow cytometric analysis with lectin staining.

BACKGROUND: This study was undertaken in mice to develop a reproducible procedure of cell permeabilization, allowing intracellular protein staining by immunofluorescence (i.e., Bcl-2) without losing surface labeling especially for lectins (i.e., B220 and peanut agglutinin [PNA]). This article reports results obtained with different permeabilization protocols. METHODS: Lymphoid cells were extracted and prepared from Peyer's patches and spleen. After surface labeling using anti-B220-Cy-chrome and PNA-biotin/streptavidin-phycoerythrin, we comparatively tested three permeabilization protocols: saponin 0.3%, methanol 70%, and the commercial kit Dako Intrastain. Final Bcl-2 staining was performed and cells were analyzed by flow cytometry. RESULTS: With 0.3% saponin as the permeabilization reagent, a significant loss of lectin labeling was observed when comparing mono PNA and triple (i.e. , B220-PNA-Bcl-2) staining (74.8% and 22.5% positive cells, respectively). Quality of PNA staining was conserved with Intrastain when comparing multiparametric versus monoparametric stainings (82. 4% of positive cells versus 78.3%, respectively). Intrastain preserved scatter characteristics (69.9% of total cells in the lymphocyte gate with Intrastain versus 13.7% with saponin 0.3% and 20.9% with methanol 70%). This protocol has been used for a preliminary multiparametric analysis in order to quantify Bcl-2 expression in PNA/B220-positive cells. CONCLUSION: This protocol may be useful to assess simultaneously lectin cell surface labeling and intracellular target staining.

Age-related alterations of somatic hypermutation and CDR3 lengths in human Vkappa4-expressing B lymphocytes.

The lower avidity and/or affinity of antibodies generated by an aged immune system could be attributed to two major changes in the antibody repertoire: a shift in germline gene usage and a decrease in the rate of immunoglobulin hypermutation. In an attempt to identify the mechanisms involved in the observed humoral immune deficiency in the elderly, we studied whether differences in the somatic diversity of a particular Vkappa region occurred with ageing. By using the polymerase chain reaction and sequencing, we analysed and compared Vkappa4-Jkappa rearrangements isolated from young (mean age 21 years) and aged (mean age 83 years) healthy adults. Mutations in the Vkappa4 gene compared with the germline sequence were determined as well as the length and structure of the CDR3 sequence. We analysed in detail various mechanisms contributing to CDR3 and Vkappa variability in rearrangements involving the Vkappa4 gene. Our data revealed that, despite strong individual variations, significantly lower levels of somatic mutation were found in the aged group, both for complementarity-determining regions (CDRs) and framework regions (FRs) encoding Vkappa4 sequences. This decrease mostly affected mutations responsible for replacements and thus resulted in a lowered somatic diversification of the encoded Vkappa4 proteins in aged individuals. Moreover, comparison of the CDR3 regions of the Vkappa4-Ckappa cDNA revealed changes in light-chain junctional diversity that correlated with age. Altogether these data suggest an impaired light-chain somatic diversity in connection with human senescence.

Modification of HLA expression on peripheral lymphocytes and monocytes during aging.

Immunosenescence involves modifications of humoral and cellular immunity. Here we report the analysis of human leukocyte antigen (HLA) expression on T lymphocytes, B lymphocytes and monocytes of 58 healthy subjects aged 23-95 years old. Using a double staining immunofluorescence and flow cytometry analysis, we have determined the percentages of cells expressing HLA class-I and HLA-DR antigens. The number of antigenic sites expressed per cell were evaluated for HLA-ABCw, HLA-A, HLA-B, HLA-DR locus with a flow cytometry quantification technique. With advancing age, we observed: (i) a significant decrease of the percentage of T cells and B cells expressing HLA-A products; (ii) a decrease of the number of HLA class-I antigenic sites expressed per cell on the three populations tested, predominantly on B cells and in a locus-dependent fashion; (iii) a decrease of the number of HLA-DR molecules expressed per T cell, although the percentage of T cells expressing DR products was increased; (iv) a significant diminution of the percentage of B cells expressing HLA-DR molecules, without changes of the number of HLA-DR antigenic sites per cells. These changes in HLA expression with increasing age could contribute to the decreased level of immunologic responsiveness observed with ageing and contribute to the modification of antigen recognition.

Differential effects of temperature on specific and nonspecific immune defences in fish.

The susceptibility of fish to disease is partly dependent on their environment, in particular on water temperature. It is generally accepted that lower temperatures adversely affect specific immune responses mediated by T helper cells. The probable mechanisms involved in such suppression in teleost fish are reviewed. Furthermore, the effects of temperature on nonspecific defences, such as phagocytosis and cytotoxicity, are described and total immune competence in teleosts at low environmental temperatures is discussed.

Effects and mechanisms of environmental temperature on carp (Cyprinus carpio) anti-DNP antibody response and non-specific cytotoxic cell activity: a kinetic study.

The effect of environmental temperatures on immune competence was investigated in carp which were subjected to changes in water temperature. The activity of non-specific cytotoxic cells (NCC) against P815 target cells, and the anti-DNP antibody response were evaluated until day 56 after transfer. Low environmental temperature (12 +/- 0.5 degrees C) enhanced NCC activity and decreased antibody production. In contrast a high environmental temperature (28 +/- 0.5 degrees C) was without effect on these parameters when compared to the standard temperature (20 +/- 0.5 degrees C). The results showed a maximum effect of low environmental temperature on day 28 and an adaptation in these immune responses 56 days following transfer. Collectively, the results indicated that non-specific immunity tends to offset specific immune suppression at low environmental temperatures. To determine the mechanism(s) by which environmental temperature affects cellular immune function, membrane fluidity measurements and sialic acid titration, as well as stress assessment by plasma cortisol measurement, were determined on day 28. Taken together, the results revealed a direct effect of temperature on cellular immune function which is modulated by membrane fluidity and sugar concentration and not by stress induction.

Serotonin modulation of lymphocyte proliferation via 5-HT1A receptors in rainbow trout (Oncorhynchus mykiss).

In this study, the effects of serotonin (5-HT) on in vitro lymphoproliferation in rainbow trout (Oncorhynchus mykiss) are investigated. Serotonin exerted immunosuppressive effects on lipopolysaccharide (LPS) and phytohemagglutinin (PHA)-stimulated proliferation of fish peripheral blood lymphocytes (PBL). 8-OH-DPAT (an agonist of 5-HT1A receptors) mimicked the inhibitory effects of serotonin on lymphocyte proliferation, whereas addition of spiperone (an antagonist of 5-HT1A and 5-HT1B receptors) reversed these inhibitory effects, indicating that 5-HT1A receptors may be implicated in serotonin-induced immunosuppression. Furthermore, in this study the serotonergic receptors present on fish peripheral lymphocytes were characterized. A Scatchard plot of serotonin binding to fish lymphocytes followed the 'bell' shape curve with a Bmax of 0.63 microM and a Kd of 1.54 x 10(-8) M/10(6) cells. These results demonstrate the presence of positive-type co-operation among receptor populations. In a displacement study, serotonin inhibited the binding of 3H-5HT to the receptor sites both in resting and LPS/PHA-stimulated trout lymphocytes. Interestingly, the agonists (8-OH-DPAT and buspirone) and antagonist (NAN-190) of the 5-HT1A receptor subtype failed to displace 3H-5HT binding to receptor sites in resting cells, whereas these agents inhibited 3H-5HT binding in LPS- and PHA-stimulated lymphocytes significantly, suggesting that after mitogenic stimulation, 5-HT1A receptors are expressed on lymphocytes. CGS-12066B (an agonist of 5-HT1B receptors) failed to influence significantly 3H-5HT binding to receptor sites both in resting and mitogen-stimulated lymphocytes, indicating that the 5-HT receptor subpopulation is not expressed either on resting or on LPS- or PHA-stimulated lymphocytes. Taken together, these results suggest that trout peripheral blood lymphocytes express functional serotonergic receptors, and 5-HT1A receptors, which are not expressed by resting lymphocytes, are expressed after mitogenic stimulation and implicated in the inhibition of mitogenic (LPS and PHA) responses.

[Antibody response, cortisolemia and prolactinemia in rainbow trouts]. / Réponse anticorps, cortisolémie et prolactinémie chez la truite arc-en-ciel.

We have studied the humoral immune response (production of anti-Yersinia ruckeri antibodies), and measured the levels of plasmatic hormones (cortisol and prolactin) in rainbow trout (Oncorhynchus mykiss) subjected to hyperosmotic stress (NaCl, 22 / 1000). Under acute stressful conditions (saline stress during 7 days), high blood cortisol and prolactin (PRL) levels were correlated with a weak anti-Yersinia ruckeri antibody response, as evidenced by late and low antibody titres as compared to normal fish. Interestingly, the group of fish subjected to chronic stress (till 30 days) exhibited no significant differences in blood cortisol and prolactin levels despite low antibody titres as compared to control group. Hence, it is possible that in acute stress, cortisol and prolactin levels might exert immunosuppressive effects on antibody production, whereas in chronic stress other neuroendocrine hormones might result in curtailed humoral immunity.

Structure-activity relationship of phenolic compounds (phenol, pyrocatechol and hydroquinone) on natural lymphocytotoxicity of carp (Cyprinus carpio).

We have tested the effects of phenol and two diphenols (pyrocatechol and hydroquinone) on a non-specific immune response, i.e. the natural cytotoxic activity, of the carp. After a 12-day exposure of fish, at a concentration of 0.1 mg/l, hydroquinone appeared to exert the most immunotoxic effect in vivo. In vitro, after a preincubation of 1 h, phenol (10(-2) M), pyrocatechol (4.25 x 10(-4) M) and hydroquinone (4.25 x 10(-5) M) decreased the natural cytotoxic activity of lymphoid cell suspension. In vivo and in vitro experiments show that hydroquinone is the most toxic compound, whereas diphenols are more toxic than phenol. These results demonstrate that the study of immune systems can reveal the presence of toxic substances with varying degrees of toxicity. Also, the position of a second hydroxic group on the benzonic nucleus seems to influence the compound toxicity.

Effects of temperature on carp leukocyte mitogen-induced proliferation and nonspecific cytotoxic activity.

The effect of environmental temperature on immune competence was investigated in carp subjected to abrupt changes in water temperature. The activity of nonspecific cytotoxic cells (NCC) against P815 target cells and the lymphoproliferation induced by PHA and ConA were evaluated. Low in vivo temperature (12 +/- 0.5 degrees C) enhances NCC activity and decreases the mitogen effect of PHA, respectively; by contrast high in vivo temperature (28 +/- 0.5 degrees C) is without effect on these parameters as compared to the standard temperature (20 +/- 0.5 degrees C). The stress induced by environmental temperature variation was evaluated by plasmatic cortisol measurement. Results indicate a significant increase (p < 0.05) of cortisol levels 2 hours after transfer at low but not at high temperature as compared to the standard thermic control (60.96 +/- 17.08 vs. 16.74 +/- 4.32 ng/mL, respectively). Because only approximately 20 minutes are required before carp body and environmental temperatures are identical, the same experiments were reproduced in vitro. Results show trends similar to those found in vivo. Taken together, these data reveal a direct effect of temperature on immune cellular functions and indicate that nonspecific immunity tends to offset specific immune suppression at low environmental temperature.

Nutritional influences on in vitro splenic lymphocyte proliferation in Psammomys obesus (Rodentia Gerbillidae).

The standard laboratory diet administered to sand rat (Psammomys obesus) induces the following physiological and immunological changes: hyperglycemia and hypercholesterolemia involving mainly the free fraction of cholesterol, with an elevation of high-density-lipoprotein levels and a decrease in B and T splenic lymphocyte proliferation in the presence of different mitogens PHA-P, Con A and LPS. These results demonstrate the important modification that could be induced in sand rat by the standard laboratory diet as compared with natural diet, and thus the sand rat (P. obesus) appears to be an interesting model for studies on experimental diabetes mellitus.

In vitro effects of substance P and somatostatin on lymphoproliferation in rainbow trout (Salmo gairdneri).

Lymphocytes from peripheral blood of rainbow trout are put in the presence of increasing concentrations of substance P (SP) and somatostatin (SOM). We have shown that SP stimulates and SOM inhibits lymphoproliferation and that the effects are dose dependent. These results suggest that SP and SOM receptors may exist on fish peripheral blood lymphocytes. When cells are stimulated by PHA or LPS, the presence of SP enhances the response to PHA whereas it only modifies the response to LPS to a slight extent. The presence of SOM inhibits PHA- or LPS-induced stimulation. The inhibition of the proliferation is higher in the case of LPS-stimulated cells. These results suggest that there is an unequal distribution of neuropeptide receptors among the various lymphocyte subpopulations.

Testosterone inhibits the immunostimulant effect of thymosin fraction 5 on secondary immune response in mice.

The purpose of the present investigation was to examine the in vivo influence of testosterone on the immune properties of a thymic factor (thymosin fraction 5, TF5) a partially purified thymic preparation in male Swiss IOPS/OF1 mice (5-10 weeks old). Testosterone administration (100 micrograms/ml) significantly inhibited the enhanced anti-sheep red blood cell antibody response induced by TF5 (100 micrograms/ml); this inhibition was only observed on the secondary antibody response and not on the primary. These results suggest that gonadal steroids can affect the immune response by modulating the activity of thymic factors.

Modulatory effect of metal ions on the immune response of fish: in vivo and in vitro influence of MnCl2 on NK activity of carp pronephros cells.

The in vivo and in vitro influence of MnCl2 on carp pronephros cells was investigated. Increased cytotoxicity against both YAC-1 and P 815 target cells was observed following an intraperitoneal injection of 40, 80, or 120 micrograms MnCl2/g body wt administrated 24 hr prior to the in vitro 51Cr release assay. Similarly, in vitro treatment of carp pronephros cells, at a final concentration of 60 micrograms/culture, resulted in an increase of NK cell activity in both YAC-1 and P 815 target cell lines. However, a significant decrease in this activity was shown with lower doses of MnCl2 (40 and 20 micrograms/culture).