Mouse Spleen Cell Cultures Research Articles

Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban

The Cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10 −7−10 3 M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10 −3 M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 μg/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 μg/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 ±0.14/cell and 13.40± 0.20/cell after treatment with Gardona (2 μg/ml) and Dursban (4 /gmg/ml), respectively, compared with 8.2 ± 0.19/cell and 7.6 ± 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.

Characterization of αβ + CD4 − CD8 − CTL lines isolated from mixed lymphocyte cultures of adult mouse spleen cells

Several CD4 − CD8 − and CD4 − CD8 + T cell lines and clones have been isolated from the same mixed lymphocyte cultures. They express the αβ T cell receptor, are CD3 +, V β8 − and heat stable antigen-, and exhibit highly specific cytolytic activity mapping to H2D b. However, monoclonal antibody against H2D b failed to block lysis by CD4 − CD8 − cytotoxic T lymphocytes (CTL). One possible explanation is that the “double negative” cytotoxic T lymphocytes may recognize H2D b in the context of other class I molecules ( Qa Tla ). CD4 and CD8 could be detected on some lines cloned in high concentrations of EL4 supernatant and the cell surface expression was influenced by growth conditions. The αβ + CD4 − CD8 − CTL described in this report are clearly digerent from the so-called “autoimmune double negatives” which suggests a function for them in normal immune responses.

Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium

A simple method is described for the preparation of large numbers of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4–5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 20 days. Using this method, 2 × 10 6 mast cells could be induced from 1 × 10 7 nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thy1,2, L3T4, Ly-2, Ig, B220, Asialo G M1, and WGA receptors were all negative but functional IgE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 ± 0.024 μg stored histamine in 1 × 10 6 of the cells. Histamine was released from the cells in an antigen-induced and IgE-mediated process. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.

Zinc status in proliferative response of T lymphocytes.

The in vitro proliferation of T lymphocytes are highly susceptible to Zn deprivation and Cd addition. In order to define Zn status in T cell proliferation, toxicological and biochemical situation of Zn in proliferative response of T cells was investigated by use of mouse spleen cell cultures stimulated by T cell-mitogen, Con A. The inhibitory effect of Cd on T cell proliferation was protected specifically by Zn. The protection by Zn was effective when Zn was added within 16 h after Con A stimulation. Zn addition affected neither Cd content in cells nor induction of Cd-thionein. These findings indicate that Zn-dependent processes expressed before the S phase of cell cycle are critical for T cell proliferation and are targets susceptible to Cd. Moreover, electrophoretical analysis showed two unknown Zn-binding proteins (49 and 100 KD) induced in spleen cells incubated with Con A and radioactive Zn. These Zn-binding proteins may be associated with the Zn-dependent processes critical for T cell proliferation.

Inhibition of Antigen‐ and Mitogen‐Induced Activation of Mouse Splenocytes by Uterine Secretions From Pregnant and Progesterone‐Treated Ewes

Pregnant and progesterone-treated, ovariectomized ewes accumulate secretory products in the uterus that are immunosuppressive in mitogen-stimulated and mixed lymphocyte sheep cell cultures. In this study, uterine secretions from pregnant (Preg-UTM) and progesterone-treated, ovariectomized (P-UTM) ewes were equally effective in suppressing 3H-Tdr incorporation in mouse spleen cells stimulated with PHA. P-UTM inhibited PHA-stimulated, purified T-cells and separated L3T4+ and Lyt2+ T-cell subpopulations more than Preg-UTM, however. Both fluids were slightly inhibitory to conA-stimulated mouse spleen cells, enriched T-cells, and Lyt2+ T-cells but neither inhibited L3T4+ T-cells. For LPS-stimulated cells, P-UTM caused more suppression than Preg-UTM of enriched B-cells; however, suppression was similar for the two fluids on unseparated splenic cells. In antigen-stimulated mouse spleen cell cultures, both fluids inhibited antibody-forming cell responses to sheep erythrocytes, a thymus-dependent antigen, but neither suppressed antibody-forming cell responses to TNP-Ficoll, a thymus-independent antigen. These data indicate that uterine secretions in the ewe produced under the influence of progesterone or pregnancy contain immunoregulatory molecule(s) which modulate the activity of both homologous sheep and unrelated mouse lymphocytes. These studies establish the mouse as a useful in vivo model for studying the biological actions of these molecules.

Synergistic effect on blastogenesis in murine spleen cells of lipopolysaccharide, lipid A, and acid-degraded polysaccharide fromFusobacterium nucleatum

Interchangeable combinations of Fusobacterium nucleatum Fev1 lipopolysaccharide (LPS) with its split products by acetic acid hydrolysis, i.e. lipid A (LA) and degraded polysaccharide (PS), amplified the blastogenic response in murine spleen cell cultures as measured by [3H]thymidine uptake. Athymic murine spleen cells precultured with LPS-Fev1 for 48 h (stage 1), washed twice and cultured together with fresh cells and either LA or PS for 72 h (stage 2) gave a synergistic response over that found in spleen cell cultures of thymic mice. Spleen cells pre-cultured with LA or PS and with fresh cells and LPS-Fev1 added to stage 2 cultures gave less significant amplification compared with precultures of LPS and either LA or PS together with fresh cells added to stage 2. Precultures with LA, PS or LPS-Fev1 and with pokeweed mitogen (PWM) and fresh cells added produced an additional increment of synergy which was most pronounced in spleen cell cultures of normal mice.

Synovial fluid from rheumatoid arthritis patients induces polyclonal antibody formation in vivo.

Our previous studies demonstrated the presence of a T-cell replacing factor in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) and that RA-SF can activate, selectively, the induction of IgG2b antibody secreting cells in lipopolysaccharide (LPS)-pretreated mouse spleen cell cultures. In the present study the effect of RA-SF was tested in vivo in mice. Injection of the polyclonal activator LPS induced the production of IgM and IgG3 secreting cells in normal mice. However, the addition of RA-SF led to a selective increase in the production of IgG2b with a peak response on day 5 and IgG1 plaque-forming cells (PFC) with a peak on day 7. Neither the IgG2b nor IgG1 responses were caused by specific immunity against heterologous proteins present in RA-SF, as injection of in vitro inactive RA-SF samples did not induce PFC. The effect on B cells of RA-SF was further evaluated by injection of RA-SF in combination with LPS to the Xid B-cell deficient CBA/N mice. RA-SF had identical effects in CBA/N as in normal mice. The biological implication of these findings is discussed. Our earlier results support the idea that B cells are endogenously activated in RA patients. We have speculated that this activation is caused by the B-cell differentiation factor which is present in SF. Therefore, we also tested whether RA-SF could influence antibody-forming cells in mice that spontaneously develop autoimmunity. We found that injection of RA-SF alone, in the absence of any other activating substance, induced a very marked increase of IgG producing cells in (NZW x NZB) F1 hybrid mice. From a relatively high background level the RA-SF could still induce an up to 100-fold increase in the numbers of PFC in spleens of such mice.

Distinct effects of aminopterin on stimulation of T and B lymphocytes with mitogen

Aminopterin, a folate antagonist, added to cultures of mouse spleen cells stimulated with T-cell mitogens markedly inhibited, in a dose-dependent manner, [3H]thymidine incorporation into the cells. Contrary to this inhibitory effect aminopterin added at the same concentrations to cell cultures stimulated with B-cell mitogens significantly increased [3H]thymidine incorporation. The inhibitory effect of aminopterin of T-cell activation was completely reversed by adding hypoxanhine to the cultures. Analysis of the mechanism of aminopterin action showed direct, mutually opposite, effects of this drug on T- and B-cell populations. The results thus not only demonstrate that polyclonally activated T and B lymphocytes differ in their sensitivity to the toxic action of aminopterin, but simultaneously support early findings suggesting differences in nucleic acid metabolic pathways between T- and B-cell populations.

Histamine-releasing activity of mouse spleen cell culture supernatants

Histamine releasing factor (HRF) generated in vitro by spleen cells of immunized mice activates homologous peritoneal mast cells (both normal and sensitized) for small but consistent, dose-dependent histamine and 5-hydroxytryptamine release. Both the time-course of this release and its susceptibility to the enhancing effect of D2O were similar to those observed in IgE-induced release; however, simultaneous challenge of sensitized mast cells with HRF and specific antigen or anti-IgE showed an additive effect, suggesting that mediator release by HRF and by the IgE system are independent of one another.

Inhibition of lymphokine-activated killer cell-mediated cytotoxicity by phorbol ester.

As previously reported, the culture of mouse spleen cells in the presence of high amounts of human rIL-2 for 4 days caused proliferation and generation of lymphokine-activated killer (LAK) cells, which could lyse a variety of tumor cells. However, an addition of PMA to the culture resulted in a striking inhibition of the generation of LAK cells. In contrast, IL-2-induced cell proliferation, IL-2R expression, and LFA-1 expression were enhanced by the addition of PMA. Kinetic studies revealed that the addition of PMA during the final 24 h, but not 4 h, of the culture was sufficient to inhibit the generation of LAK cells. The same inhibition of LAK activity was observed when 4-day cultured LAK cells were pretreated with PMA for over 12 h before cytotoxicity assay. Flow cytometry analysis showed that PMA pretreatment had no effect on the binding of LAK cells to target cells. PMA pretreatment of LAK cells caused total disappearance of protein kinase C (PKC) activity from LAK cells concomitant with the loss of LAK activity. However, PMA-pretreated LAK cells cultured for another 24 h in the absence of PMA revealed levels of PKC activity and cytotoxicity identical with untreated LAK cells. These results strongly suggest that PMA-induced down-regulation of LAK cell-mediated cytotoxicity is due to the inactivation of PKC-dependent transduction systems that are essential post LAK cell-target cell binding.

Regulation of lymphocyte blastogenesis by histamine produced in the system per se.

The culture of mouse spleen cells with concana valin A (Con A) or phytohemagglutinin (PHA) increased histidine decarboxylase (HDC) activity as a function of incubation time. The histamine level of the medium increased in parallel with the increase in HDC activity. Lymphocyte proliferation induced by Con A was enhanced by cimetidine, a histamine H2-antagonist, in the absence of exogenous histamine. Dimaprit, an H2-agonist, blocked lymphocyte blastogenesis at high doses (> 10 ”5 m). Diphenhydramine, an Hj -antagonist, abolished mitogenesis induced by Con A. At low doses (10-9~ 10-7 m), the H^agonist 2-pyridylethylamine augumented mitogenesis mediated by Con A. The addition of alphafluoromethylhistidine or histaminase (EC 1.4.3.6), at low doses, to the culture enhanced Con-A-dependent lymphocyte proliferation. At high doses they inhibited the reaction. Histamine itself augmented [3H]thymidine uptake by lymphocytes induced by Con A at the low doses of 10~13~10 11 M; at higher doses (> 10”5 m), it suppressed t...

Depletion of catecholamines with α-methyl- p-tyrosine suppresses splenic NK cell activity

The effects of depleting the tissue catecholamines with α-methyl- p-tyrosine (AMPT, an inhibitor of catecholamine synthesis) on the splenic NK cell cytotoxic activity were assessed both in vivo and in vitro. Treatment with i.p. doses of AMPT (3–500 mg/kg) produced a dose-related suppression in the splenic NK cytotoxic activity in many mouse strains. For an i.p. dose of 300 mg/kg of AMPT, the splenic NK cell activity began to fall 1 h after AMPT injection. The splenic NK cell activity reached its maximal level at 3 h, accompanied by a severe depletion of norepinephrine contents in the spleen. Both the NK activity and the splenic norepinephrine content returned to their control levels at 24 h. Direct addition of AMPT (up to 2500 μg/ml) to the cultured mouse spleen cells in vitro resulted in no NK suppression. However, when the serum obtained from the AMPT-treated mice was added to the cultured mouse spleen cells of the AMPT-untreated mice, the splenic NK cytotoxic activity was greatly suppressed. In addition, natural killing by spleen cells from AMPT-untreated mice was not reduced by the addition of spleen cells from AMPT-treated mice. No evidence of AMPT-induced cellular suppressors of natural killing could be detected. It was also found that both the splenic NK cell activity and the effector-target cell conjugation activity were suppressed by treatment with AMPT. These observations indicate that depleting the tissue catecholamines with AMPT results in the release of certain humoral factors which can suppress both the effector—target cell conjugation activity and the splenic NK cell activity in mice.

Synthesis and secretion of immunoglobulin G by lymphocytes from cultured mouse spleen cells is not affected by heat shock.

Short-term hyperthermic episodes (in vivo and in vitro) alter gene expression in mammalian lymphocytes, resulting in the enhanced synthesis of a select group of polypeptides--the heat-shock proteins--and the depressed synthesis of many normally synthesized polypeptides. Such alterations could have profound implications to an individual if the appropriate functioning of lymphocytes within the immune response was compromised by a depression in immunoglobulin synthesis during naturally occurring periods of hyperthermia, such as fever. In the present study we asked if heat-shock affects the facultative synthesis and secretion of immunoglobulin G by cultured mouse lymphocytes. We found that the quantity of immunoglobulin G synthesized and secreted by these cells is not affected by heat-shock treatments sufficient to induce the synthesis of heat-shock proteins.

Circulating autoantibodies to mammalian tissue kallikreins.

Autoantibodies to tissue kallikrein (EC 3.4.21.35) were discovered in normal human, rat, mouse, and guinea pig sera. Three independent methods--binding of iodolabeled antigen, enzyme-linked immunosorbent assay (ELISA), and immunoblotting--were used to demonstrate these kallikrein autoantibodies. Autoantibodies from rat and human sera were purified, using rat and human tissue kallikrein-affinity chromatography, respectively. Purified rat kallikrein autoantibody bound 50% of 125I-labeled rat urinary kallikrein upon incubation of antibody at 2.5 X 10(-10) M. The subtypes of rat and human kallikrein autoantibodies were determined by an ELISA, using antisera to immunoglobulin subclasses. In both species, autoantibody was predominantly IgG (approximately 80%) and some IgM (approximately 20%). Purified autoantibodies from rat and human sera were separated on sodium deodecyl sulfate-polyacrylamide gels, and their subunits were identified by Western blot analyses, using anti-rat and anti-human IgG antibodies, respectively. When primary cultures of mouse spleen cells were incubated for 1 to 5 days with lipopolysaccharide (1 to 5 micrograms/ml), the anti-kallikrein antibodies in the media increased up to seven-fold. We have demonstrated circulating autoantibodies that recognize and bind both autologous and heterologous kallikrein; however, their significance to the function of the tissue kallikrein-kinin system in normal and disease states remains to be explored.

7] Purification of staphylococcal enterotoxins

Publisher Summary This chapter describes the purification of Staphylococcal Enterotoxins. Six distinct enterotoxins (A, B, C l , C 2 , D, and E) are produced by Staphylococcus aureus and account for the majority of food poisoning in the United States. The purification of the enterotoxins is based on classical ion-exchange and gel filtration techniques. However, newer methodologies, such as fast-protein liquid chromatography (FPLC), are applied to the purification of these toxins. Although these toxins are termed enterotoxins, it is a misnomer, because none produces the classic ileal loop response. The enterotoxins are also shown to be powerful mitogens and are able to induce DNA synthesis and interferon synthesis in lymphocytes. Enterotoxin A is among the most effective T-cell mitogens that stimulate interferon production in mouse spleen cell cultures. However, it seems clear that these mitogenic activities are not associated with either major antigenic regions of the molecules or with their site of action for emesis and diarrhea.

Phospholipase A2 treatment of lymphocytes provides helper signal for interferon-gamma induction. Evidence for second messenger role of endogenous arachidonic acid.

To examine the role of endogenous arachidonic acid (AA) as the possible second messenger signal in interferon-gamma (IFN-gamma) production, helper cell-depleted mouse spleen cell cultures were treated with the enzyme phospholipase A2 (PLA2). Treatment with PLA2 from several different animal sources at concentrations between 10 and 300 U/ml resulted in complete, dose-dependent restoration of competence for IFN-gamma production. By comparison, phospholipase C (PLC) from several different species failed to restore competence at concentrations between 0.3 and 30 U/ml; the inability of PLC to provide the helper signal for induction of IFN-gamma was not due to cytotoxicity. Since PLA2 provides competence for IFN-gamma production by sn-2 hydrolysis, it was of interest to identify eicosanoids and other lipids released from [3H]-AA labeled cells by PLA2 and PLC. Treatment of spleen cells with PLA2, but not PLC, resulted in the appreciable release of AA only. Sufficient AA was released from spleen cells for restoration of competence for production of IFN-gamma. All glycerol-derived cell membrane phospholipids examined (phosphatidylethanolamine, -inositol, -choline, and -serine) incorporated labeled AA which was releasable by treatment with PLA2. The data support and extend previous studies which suggested that AA plays a pivotal role in mediation of the interleukin 2 helper signal for IFN-gamma production.

Inhibition by interleukin‐1 of the action of erythropoietin on erythroid precursors and its possible role in the pathogenesis of hypoplastic anaemias

Summary. Highly purified and cloned preparations of interleukin‐1 (IL‐1) were found to antagonize the capacity of erythropoietin (Epo) to stimulate the proliferation of mouse spleen and bone marrow erythroid precursor cells (EPC) in culture. Cloned murine IL‐1 and purified and cloned human IL‐1α and IL‐1β were approximately equipotent in this assay. IL‐1 inhibited the proliferation response of EPC even when added as long as 17h after Epo, suggesting that IL‐1 does not affect binding of Epo to receptors or biochemical events following shortly thereafter. Indomethacin did not influence the inhibitory effect of IL‐1 on Epo‐induced proliferation, and PGE2 had no demonstrable effect on the process. Tumornecrosis factor‐α and interferons β1, and γ did not affect Epoinduced proliferation. It is suggested that IL‐1 mediated antagonism of the effects of Epo on erythroid precursors is a factor in the pathogenesis of many types of hypoplastic anaemia, including those associated with infections, rheumatoid arthritis and systemic lupus erythematosus, giant‐cell arteritis, graft‐versus‐host disease and disorders associated with lymphocyte‐mediated suppression of erythropoiesis.

Inhibition by interleukin-1 of the action of erythropoietin on erythroid precursors and its possible role in the pathogenesis of hypoplastic anaemias.

Highly purified and cloned preparations of interleukin-1 (IL-1) were found to antagonize the capacity of erythropoietin (Epo) to stimulate the proliferation of mouse spleen and bone marrow erythroid precursor cells (EPC) in culture. Cloned murine IL-1 and purified and cloned human IL-1 alpha and IL-1 beta were approximately equipotent in this assay. IL-1 inhibited the proliferation response of EPC even when added as long as 17 h after Epo, suggesting that IL-1 does not affect binding of Epo to receptors or biochemical events following shortly thereafter. Indomethacin did not influence the inhibitory effect of IL-1 on Epo-induced proliferation, and PGE2 had no demonstrable effect on the process. Tumor-necrosis factor-alpha and interferons beta 1, and gamma did not affect Epo-induced proliferation. It is suggested that IL-1 mediated antagonism of the effects of Epo on erythroid precursors is a factor in the pathogenesis of many types of hypoplastic anaemia, including those associated with infections, rheumatoid arthritis and systemic lupus erythematosus, giant-cell arteritis, graft-versus-host disease and disorders associated with lymphocyte-mediated suppression of erythropoiesis.

Dependence on intact cells for the in vitro activation of dimethylnitrosamine to an immunosuppressive form

The in vitro activation of dimethylnitrosamine (DMN) to an immunosuppressive form was studied utilizing liver-enzyme fractions and intact hepatocytes. The N-demethylation of DMN by mouse S9 and microsome preparations was confirmed by determination of formaldehyde generation. S9 fractions from both phenobarbital(PB)- and isopropanol(iso)-pretreated mice displayed significantly greater demethylase activity than uninduced S9 fractions. However, when incubated with spleen cells, neither S9 preparation was capable of activating DMN to a form capable of suppressing antibody responses by recovered spleen cells. In contrast, the positive control, cyclophosphamide, was activated to a markedly immunosuppressive form. S9 fractions failed to activate DMN to an immunosuppressive form regardless of S9 concentration, time of preincubation, or rocking speed. Liver microsomes from PB-pretreated mice displayed significantly greater N-demethylase activity than S9 fractions yet were unable to activate DMN to an immunosuppressive form. In contrast, the addition of DMN to mixed cultures of mouse hepatocytes and mouse spleen cells resulted in activation of DMN and marked suppression of antibody responses. The separation of spleen cells from the hepatocyte monolayer by an agar layer less than 1 mm thick resulted in complete reversal of the immunosuppressive effect of DMN. Unlike the metabolism of DMN to a mutagenic form, the in vitro activation of DMN to an immunosuppressive form was therefore dependent on intact cells. Furthermore, the activation by intact hepatocytes was shown to be dependent on cell-cell contact or close proximity of activating and target cells.

Monoclonal antibodies to human parathyroid hormone (1-34) and their use in the immunocytochemical detection of parathyroid tumours.

Monoclonal antibodies against the biologically active N-terminal fragment of human parathyroid hormone, hPTH (1-34), were produced. The procedure included the use of novel secondary immunization in vitro of mouse spleen cell cultures. Dissociated spleen cells from primary immunized Balb/c mice, were cultured for five days in the presence of thymocyte conditioned media (TCM) and synthetic hPTH (1-34). Contrary to previous findings by other workers, in our hands Balb/c mice responded well. Following immunization the spleen cells were fused with NSl myeloma cells and cultured for eleven days before screening for antibody. Using an enzyme linked immunosorbent assay (ELISA) a number of positive clones were detected. Positive cells were cloned by limiting dilution and fifteen specific monoclonal hybridomas were produced. The immunoglobulin class of the different monoclonal antibodies was found to be IgGl. The immunocytochemical reaction was tested with chief cell carcinoma tissue and found to be clearly positive.