SA3443, a novel cyclic disulfide compound, depresses anti-SRBC antibody-forming cell responses in the mouse through inhibition of antigen-presenting cell activities.

(4R)-Hexahydro-7,7-dimethyl-6-oxo-1,2,5-dithiazocine-4-carboxylic acid (SA3443) is a newly synthesized cyclic disulfide compound which has potential hepatoprotective properties. The effect of SA3443 on the induction of anti-sheep red blood cell (SRBC) plaque (antibody) forming cell (PFC) responses was investigated in vivo and in vitro. SA3443 (approximately 3 mg/kg/day) remarkably decreased the number of anti-SRBC PFC in the spleens of mice immunized with a high dose of SRBC in vivo. The addition of SA3443 (approximately 1 x 10(-7) M) at the initiation of mouse spleen cell cultures in vitro also exerted a significant inhibitory effect on subsequent PFC response to SRBC, and removal of SA3443 after 24 h did not reverse its inhibitory effect. Pre-incubation of isolated adherent spleen cells with SRBC and SA3443 resulted in a similar inhibition of subsequent PFC response, but a pre-incubation of macrophage-depleted cells with SRBC and SA3443 or a pre-incubation of the unseparated spleen cells with SA3443 in the absence of SRBC had no effect. These findings have suggested that SA3443 may depress antibody response through inhibition of macrophage antigen-presenting cell activity.     

Effects of Tremella polysaccharides on immune function in mice

It was found in vitro that Tremella polysaccharides (TP) (50, 100, 150 and 200 micrograms/ml) augmented lymphocyte proliferation induced by Con A and did not antagonize the suppressive effect of hydrocortisone on lymphocyte proliferation. In vivo TP promoted the plaque-forming cell (PFC) response to SRBC in mice. TP 50 and 100 mg/kg ip for 5 d produced 77.6% and 81.8% increases in PFC response respectively. At the doses of 150 and 200 micrograms/ml, TP decreased the interleukin 2 (IL-2) activities in the supernatant of culture media of mouse spleen cells. TP (50 micrograms/ml) enhanced the lymphocyte proliferation induced by Con A and increased the PFC response to SRBC by 47.1% in 14-month-old mice.     

Suppression of the antibody response by phorbol esters in the mouse is due to an effect on the nylon wool adherent cell population

Phorbol esters, in particular 12-O-tetradecanoyl-phorbol-13-acetate (TPA), have been shown to have profound effects on most biological systems including tumor promotion. Presented here are studies on the acute toxic effects of TPA, and the effects of phorbol esters on the in vivo and in vitro, T cell-dependent, antigen-specific antibody response in the mouse. The LD50 of a single i.v. dose of TPA in the mouse was 309 micrograms/kg. Acute toxic effects included lethargy, hypothermia and enlarged, hemorrhagic spleens at the higher doses. TPA was shown to be a potent inhibitor of the in vivo primary antibody response as measured by the IgM antibody-forming cell (AFC) response to sheep red blood cells (sRBC). The ED50 of a cumulative i.v. dose was 145 micrograms/kg administered the day before and the day of immunization (72.5 micrograms/kg/day). A cumulative dose of 500 micrograms/kg (250 micrograms/kg/day) resulted in a 100% suppression of the response. This in vivo exposure to TPA did not alter B cell/T cell ratio in the spleen. Phorbol ester analogs inactive in other biological systems were also inactive in the in vivo AFC response. The in vitro AFC assay was used to determine what cell type was being affected by TPA. Separation of the adherent spleen cells into B and T cell populations was done using nylon wool columns and anti-theta plus complement treatment. Experiments with these cell populations indicated that TPA produced suppression of the response due to an effect on the nylon wool adherent cell population.     

Ketotifen-induced histamine release, inhibition of histamine secretion and modulation of immune response

In concentrations above 0.1 mM Ketotifen (KT) induced secretion of histamine from rat and mouse mast cells, but not from human basophils. In the same concentrations KT inhibited histamine release from rat mast cells, induced by compound 48/80 and histamine release from basophils, induced by anti-IgE-antibodies and concanavalin A. At low concentration (0.05-0.005 mM) KT enhanced in vitro phytohemagglutinin-induced proliferative response of human lymphocytes. The immunostimulating effect of KT was confirmed in vivo. In doses 0.18-1.4 micrograms/mouse KT significantly increased the number of antibody-producing cells (APC) in spleens of mice immunized by sheep red blood cells (SRBC). KT stimulated both IgM- and IgG-immune response to SRBC.     

Enhancement of immune responses and possible inhibition of suppressor cells by aclacinomycin A

Antitumor antibiotics were examined as possible candidates that possess activity which inhibits preferentially suppressor cells in comparison with effector cells. In screening for such compounds among known antibiotics, aclacinomycin was found to augment antibody formation and delayed-type hypersensitivity in mice over a wide concentration range. The addition of aclacinomycin to mouse spleen cell cultures also enhanced antibody formation in vitro. The generation of suppressor cells or the suppressor activity per se in mice immunized with high doses of SRBC was reduced by aclacinomycin. These results suggest that the drug may possibly inhibit suppressor cells selectively. The administration of aclacinomycin at ow doses exhibited antitumor effects on IMC carcinoma; the effect was not dose-dependent.     

Direct suppression of cultured spleen cell responses by chlordane and the basis for differential effects on in vivo and in vitro immunocompetence

Immunosuppression by gamma-chlordane was examined by the direct addition of chlordane to cultured spleen cells from untreated B6C3F1 mice. Both cell-mediated and humoral immune responses were markedly suppressed upon in vitro exposure. The mixed lymphocyte response and the proliferative response to both B- and T-cell mitogens were significantly suppressed at micromolar concentrations of chlordane. The antibody response to sheep erythrocytes (SRBC) was suppressed 90% at 10 microM chlordane. The kinetics of the SRBC response were not altered by chlordane. Addition of chlordane to the antibody cultures on various days indicated an effect at the early stages of the response. Previous studies with chlordane failed to demonstrate immunosuppression following in vivo exposure. The possibility that chlordane was metabolized in vivo to a less immunosuppressive form was studied by examining the effect of the major metabolite, oxychlordane, on the in vitro antibody response and by incubating splenocytes with chlordane and a liver S9 preparation prior to culture with SRBC. Oxychlordane was immunosuppressive by itself, and the activity of chlordane was unaltered in the co-culture experiments. The association of chlordane with serum components was evaluated in vitro in cultures of mouse bone-marrow cells (BMC). The chlordane-induced suppression of [3H]thymidine incorporation by BMC was reversed by the addition of mouse or human serum. In summary, chlordane produces marked suppression of in vitro immune responses via an apparent antiproliferative action. The failure of chlordane to produce in vivo immunosuppression may be related to extensive association of chlordane with serum components.     

Augmentation of the antibody response by lipoic acid in mice. I. Analysis of the mode of action in an in vitro cultures system

Lipoic acid (Lip), a naturally occurring disulfide compound, was found to augment markedly in vitro antibody responses to sheep erythrocytes (SRBC), dinitrophenyl-Ficoll and trinitrophenyl-lipopolysaccharide (TNP-LPS) as effectively as 2-mercaptoethanol (2-ME) in murine lymphocytes. The mitogenic response to LPS or concanavalin A (Con A) was augmented by Lip only slightly. 2-ME has been reported to facilitate cystine utilization by the lymphocytes, but Lip did not, indicating that the mode of action of Lip is different from that of 2-ME. Lip-augmentation of anti-SRBC response was markedly abrogated when murine lymphocytes were depleted of T cells and cultured in the presence of Con A-conditioned medium containing T cell-replacing factor. The effect of Lip was also diminished in the response to TNP-LPS when the spleen cells were depleted of T cells. These observations suggest that Lip could augment the antibody response by stimulating a T cell subpopulation. This idea was confirmed by the experiment that Lip could enhance helper T cell activity which was induced by culturing murine lymphocytes with the antigen.     

The effect of salazosulfapyridine on the in vitro antibody production in murine spleen cells

The effect of salazosulfapyridine (SASP) on the antibody response of murine spleen cells in vitro was studied. SASP inhibited the response to sheep red blood cells (SRBC), a T-cell-dependent (TD) antigen, dose-dependently and was most effective at a dose of 2 x 10(-4) M without cell toxicity. No remarkable inhibition was seen with the main metabolites of SASP, 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP). SASP failed to inhibit antibody production to T-cell-independent antigens such as dinitrophenyl-Ficoll or trinitrophenyl (TNP)-lipopolysaccharides, although the response to TNP-keyhole limpet hemocyanin, another TD antigen like SRBC, was inhibited. Further, this drug did not show any depression of the anti-SRBC plaque-forming cell (PFC) response in spleen cells treated with anti-Thy1.2 antibody plus complement. The inhibition of anti-SRBC PFC response by SASP was accompanied by a reduction of interleukin 2 (IL-2) secretion. Our results suggest that SASP may act on T cell populations and may inhibit the T-cell-dependent antibody response partly through a depression of IL-2 production. The active compound appears to be SASP itself, rather than its metabolites.     

Effect of benzodiazepine derivatives: I. Augmentation of T cell-dependent antibody response by diazepam in mouse spleen cells

Oral administration of diazepam at doses of 5-10 mg/kg to restraint-stressed mice resulted in almost complete recovery in the stress-induced suppression of the antibody response to sheep red blood cell (SRBC). Moreover, this compound restored the suppression of antibody response to SRBC in cyclophosphamide-treated mice. Diazepam treatment also enhanced the antibody response against SRBC in normal mice only when the animals were immunized with the reduced amount of antigen. It was demonstrated that antigen specific helper T cell activity was promoted by diazepam administration in mice. Addition of diazepam augmented the in vitro anti-SRBC hemolytic plaque-forming cell (PFC) response in mouse splenocytes without altering kinetics of the response. However, the enhancing effect was observed only when the drug was added to the medium at the culture initiation. On the other hand, antibody response to T cell-independent antigens such as trinitrophenylated (TNP)-Ficoll and TNP-lipopolysaccharide were not enhanced by diazepam. Concanavalin A or LPS-induced 3H-thymidine uptake into splenocytes were not stimulated by diazepam. These results suggest that diazepam promotes the antibody response through stimulating helper T cell functions.     

Assessment of the immunotoxic potential of the fungicide dinocap in mice.

The immunotoxic potential of dinocap was evaluated in female C57BL/6J mice following in vivo and in vitro exposure to this fungicide. In in vivo studies, groups of mice were dosed by gavage with technical grade dinocap at dosages ranging from 12.5 to 50 mg/kg per day for 7 or 12 days and selected immune functions examined. Mice dosed at 50 mg/kg per day dinocap died after 4 days of dosing. Twelve days of dosing with dinocap at 25 mg/kg per day resulted in decreased thymus weights and cellularity, and increased spleen weights. No changes were observed in body weight, absolute differential peripheral leukocyte counts, the lymphoproliferative responses to B- or T-cell mitogens, the mixed lymphocyte reaction, or natural killer (NK) cell activity of spleen cells from mice exposed to dinocap. Lymphoproliferative responses to concanavalin A (Con A) and phytohemagglutinin (PHA), however, were reduced in thymocytes from mice dosed at 25 mg/kg per day dinocap. The cytotoxic T lymphocyte (CTL) response to P815 mastocytoma cells was enhanced in mice exposed for 7 days to 25 mg/kg per day dinocap. Exposure of mice for 7 days to 25 mg/kg per day dinocap also caused a significant reduction in the IgM and IgG plaque-forming cell (PFC) response to sheep red blood cells (SRBC). A time-course study indicated that dinocap-induced suppression of the IgM PFC response was due to a delay in the peak PFC response to SRBC. In vitro studies using murine thymocytes cultured with dinocap (10 micrograms/ml for 72 h) resulted in suppression of the proliferative response to Con A and PHA. Exposure of thymocytes to dinocap in vitro for as little as 30 min resulted in suppression of the mitogen-stimulated response in the absence of any apparent direct cytotoxic effect. These results suggest that dinocap alters the immune system of the mouse, however, these effects are relatively modest in terms of adverse immune function and are only seen at relatively high exposure levels.     

Modulatory effect of isoprinosine on lymphocyte proliferative response under immunodepressive conditions

In the present work the effect of Isoprinosine on the mitogenic responses of T and B lymphocytes has been studied. We have found that Isoprinosine can enhance in vitro the response to Concanavalin A. This enhancement was more apparent in cell cultures showing an initially low blastogenic response. In low responses artificially induced by treatments in vivo with cyclophosphamide, our results indicate that Isoprinosine, administered in vivo, does not enhance the response to Con A of treated mice. However, addition of Isoprinosine (75 micrograms/ml) to cultures of spleen cells from mice previously treated with cyclophosphamide enhanced the suppressed response up to normal levels. Neither in vivo nor in vitro Isoprinosine treatments increased the response of lymphocytes to lipopolysaccharide, but usually inhibited the blastogenesis of B cells.     

Immunological reactivity to a mycobacterial fraction is associated with nonspecific suppression of immunological responsiveness in vivo

Previously reported studies revealed that spleen cells from BALB/c mice immunized against a methanol extraction residue (MER) fraction of tubercle bacilli are defective in the in vitro generation of antibodies to SRBC and in allogeneic responsiveness against C57BL spleen cells. We now show that mice repeatedly immunized with MER also exhibit a depressed capacity to respond to antigenic stimulation in vivo. Thus mice repeatedly injected with MER were impaired in their ability to react to antigenic stimulation by SRBC and by C57BL spleen cells. Impairment in the response to SRBC immunization was expressed at the level of delayed-type hypersensitivity (DTH) as well as of antibody production. The response of MER hyperimmunized mice to contact sensitization with dinitrofluorobenzene (DNFB) was not impaired, but the lymph node cells of DNFB-sensitized animals had a depressed ability to respond to in vitro stimulation by the monovalent hapten dinitrobenzene sulfonate (DNBS). The present findings indicate that extensive exposure to an immunogenic immunomodulating mycobacterial fraction can lead to a depressed responsiveness to unrelated antigenic stimulation.     

Immunopharmacologic studies of D-penicillamine-L-cysteine disulfide

Effects of D-penicillamine-L-cysteine disulfide (P-C) on some immunological parameters were examined in normal and immunity-impaired mice and rats. P-C enhanced the DNA synthesis in concanavalin A-stimulated mouse spleen cell cultures in vitro. In vivo, administration of P-C produced either enhancement or depression of plaque forming cell (PFC) response and delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) in low responder mice to SRBC, depending on the dose of P-C. P-C restored the impaired PFC response in hydrocortisone-pretreated mice. The enhancing effect of P-C was not shown in high responder mice to SRBC, but an inhibiting effect was observed. P-C inhibited the suppressor cell induction on PFC response in mice immunized with a supraoptimum dose of antigen. In adjuvant arthritic rats, P-C induced severe arthritis by eliminating the suppressor cells regulating this disease process. The relevance of these findings and mode of action of D-penicillamine in rheumatoid arthritis is discussed.     

Suppression of the antibody response by a formamidine pesticide: dependence on the route of exposure

These studies investigated the effects of exposure to chlordimeform (CDM), a formamidine pesticide, on selected in vivo immune parameters in the random bred CD-1 mouse. Further studies were done on the effects of this compound on the in vitro PFC response in C57BL/6 mice. Acute and 14-d exposure to CDM via the i.p. route resulted in a decrease in IgM antibody-forming (plaque-forming) cells (PFC) directed at the sheep red blood cell (sRBC) antigen when measured 4 d after i.p. immunization. This suppression was seen at doses as low as 20 mg/kg . d for 14 d. These same doses of CDM did not result in any alteration of cell-mediated immunity as measured by the delayed hypersensitivity response (DHR) to both keyhole limpet hemocyanin (KLH) and sRBC. Lymphocyte blastogenesis was increased in spleen cells from mice exposed to 40 mg/kg . d CDM in response to media alone, concanavalin A (Con A), and lipopolysaccharide (LPS). The in vitro PFC response by C57BL/6 mice was utilized to determine if CDM could suppress the antibody response due to a direct effect on the immune cells. CDM suppressed the in vitro PFC response only at concentrations that were directly cytolytic. A direct cytolytic effect was considered unlikely following exposure in the whole animal, since the suppression of the antibody response occurred in the absence of any effects on spleen cell number or spleen weight. To determine if route of exposure was a factor in the suppressive effects of CDM, 14-d studies were conducted administering CDM orally at doses up to 120 mg/kg . d. Both the CD-1 and C57BL/6 mouse were used to verify that a strain difference was not a factor. There was no effect on either the d 4 or d 5 antibody response, even though 43% of the mice exposed to 120 mg/kg died from the acute toxicity that can characterize this chemical. From an operational standpoint, these results indicate that the route of exposure of a compound relative to the route of administration of an antigen is an important consideration when determining the effects of that compound on an immune response. From an environmental standpoint, these results indicate that relatively high doses of chlordimeform do not result in consistent immunotoxicity as determined by the assays utilized.     

Immunocompetence status as related to growth progression of mouse MOPC-315 plasmacytoma

The immunocompetence status of mice bearing MOPC-315 plasmacytoma was determined at various days after tumor inoculation. Changes in T and B-cell functions appeared gradually. The allogeneic response of spleen cells from BALB/c tumor-bearing mice against C57BL spleen cells was impaired from the 4th day after the tumor inoculation (nonpalpable tumor stage). The primary antibody response in vitro against SRBC was depressed at 18 days, and the mitogenic response of splenic cells to PHA and to LPS was depressed at 25 days after the tumor inoculation. T cells taken from day 18 tumor-bearing mice partially suppressed the MLR response of normal splenocytes. Mice bearing large MOPC-315 tumors responded less to SRBC immunization than normal, noninoculated mice. The relative percentage of Lyt 1, Lyt 2 and L3T4 T-cell subsets decreased starting from the 11th day after tumor inoculation.     

Augmentation of the antibody response by lipoic acid in mice. II. Restoration of the antibody response in immunosuppressed mice

Lipoic acid (Lip) was examined for its effect on the in vivo antibody response in mice. It was found that Lip significantly restored the suppressed antibody response to sheep erythrocytes (SRBC) in cyclophosphamide-injected mice when it was orally administered at 25 mg/kg twice a day for 4 consecutive days after immunization. On the other hand, Lip-administration did not affect the antibody response in normal mice immunized with SRBC. Normal or cyclophosphamide-treated mice were primed with horse erythrocytes (HRBC) and were given either saline or Lip for 3 consecutive days. HRBC-specific helper T cell activity of their spleen cells were compared by coculturing these cells with trinitrophenyl (TNP)-keyhole limpet hemocyanin-primed spleen cells in the presence of TNP-HRBC as the antigen. A significant restoration of the suppressed helper T cell activity was observed in the Lip-administered group. However, the helper T cell activity was not affected significantly by Lip-administration in normal mice. Lip could also partially restore the helper T cell activities that were suppressed by the treatment with hydrocortisone or X-ray irradiation.     

Effect of L-alanosine on immune response

L-alanosine is an antitumor compound which has recently entered clinical trials. Single i.p. doses of this drug inhibit antibody production to thymus-dependent and thymus-independent antigens as well as delayed hypersensitivity to SRBC in mice. Moreover, the in vitro lymphoproliferative responses of splenocytes to Concanavalin A and bacterial Lipopolysaccharides are also affected when the drug is added upon setting up the cultures. On the other hand, in vivo treatment with L-alanosine does not impair spleen natural killer (NK) activity. The results are discussed in an effort to characterize the immuno-suppressive activity of L-alanosine.     

Inhibition of the antibody production by acetaminophen independent of liver injury in mice

The causal relationship between the inhibition of antibody production and liver injury induced by single doses of acetaminophen (APAP) was investigated in mice. The liver injury and antibody production were evaluated using the serum transaminase activity and the number of antibody forming cells against sheep red blood cells (SRBC), respectively. The relevance of APAP hepatotoxicity with inhibiting antibody production was elucidated in fasted and fed mice treated with a single oral administration of APAP. In fasted mice, the oral administration of APAP produced serious liver injury, while it was not the case in the fed mice. As the antibody production was measured under these conditions, APAP significantly depressed the antibody production in fed mice as well as in fasted mice. The rate of B220 positive cells in the splenocytes was significantly decreased by APAP administration in both the fasted and fed mice. Splenocytes proliferative responses following mitogenic stimulation with concanavalin A or lipopolysaccharide were inhibited by APAP. Moreover, APAP added directly to the splenocyte culture also inhibited the in vitro antibody-producing response to SRBC. These findings indicate that the APAP-induced depression of antibody production may not be a secondary response to APAP-hepatitis, but may be a primary response to APAP.     

Immunosuppressive effects of 2-acetylaminofluorene and 2-aminofluorene on murine splenocytes culture

The immunosuppressive effects of 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) were studied in BALB/c mouse splenocyte culture. Direct addition of AAF and AF to the splenocyte culture produced a dose-related suppression of the in vitro antibody response to sheep erythrocytes(SRBC), DNP-Ficoll, and lipopolysaccharide(LPS). AAF and AF also produced suppression on lymphoproliferative responses to LPS and concanavalin A(Con A). The immunosuppressive effects of AAF and AF, however, were diminished when AAF and AF were incubated in the splenocyte-hepatocyte coculture system for 4 hr. When hepatocyte cultures were pretreated with SKF 525A, a cytochrome P-450 inhibitor, before coculture with spleen cell along with AAF and AF, the suppression of in vitro antibody response reappeared. Meanwhile, both AAF and AF produced a dose-related DNA single-strand breaks in spleen cells only if AAF and AF were treated to spleen cells cocultured with hepatocytes. These results indicate that the immunosuppression by AAF and AF is not mediated by the reactive metabolites implicated to DNA damage.     

Suppressive effects of chlorphenesin on lymphocyte function in mice and humans.

The immunosuppressive action of chlorphenesin was investigated in a wide variety of in vitro assays for cellular immunity in humans and mice. Chlorphenesin, at doses of 20-50 micrograms/ml, inhibited mitogenic responses of both mouse and human B and T cells. These doses did not kill cells exposed to the drug for 72 hr. Mixed lymphocyte reactions in inbred strains of mice and in unrelated humans were also inhibited at concentrations of about 50 micrograms/ml. However, the generation of cytotoxic T cells in cell-mediated lympholysis assays was not inhibited to the same degree as proliferation in mixed lymphocyte reaction and the cytotoxic potential of presensitized mouse T cells for allogeneic targets was totally unaffected. These studies suggest that chlorphenesin may have a broad spectrum of suppressive effects both on T and B cells and that the predominant inhibition of proliferative responses in these cells may reduce the expansion of clones of immunocompetent cells in vivo.