Influence of Arecoline on Immune System: II. Suppression of Thymus-Dependent Immune Responses and Parameter of Non-specific Resistance After Short-Term Exposure

Abstract

Arecoline, a major alkaloid of arecanut was screened to explore its modulatory influence on cell-mediated immune response in a murine model system. the in viva and in vitro effects were evaluated at subtoxic concentrations of arecoline. Delayed type hypersensitivity (DTH) reactions to sheep red blood cells (SRBC) were evaluated in male mice. When treated subcutaneously with 20 mg/kg bw (1/5 of LD50) dose of arecoline for 1, 2 or 3 weeks, the DTH reactions were significantly suppressed. At arecoline concentration of 10 mg/kg bw, there was a moderate reduction in DTH response, while no appreciable change was observed at a dosage of 5 mg/kg bw. the effects were not dependent on the duration of treatment. In contrast, treating with arecoline continuously for 4 days following SRBC immunization showed significant suppression in DTH reactions at both 10 and 20 mg/kg bw doses. When treated after 12 h following immunization with 20 mg/kg bw arecoline, significant reduction in DTH reactions were seen. While moderate reduction in response was observed with arecoline dosage of 10 mg/kg bw, there was no alteration in response at the dose level of 5mg/kg bw. Recovery experiments in mice revealed that arecoline mediated effects are of a reversible nature. Arecoline treatment did not appreciably alter the host resistance to endotoxin shock. In vitro experiments revealed both dose-dependent and time-dependent cytotoxic effects of arecoline when spleen cells were incubated with varying concentrations of arecoline. Concomitant exposure of arecoline at concentrations of 10–6–10–4 m with con A, markedly suppressed both 3H-thymidine incorporation and interleukin-2 production of splenic cells. In contrast, concomitant exposure of arecoline with IL-2 did not alter 3H-thymidine incorporation in the IL-2 dependent cytolytic T-lymphocyte line (CTLL), except at the concentration of 10–4 m arecoline. From these studies it is concluded that the dose-dependent suppressive effects of arecoline on DTH response to SRBC and on certain in vitro lymphocyte functions are more clear than the host resistance to endotoxin shock.     

Influence of Arecoline on Immune System: III. Suppression of B Cell-Mediated Immune Response in Mice After Short-Term Exposure

Abstract

Arecoline, a major alkaloid of arecanut was examined to explore its modulatory influence on B cell-mediated immune response in a murine model system. The in vivo and in vitro effects were evaluated at sub-toxic concentrations of arecoline. The number of primary antibody forming cells (AFC) and hemagglutinating and hemolysis antibody titers to Sheep Red Blood Cells (SRBC) were evaluated in male mice. Arecoline exposure for a week invoked dose-dependent effect on primary antibody forming cells to SRBC with a maximum reduction at the dosage of 20 mg/kg bw, a moderate reduction at 10 mg/kg bw and no effect at 5 mg/kg bw dose level. HA and HL antibody titers to SRBC were suppressed markedly at arecoline dosage of 20 mg/kg bw and moderately at a dose of 10 mg/kg bw, given daily for 1, 2 or 3 weeks. The inhibitory effect of arecoline was not dependent on the duration of treatment. Like the primary antibody response, the secondary HA and HL antibody titers were also decreased after arecoline exposure. The administration of arecoline dosages 10 and 20 mg/kg bw daily for 4 days following SRBC immunization also, exerted dose-dependent suppression of primary antibody response. Similarly, when treated after 12 h following immunization, significant reduction in response was observed with arecoline dosage of 20 mg/kg bw. While moderate suppression of antibody response was noticed at the dose level of 10 mg/kg bw, there was no alteration in response at a dosage of 5 mg/kg bw. In contrast, arecoline dosages 5, 10 or 20 mg/kg bw given after 1, 2 or 4 days following immunization did not alter the HA and HL antibody titers to SRBC. Recovery experiments in mice revealed that arecoline-mediated suppression of antibody response is of a reversible nature. Concomitant exposure of arecoline at the concentrations of 10^?5 – 10^?4 M with PWM suppressed ³H-thymidine incorporation of splenic cells in vitro. Taken together, the findings reported in this paper suggest that the intensity of arecoline-mediated suppression of antibody response to SRBC and PWM-induced splenic cell proliferation is dependent upon the dosage and the mode of treatment.     

Influence of Arecoline on Immune System: I. Short Term Effects on General Parameters and on the Adrenal and Lymphoid Organs

Arecoline, a suspected carcinogenic/cocarcinogenic alkaloid was screened to explore in detail its immunomodulatory influence in murine model system. The oral LD₅₀ value for male mice was 371 mg/kg bw whereas it was 309 mg/kg bw for female mice. The subcutaneous LD₅₀ value for both sexes was 97 mg/kg bw. Only a marginal difference was observed in intraperitoneal LD₅₀ values between male (120 mg/kg bw) and female (109 mg/kg bw) mice. Arecoline was administered subcutaneously to male mice at subtoxic dose levels (5, 10, and 20 mg/kg bw) for 1, 2 and 3 weeks on a daily basis. In groups where significant decreases in body weight were present (at 20 mg/kg bw for both sexes), reductions in thymus weight were also noted. Spleen, mesenteric lymph nodes (MLN), liver, and kidney showed moderate reductions in their weights. Histopathological effects at 20 mg/kg bw included lymphocyte depletion of the thymic cortex, and the B and T lymphocyte areas in spleen and MLN. In concordance with the zona fasciculate hypertrophy of adrenals, corticosterone concentration in serum increased depending on the dose with a significant elevation at 20 mg/kg bw. While total protein, albumin, glucose, acid phosphatase and hemoglobin concentrations were not altered, increases in SG0T and SGPT levels were observed at the high dose. The white and red blood cell counts decreased in a dose-dependent manner. Marked reduction in cell number of thymus, and moderate effect on cellularity of spleen and MLN, were observed at 20 mg/kg bw. In vitro exposure of rat thymocytes to arecoline resulted in a biphasic oxygen consumption response with progressive increase in oxygen consumption, reaching a maximum value at 10^-5 M and decreasing sharply at 10^-3 M, Exogenously added substrates such as glucose, pyruvic acid and lactic acid retarded the fall in the oxygen consumption induced at 10^-3 M arecoline. These observations demonstrate the effects of arecoline on lymphoid organs, which may be due to its direct action or through the elevation of corticosterone.     

Influence of Arecoline on Immune System: I. Short Term Effects on General Parameters and on the Adrenal and Lymphoid Organs

Abstract

Arecoline, a suspected carcinogenic/cocarcinogenic alkaloid was screened to explore in detail its immunomodulatory influence in murine model system. The oral LD₅₀ value for male mice was 371 mg/kg bw whereas it was 309 mg/kg bw for female mice. The subcutaneous LD₅₀ value for both sexes was 97 mg/kg bw. Only a marginal difference was observed in intraperitoneal LD₅₀ values between male (120 mg/kg bw) and female (109 mg/kg bw) mice. Arecoline was administered subcutaneously to male mice at subtoxic dose levels (5, 10, and 20 mg/kg bw) for 1, 2 and 3 weeks on a daily basis. In groups where significant decreases in body weight were present (at 20 mg/kg bw for both sexes), reductions in thymus weight were also noted. Spleen, mesenteric lymph nodes (MLN), liver, and kidney showed moderate reductions in their weights. Histopathological effects at 20 mg/kg bw included lymphocyte depletion of the thymic cortex, and the B and T lymphocyte areas in spleen and MLN. In concordance with the zona fasciculate hypertrophy of adrenals, corticosterone concentration in serum increased depending on the dose with a significant elevation at 20 mg/kg bw. While total protein, albumin, glucose, acid phosphatase and hemoglobin concentrations were not altered, increases in SG0T and SGPT levels were observed at the high dose. The white and red blood cell counts decreased in a dose-dependent manner. Marked reduction in cell number of thymus, and moderate effect on cellularity of spleen and MLN, were observed at 20 mg/kg bw. In vitro exposure of rat thymocytes to arecoline resulted in a biphasic oxygen consumption response with progressive increase in oxygen consumption, reaching a maximum value at 10^?5 M and decreasing sharply at 10^?3 M, Exogenously added substrates such as glucose, pyruvic acid and lactic acid retarded the fall in the oxygen consumption induced at 10^?3 M arecoline. These observations demonstrate the effects of arecoline on lymphoid organs, which may be due to its direct action or through the elevation of corticosterone.     

Influence of arecoline on immune system: I. Short term effects on general parameters and on the adrenal and lymphoid organs

Arecoline, a suspected carcinogenic/cocarcinogenic alkaloid was screened to explore in detail its immunomodulatory influence in murine model system. The oral LD50 value for male mice was 371 mg/kg bw whereas it was 309 mg/kg bw for female mice. The subcutaneous LD50 value for both sexes was 97 mg/kg bw. Only a marginal difference was observed in intraperitoneal LD50 values between male (120 mg/kg bw) and female (109 mg/kg bw) mice. Arecoline was administered subcutaneously to male mice at subtoxic dose levels (5, 10, and 20 mg/kg bw) for 1, 2 and 3 weeks on a daily basis. In groups where significant decreases in body weight were present (at 20 mg/kg bw for both sexes), reductions in thymus weight were also noted. Spleen, mesenteric lymph nodes (MLN), liver, and kidney showed moderate reductions in their weights. Histopathological effects at 20 mg/kg bw included lymphocyte depletion of the thymic cortex, and the B and T lymphocyte areas in spleen and MLN. In concordance with the zona fasciculata hypertrophy of adrenals, corticosterone concentration in serum increased depending on the dose with a significant elevation at 20 mg/kg bw. While total protein, albumin, glucose, acid phosphatase and hemoglobin concentrations were not altered, increases in SGOT and SGPT levels were observed at the high dose. The white and red blood cell counts decreased in a dose-dependent manner. Marked reduction in cell number of thymus, and moderate effect on cellularity of spleen and MLN, were observed at 20 mg/kg bw. In vitro exposure of rat thymocytes to arecoline resulted in a biphasic oxygen consumption response with progressive increase in oxygen consumption, reaching a maximum value at 10(-5) M and decreasing sharply at 10(-3) M. Exogenously added substrates such as glucose, pyruvic acid and lactic acid retarded the fall in the oxygen consumption induced at 10(-3) M arecoline. These observations demonstrate the effects of arecoline on lymphoid organs, which may be due to its direct action or through the elevation of corticosterone.     

Modification of Immune Response by Cinnarizine

Effects of cinnarizine on immune response in mice were investigated. Mice were orally administered with cinnarizine and were immunized with sheep red blood cells (SRBC) intravenously. Numbers of plaque forming cells (PFC) to SRBC in spleen of these mice were assayed and delayed-type hypersensitivity (DTH) response to SRBC was measured. 1) PFC response in immunization with 5 × 10⁶ cells/mouse of SRBC was enhanced by administration of 25 mg/kg of cinnarizine, while the response in immunization with 5 × 10⁸ cells/mouse was suppressed by 25 to 200 mg/kg of cinnarizine. 2) From study on timing of administration, suppression of PFC response by 6.25 to 200 mg/kg of cinnarizine was observed at 24 hr. after the immunization. 3) 12.5 to 200 mg/kg of cinnarizine suppressed polyclonal B cell activation induced by lipopolysaccharide (LPS). 4) Colchicine induced suppressor T cell inactivation was prevented by administration of 50 mg/kg of cinnarizine and it was suggested that cinnarizine may induce suppressor T cells from the study of adoptive cell transfer system. 5) 50 mg/kg of cinnarizine showed the suppression of DTH response in expression phase, but not in induction phase. It was concluded that immune responses in mice were modified by cinnarizine.     

Modification of Immune Response by Cinnarizine

Abstract

Effects of cinnarizine on immune response in mice were investigated. Mice were orally administered with cinnarizine and were immunized with sheep red blood cells (SRBC) intravenously. Numbers of plaque forming cells (PFC) to SRBC in spleen of these mice were assayed and delayed-type hypersensitivity (DTH) response to SRBC was measured. 1) PFC response in immunization with 5 × 10⁶ cells/mouse of SRBC was enhanced by administration of 25 mg/kg of cinnarizine, while the response in immunization with 5 × 10⁸ cells/mouse was suppressed by 25 to 200 mg/kg of cinnarizine. 2) From study on timing of administration, suppression of PFC response by 6.25 to 200 mg/kg of cinnarizine was observed at 24 hr. after the immunization. 3) 12.5 to 200 mg/kg of cinnarizine suppressed polyclonal B cell activation induced by lipopolysaccharide (LPS). 4) Colchicine induced suppressor T cell inactivation was prevented by administration of 50 mg/kg of cinnarizine and it was suggested that cinnarizine may induce suppressor T cells from the study of adoptive cell transfer system. 5) 50 mg/kg of cinnarizine showed the suppression of DTH response in expression phase, but not in induction phase. It was concluded that immune responses in mice were modified by cinnarizine.     

Cyclosporin A prevents suppression of delayed-type hypersensitivity in mice immunized with high-dose sheep erythrocytes.

When administered intraperitoneally to mice 2 days before immunization with a tolerogenic dose (10(9)) of sheep red blood cells (SRBC), cyclosporin A (CsA; 200 mg/kg) strikingly augmented 4-day delayed-type hypersensitivity (DTH) footpad reactions. These enhanced responses were similar in magnitude to those seen in animals sensitized with an immunogenic, low-dose (10(6)) SRBC. The stimulatory effect of CsA was observed over the dose range of 5-200 mg/kg and was obtained in animals given the drug in one injection, up to 7 days before sensitization. The augmentation of DTH was characterized by footpad swelling, intense mononuclear cell infiltration and increased deposition of 125I-fibrinogen within the challenge site. In addition, increased expression of procoagulant activity by spleen cells in response to antigen was observed. Cell transfer experiments showed that the CsA-enhanced DTH could be adoptively transferred to naive recipients. Additional transfers conducted at the time of antigen challenge suggested that, under the conditions described, CsA inhibited the action of a population of suppressor cells normally effective during DTH reactions.     

Enhancement of the Arthus reaction and suppression of delayed type hypersensitivity (DTH) by pluronic F-68, a detergent frequently used to prepare perfluorocarbon emulsions

The effect of a 20% w/v RM101 (perfluorobutyltetrahydrofuran) emulsion containing 5% w/v of the detergent Pluronic F-68 or 5% w/v Pluronic F-68 given alone on the Arthus reaction and on delayed type hypersensitivity (DTH) were evaluated in female A/J mice. The test substances were administered i.v. at 1% body weight at 0,4,7,14 and 28 days prior to the i.p. immunization with 10(7) sheep red blood cells (SRBC). The increase in footpad swelling at 4 h (Arthus reaction) and at 24 h (DTH) after elicitation with the s.c. administration of 10(8) SRBC into the left footpad was used to assess immune competence. Pluronic F-68 given alone enhanced the Arthus reaction only when administered on day 0 of immunization. Pluronic F-68 given alone, as well as the perfluorocarbon emulsion containing Pluronic F-68, suppressed the 24 h DTH for as long as 4 days prior to immunization. Nonemulsified perfluorocarbon, on the other hand, had no effect on either the Arthus reaction or on DTH. The immunostimulatory agent, levamisole, administered (10 mg/kg i.p.) 1.5-2 h prior to immunization with SRBC counteracted both the Arthus reaction and the DTH response produced by Pluronic F-68. The present data clearly demonstrate that the changes in Arthus reaction and the DTH response are due to the Pluronic F-68 used to emulsify the RM101 perfluorocarbon; the changes induced by the detergent in these two immune parameters probably involve separate mechanisms.     

Effect of 1-thiocarbamoyl-2-imidazolidinone on the generation of plaque forming cell responses

Although 1-thiocarbamoyl-2-imidazolidinone (TCI) is a highly potent modulator of cellular immunity, its effects on humoral immunity have not been investigated. Given orally to mice prior to immunization with sheep red blood cells (SRBC), low TCI doses (10(-14) to 10(-10) g/kg) suppressed primary plaque forming cell (PFC) responses of spleen cells by 50-75%. TCI effects in vivo were dependent on drug dose, antigen dose and time of drug administration relative to immunization. The kinetics of this response were not appreciably altered by TCI. Higher TCI doses, immunization with higher levels of SRBC than required to produce a maximal response or administration of TCI later than 48 hours after immunization resulted in drug effects ranging from slight suppression to mild enhancement of the primary PFC response. TCI given in vivo enhanced primary PFC responses to the T-independent antigen DNP-Ficoll by greater than 700%+. TCI given before a primary immunization suppressed a secondary PFC response to SRBC elicited 28 days later. However, when TCI was given 24 h prior to a secondary immunization, doses greater than 10(-5) g/kg were necessary to suppress the PFC response. The effect of TCI on in vitro immunized spleen cell cultures was similar to that found for in vivo immunized mice. TCI at concentrations up to 10(-1) g/l did not cause a loss of lymphocyte viability or inhibit plaque production by antibody producing cells. Effects of TCI on PFC responses in vitro were reversible if cells briefly exposed to an optimal concentration of drug were washed extensively prior to immunization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of immune responses in mice by a new inosine derivative--methyl inosine monophosphate (MIMP).

Inosine 5'-methyl monophosphate (MIMP) is a new immunomodulator designed to improve upon the activity of other thymomimetic purines. In Balb/c mice, MIMP was assessed for toxicity and activity on immune responses. The lethal dose for half the mice (LD50) exceeded 500 mg/kg of body weight by both the parenteral and oral routes. At doses of 1-100 mg/kg, the mice showed no visible untoward effects. The antibody response of splenocytes to sheep erythrocytes (SRBC) was measured by IgM plaque-forming cells (PFC) in soft agar under optimal conditions of immunization and challenge. MIMP (1-100 mg/kg) was given by both the intraperitoneal and oral routes (gavage) at the time of SRBC injection and 4 days thereafter. The PFC response was found to be significantly augmented. The maximum effect (approximately 2x) was observed at 50 and 100 mg/kg, via intraperitoneal (i.p.) and oral routes, respectively. Increases (maximally 1.5x) in the responses of splenic lymphocytes to mitogen stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A) were observed under similar conditions of MIMP treatment. SRBC-induced delayed-hypersensitivity (DTH) was also measured under optimal conditions. By both i.p. and oral routes, enhancement of DTH response was produced by the lower doses of MIMP (0.01-1 mg/kg). Again, a second peak of optimum stimulation of DTH response was produced by 50 mg/kg of MIMP when administered by both routes. The effect was observed mainly on the sensitization rather than on the expression phase. MIMP qualifies as an effective immunopotentiator in normal mice.     

Sodium Valproate Does not Alter Immune Competence in Mice

Spleen and thymus weight, delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC), contact hypersensitivity to picryl chloride, anti-sheep red blood cell hemagglutinin titers and plaque-forming cells (PFC), resistance against experimental toxoplasmosis as well as serum total and intestinal specific IgA were investigated in Swiss mice given sodium valproate (250 mg/kg/day) orally for 21 consecutive days. Sodium valproate (SV) did not exert marked effects as only DTH to SRBC and PFC numbers were found to be slightly altered. These results suggest that SV is unlikely to alter immune competence markedly.     

RM-11, a new izoxasole derivative, is a potent stimulator of the humoral and cellular immune responses in mice

In this report we describe immunostimulatory properties of RM-11 in several in vivo and in vitro tests in the murine model. We found that RM-11 significantly stimulated the humoral immune response to sheep erythrocytes (SRBC) when given intraperitoneally (i.p.) at doses of 10 and 100 microg or per os (doses of 20 and 200 microg) 3 h before immunization. The compound was also stimulatory with regard to generation of delayed type hypersensitivity (DTH) to SRBC when given i.p. or per os (doses of 10, 100 and 500 microg/mouse). The described immunostimulatory activities of RM-11 were higher compared to that of the reference drug, levamisole. RM-11 stimulated, in addition, concanavalin A (ConA)-induced splenocyte proliferation. Lastly, we showed that RM-11 was not toxic when given to mice per os at doses 250 mg/kg body weight. Taken together, RM-11 appeared to be a universal stimulator of the immune response in mice. Lack of toxicity and the ability to stimulate the immune response, when administered per os, predispose the compound for further preclinical studies.     

Validation of the Candida albicans delayed-type hypersensitivity (DTH) model in the female B₆C₃F₁ mouse for use in immunotoxicological investigations

Although numerous models are used to evaluate the immunotoxic effects of xenobiotics on cell-mediated immunity (CMI), no holistic model for evaluating such effects on the delayed-type hypersensitivity (DTH) response has gained widespread acceptance. Due to a lack of interference from antigen-specific antibody production, the Candida albicans DTH model has recently been demonstrated to be a more appropriate model for assessing effects on CMI than other DTH models that utilize different sensitizing antigens, such as sheep erythrocytes (SRBC) or keyhole limpet hemocyanin (KLH). The present studies were conducted to validate the C. albicans DTH model for its ability to detect suppression (or the lack thereof) of CMI following exposure for 28 days to well-characterized immunosuppressive drugs, each having a different mechanism of action. The compounds evaluated included azathioprine (AZA), cyclophosphamide (CPS), cyclosporin A (CSA), dexamethasone (DEX), and the non-immunotoxic compound, benzo[e]pyrene (B[e]P). Exposure to each of the four known immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans. Footpad swelling was decreased following exposure to AZA at ≥ 20?mg/kg but not at 10?mg/kg, CPS at ≥ 10?mg/kg but not at 5?mg/kg, CSA at ≥ 3?mg/kg but not at 1?mg/kg, or DEX at ≥ 0.3?mg/kg (intermittently at 0.1?mg/kg) but not at 0.03?mg/kg. As expected, exposure to B[e]P for 28 days at doses up to 40?mg/kg had no effect on the DTH response. These results demonstrated that the C. albicans DTH assay in the B?C?F? mouse was capable of appropriately classifying each test article as to its immunotoxic effects on CMI. Furthermore, comparisons of these results with previous reports of effects on ex vivo CMI end points suggest that this DTH assay may be more sensitive than standard ex vivo assays at detecting immunosuppressive effects.     

Lack of immunosuppression by ketoconazole and itraconazole.

The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 microM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 microM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presence of 10 microM ketoconazole or 1 microM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxazolone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. II. Inflammatory helper T cells and effector T cells in mice with delayed-type hypersensitivity and in suppressed mice.

Peritoneal exudate cells were induced in mice 4 days after immunization with SRBC. A low dose of SRBC (10(6) i.v.) caused T lymphocytes to appear in inflammatory exudates. These cells, not only transferred DTH reactions, but also functioned as helper T cells in antibody production after transfer to syngeneic nu/nu recipient mice. After a high dose of SRBC (10(9) i.v.), very few helper T cells and no DTH transferring T cells were found in inflammatory exudates, although they were present in the spleen. It is postulated that T cells mediating DTH reactions and helper T cells behave similarly as far as those dose dependency of appearance in inflammatory exudates is concerned. A high dose of sensitizing antigen causes retention of helper and effector T cells in the spleen, in this way favouring antibody formation; low doses of antigen allow them to leave the spleen, thus favouring mediation of DTH reactions in the periphery.     

Validation of the Candida albicans delayed-type hypersensitivity (DTH) model in the female B6C3F1 mouse for use in immunotoxicological investigations

Although numerous models are used to evaluate the immunotoxic effects of xenobiotics on cell-mediated immunity (CMI), no holistic model for evaluating such effects on the delayed-type hypersensitivity (DTH) response has gained widespread acceptance. Due to a lack of interference from antigen-specific antibody production, the Candida albicans DTH model has recently been demonstrated to be a more appropriate model for assessing effects on CMI than other DTH models that utilize different sensitizing antigens, such as sheep erythrocytes (SRBC) or keyhole limpet hemocyanin (KLH). The present studies were conducted to validate the C. albicans DTH model for its ability to detect suppression (or the lack thereof) of CMI following exposure for 28 days to well-characterized immunosuppressive drugs, each having a different mechanism of action. The compounds evaluated included azathioprine (AZA), cyclophosphamide (CPS), cyclosporin A (CSA), dexamethasone (DEX), and the non-immunotoxic compound, benzo[e]pyrene (B[e]P). Exposure to each of the four known immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans. Footpad swelling was decreased following exposure to AZA at ≥ 20?mg/kg but not at 10?mg/kg, CPS at ≥ 10?mg/kg but not at 5?mg/kg, CSA at ≥ 3?mg/kg but not at 1?mg/kg, or DEX at ≥ 0.3?mg/kg (intermittently at 0.1?mg/kg) but not at 0.03?mg/kg. As expected, exposure to B[e]P for 28 days at doses up to 40?mg/kg had no effect on the DTH response. These results demonstrated that the C. albicans DTH assay in the B₆C₃F₁ mouse was capable of appropriately classifying each test article as to its immunotoxic effects on CMI. Furthermore, comparisons of these results with previous reports of effects on ex vivo CMI end points suggest that this DTH assay may be more sensitive than standard ex vivo assays at detecting immunosuppressive effects.     

Augmentation of delayed-type hypersensitivity to high dose sheep erythrocytes by cyclosporin A in the mouse: influence of drug dosage and route of administration and analysis of spleen cell populations.

When administered by various routes 48 h before a high systemic dose (10 degrees) of sheep red blood cells (SRBC), Cyclosporin A (CsA) prevented the suppression of delayed-type hypersensitivity (DTH) reactions elicited 4 days later. Augmentation of DTH was observed over a wide range (5-200 mg/kg) and with circulating CsA levels ranging below 45 ng/ml at the time of immunization or antigen challenge. Splenic lymphocytes from vehicle- and CsA-treated mice exhibited good proliferative responses to mitogen in vitro, but only those from CsA-treated animals responded to antigen. Expression of DTH was associated with a progressive, 2-fold increase in the absolute numbers of splenic L3T4+ cells, whereas no significant alteration in the number of Lyt-2+ lymphocytes was recorded. B cell and macrophage numbers in the spleen were unaffected by CsA. In contrast to its potentiating effects on cell-mediated immunity, CsA caused profound (up to 100%) suppression of the concomitant production of splenic anti-SRBC IgM-secreting plasma cells. Circulating anti-SRBC antibody levels were also markedly reduced. These data show that CsA can permit induction of TDTH, whilst suppressing T-dependent humoral immunity and without significant change in absolute numbers of Lyt-2+ cells.     

Effect of immunomodulators pyrimethamine and cimetidine on immunosuppression induced by sulfur mustard in mice

Despite extensive world-wide research no effective therapy has been devised for the treatment and cure of patients exposed to sulfur mustard (S-M). A severe suppression of the immune system still remains the major cause of opportunist infections, septicemia and death in patients injured by S-M. In this report we present a model of S-M contamination in mice which is suitable for immunomodulation studies. Results show that differing doses of S-M caused an overall suppression of the immune response to SRBC as indicated by agglutination titer, (DTH) tests, spleen histology and spleen weight indices. In the second stage two immunomodulating agents; pyrimethamine and cimetidine were employed and their effectiveness in augmenting immune responses after S-M induced immunosuppression was evaluated. Pyrimethamine, at all doses employed, enhanced antibody titers to SRBC, augmented DTH responses, and restored splenic follicles as compared with controls only exposed to S-M. Cimetidine augmented antibody titers and enhanced DTH responses at doses of 10 and 15 mg/kg as compared with controls. At a dose of 5 mg/kg cimetidine did not exhibit any effect on titers or DTH responses. Histological studies revealed that cimetidine restored splenic follicles and increased macrophage numbers and phagocytic activity at all three doses. Spleen weight indices were not augmented by either drug. These data provide evidence that immunomodulating drugs may prove effective in countering the immunosuppressive effects of S-M.     

A new assay system detecting antibody production and delayed-type hypersensitivity responses to trinitrophenyl hapten in an individual mouse

A new assay system detecting antibody production and delayed-type hypersensitivity (DTH) responses to trinitrophenyl hapten in an individual mouse (AS-DAD) was established. BALB/c mice were immunized intraperitoneally with varying amounts of 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC) on day 0. Venous blood was collected on days 2, 4, 6, 8 and 10. Levels of anti-TNP IgM and IgG in serum were assayed by enzyme-linked immunosorbent assay (ELISA). After series of bleeding the mice were challenged with 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution in the footpad on day 14. Footpad swelling was measured 24 or 48 h after the challenge. Peak responses of the anti-TNP IgM and IgG production were detected 4 or 6 days after the immunization with 10⁹ TNP-SRBC. Maximum DTH response was also observed with 10⁹ TNP-SRBC 24 h after the challenge on day 14. The antibody and DTH responses were also induced in other normal inbred strains such as C3H/He and DBA/1 but not BALB/c nu/nu mice. To evaluate AS-DAD in immunopharmacological studies, various immunomodulating agents were examined in BALB/c mice by subcutaneous administration on days 0, 1, 2 and 3. Cyclosporin or cyclophosphamide at 100 mg/kg/day completely inhibited not only the anti-TNP IgM and IgG production but also the TNP-specific DTH response. Prednisolone at 0.5 mg/kg/day had no significant effect on the IgM and IgG production, whereas it inhibited the TNP-specific DTH response. Interestingly, histamine-added mouse γ-globulin at 150 mg/kg/day clearly enhanced the anti-TNP IgM and IgG production, while it showed a suppressive effect on the TNP-specific DTH response. Levamisole at 5.0 mg/kg/day showed suppressive effects on the anti-TNP IgG production without affecting the IgM production and the DTH response. These results suggest that AS-DAD is useful for evaluating the immunopharmacological action of various agents.