Immunotoxicological profile of morphine sulfate in B6C3F1 female mice.

This study was undertaken to investigate a number of immune parameters which may be compromised with exposure to morphine sulfate. Mice were implanted subcutaneously with 8-, 25-, or 75-mg morphine sulfate pellets. Placebo pellets of identical makeup to the 75-mg morphine pellet (without morphine of course) were used as a control. Twenty-four hours after implantation of a 75-mg morphine pellet, blood levels reached a peak of 1610 ng/ml. Corticosterone increased in parallel with morphine and reached a peak level of 966 ng/ml 24 hr after implantation. The dose response of morphine to increase corticosterone, however, was flat. The weight of the lymphoid organs, spleen and thymus, and the liver were significantly reduced in the morphine-treated groups. Morphine treatment was associated with an increase in serum albumin, SGPT, BUN, and alkaline phosphatase indicative of hepatic damage. In contrast to increased serum proteins, the C3 component of complement was reduced in a dose-dependent manner. Leukocyte number in the peripheral blood was significantly reduced, while erythrocyte number and hematocrit were both increased. The number of B cells and T cells was decreased in morphine-treated animals. However, the percentage of T cells relative to B cells was increased. The primary IgM antibody response to the T-dependent antigen, sheep red blood cells, was decreased. Natural killer cell activity was reduced in response to morphine, as was the phagocytic capacity of Kupffer cells. Host-resistance models of Listeria monocytogenes or Streptococcus pneumoniae showed an increased resistance following administration of morphine. This increased host resistance, however, was not due to an increase in antimicrobial action of sera obtained from mice treated with morphine. The majority of morphine's effects on the immune system exhibited a flat dose response, suggesting that these effects may be mediated secondarily through corticosterone.

Splenic cell targets in gallium arsenide-induced suppression of the primary antibody response.

In vivo exposure of female B6C3F1 mice to gallium arsenide (GaAs) was evaluated for its effect on the in vitro IgM antibody-forming cell (AFC) response. In vivo exposure to a single intratracheal dose of GaAs (2.5-200 mg/kg) resulted in a dose-dependent decrease in the in vitro IgM AFC response to the T-dependent antigen sheep red blood cells (SRBC) with a 97% decrease at 200 mg/kg when compared to vehicle controls. The response to the T-independent antigen DNP-Ficoll was significantly reduced at 100 and 200 mg/kg. Spleen cellularity decreased in a dose-related manner with a 54% decrease at 200 mg/kg. Enumeration of splenic subpopulations following GaAs (200 mg/kg) indicated a 58, 61, and 30% decrease in the total number of Thy 1.2 (T cells), Ig (B cells), and F4/80 (macrophages) positive cells, respectively, with no alterations in the percentages of these cells. Mitogenic responsiveness of splenocytes from GaAs-exposed mice was unaltered. To identify the splenic cell populations targeted by GaAs, the AFC response to SRBC was evaluated following cell separation/reconstitution of splenocytes from GaAs- (200 mg/kg, 24-hr exposure) and vehicle-exposed mice. Results demonstrated AFC suppression was due to functional alterations in both adherent (AD; macrophages) and nonadherent, (both T and B lymphocytes) cell populations. Further investigation focused on alterations in the AD population. Separation/reconstitution experiments demonstrated AFC suppression to SRBC was dependent on the concentration of macrophages from GaAs-exposed mice. This macrophage-mediated suppression of the in vitro AFC response could not be attributed to the presence of suppressor macrophages or release of prostaglandins.

Effects of CI-949, a novel antiallergy agent, on immune function of male Fischer 344 rats.

CI-949 is an orally effective inhibitor of allergic mediator release as measured with in vitro and animal models. To assess the effects of CI-949 on immune function, male Fischer 344 rats were evaluated for splenic T- and B-lymphocyte populations, antibody-forming cell response to sheep red blood cells (sRBC), concanavalin A- and pokeweed mitogen-induced lymphocyte proliferation, Natural Killer cell activity and reticuloendothelial system clearance of sRBC. CI-949 was administered by gavage to rats at 25, 50 and 100 mg/kg/day for 14 consecutive days. A vehicle control and two positive controls (cyclosporine A and cyclophosphamide) were run concurrently. CI-949 at 100 mg/kg/day decreased body weight gain and was lethal to 5 of 40 rats. The deaths occurred between days 5 and 12 of study. This overtly toxic dose did not alter splenic cellularity or change the percentages of T- and B-lymphocyte subpopulations. Additionally, CI-949 did not inhibit lymphocyte proliferation or hinder clearance of sRBC by the reticuloendothelial system. Antibody-forming cell response after immunization showed a dose-related increase in the number of immunoglobulin M secreting cells. Based on the results of these assays, the immune system does not appear to be adversely affected by CI-949 even at high doses.

Contact hypersensitivity response to o-benzyl-p-chlorophenol in mice.

o-Benzyl-p-chlorophenol was evaluated for its potential as a sensitizing agent for allergic contact hypersensitivity in mice. Female B6C3F1 mice were sensitized with 1.0, 3.0, and 10.0% o-benzyl-p-chlorophenol and challenged with 20.0% o-benzyl-p-chlorophenol. Doses of o-benzyl-p-chlorophenol were selected from assays for primary irritancy. Mice received 20 microliters by direct dermal application, for 5 days, to sites prepared by shaving, dermabrading and, in some mice, with intra dermal injection of Freund's complete adjuvant. The rest period was 7 days. Measurement of the contact hypersensitivity response in mice was by radioisotopic assay two days after challenge and mouse ear swelling test one and two days after challenge. Mice demonstrated statistically significant dose-dependent contact hypersensitivity response to o-benzyl-p-chlorophenol with or without adjuvant pretreatment.

The B lymphocyte is the immune cell target for 2',3'-dideoxyadenosine.

2',3'-Dideoxyadenosine (ddA) is a nucleoside analogue with anti-HIV activity and one of its metabolic products, 2',3'-dideoxyinosine (ddI), has shown promising results in clinical trials for the treatment of acquired immunodeficiency syndrome (AIDS). Because AIDS viruses target the immune system, it is important to understand the potential effects of anti-AIDS drugs, including natural nucleosides, on the immune system. Previous immunotoxicological studies have shown that 22 treatments with ddA to female B6C3F1 mice over a period of 30 days had no effect on cell-mediated immunity, including the mixed lymphocyte reaction and response to mitogenic signals, but suppressed in vitro IgM plaque-forming cell (PFC) response to sheep red blood cells. The present studies show that suppression of the IgM PFC response was dose dependent with a 96% reduction in IgM PFCs/10(6) spleen cells at the highest dose (350 mg/kg). The in vivo IgM PFC response to DNP-Ficoll and the in vitro IgM PFC response to lipopolysaccharide, both T-independent antigens, were also suppressed in the spleens of ddA-treated mice. The analysis of splenocyte subtypes shows no change in the percentage of B cells (surface immunoglobulin positive cells), T helper cells (L3T4 positive cells), and T suppressor cells or T cytotoxic cells (Lyt-2 positive cells) in the spleens of ddA-treated mice. In vitro separation and reconstitution studies in which the IgM PFC response was monitored indicated that the B lymphocyte rather than the T lymphocyte or antigen-presenting cell is the primary cell targeted by ddA. This information provides a data base for further mechanistic study and may reflect on the clinical use of other nucleoside analogues, e.g., ddI by providing the clinician with information indicating the potential decrease in humoral immunity.

Contact hypersensitivity response to isophorone diisocyanate in mice.

Isophorone diisocyanate was evaluated for its potential as a sensitizing agent for allergic contact hypersensitivity in mice. Female B6C3F1 mice were sensitized with 0.1, 0.3, and 1.0% isophorone diisocyanate and challenged with 3.0% isophorone diisocyanate. Doses of isophorone diisocyanate were selected from assays for primary irritancy. Mice received 20 microliters by direct dermal application, for 5 days, to sites prepared by shaving, dermabrading and, in some mice, with intra dermal injection of complete Freund's adjuvant. The rest period was 7 days. Measurement of the contact hypersensitivity response in mice was by radioisotopic assay two days after challenge and mouse ear swelling one and two days after challenge. Mice demonstrated statistically significant dose-dependent contact hypersensitivity responses to isophorone diisocyanate with or without adjuvant pretreatment.

Effects of subacute administration of O,S,S-trimethyl phosphorodithioate on cellular and humoral immune response systems.

The effects of 14-day treatment with low doses of O,S,S-trimethyl phosphorodithioate (OSS-TMP), an impurity in technical malathion, on the generation of cell-mediated and humoral immune responses were examined in female C57BL/6 mice. At a dose of 2.0 mg/kg per day OSS-TMP, the generation of antibody-secreting cells to sheep red blood cells, the generation of cytotoxic T lymphocytes (CTL) to alloantigen and the production of Interleukin-2 were elevated approximately 2-3 fold, while no changes were observed in the proliferative responses to the polyclonal activators, Concanavalin A, lipopolysaccharide, or phytohemagglutinin. In contrast, at 5.0 mg/kg per day OSS-TMP, both the CTL and specific antibody responses were suppressed, while all other immune parameters examined were unchanged. Data from cell separation and reconstitution experiments indicated that both T and B lymphocytes were affected by these treatment regimes. These data suggest that long-term exposure to low doses of OSS-TMP may enhance the ability of an animal to generate an immune response while higher doses of OSS-TMP may suppress the generation of an immune response.

Contact hypersensitivity response to glutaraldehyde in guinea pigs and mice.

Glutaraldehyde has a wide spectrum of uses which can result in dermal contact with the agent. The low number of reports of hypersensitive reactions to glutaraldehyde indicates a low incidence of sensitization. This paper describes the contact hypersensitivity response to glutaraldehyde in the guinea pig and the mouse. Female albino Hartley strain guinea pigs and female B6C3F1 mice were sensitized with 0.3, 1.0 and 3.0% glutaraldehyde and challenged with 10% glutaraldehyde. Doses of glutaraldehyde were selected from assays for primary irritancy. Guinea pigs received 100 microliters by direct dermal application, for 14 consecutive days, and mice received 20 microliters by direct dermal application, for 5 or 14 consecutive days, to sites prepared by shaving and dermabrading. Rest periods were 7 or 14 days for guinea pigs and 4 or 7 days for mice. Measurement of the contact hypersensitivity response in guinea pigs was both visual evaluation (scoring) at 24 and 48 hours following challenge and radioisotopic assay at 48 hours, and in mice by radioisotopic assay 48 hours after challenge. Both guinea pigs and mice demonstrated dose-dependent contact hypersensitivity responses to glutaraldehyde. The radioisotopic assay appeared to be more sensitive than visual evaluation in detecting contact allergic hypersensitivity to glutaraldehyde.

Human T cell hybridomas producing cytotoxic lymphokines: induction of lymphotoxin release and killer cell activity by anti-CD3 monoclonal antibody or lectins and phorbol ester.

Human T cell hybrids were constructed between lectin-activated peripheral blood lymphocytes and CEM.TET1, a variant of the CEM T lymphoblastoid cell line. Hybrids were screened for their ability to produce cytotoxic lymphokines and interleukin-2 (IL-2) following lectin activation. Five hybrids were identified that released significant levels (greater than 75 units/ml) of cytotoxic activity that was detected on the murine L929 target cells; three hybrids were identified that produced IL-2. Cell surface phenotype of the T cell hybrids did not correlate with their ability to produce these lymphokines. The II-23 hybrid (CD3+;4+) produced an inducible cytotoxic activity that was fully neutralized by anti-lymphotoxin antibodies. The release of lymphotoxin (LT) rapidly attained maximum levels 12 hrs to 24 hrs after lectin activation; IL-2 production was maximum at 48 hrs. The induction of LT and IL-2 release required similar membrane stimulating agents. The phorbol ester, phorbol 12-myristate 13-acetate (PMA) in combination with phytohemagglutinin or concanavalin A, were required for maximal release of LT and IL-2. Anti-CD3 monoclonal antibodies coupled to agarose, but not anti-T200-agarose, induced the release of high levels of LT and IL-2. Soluble anti-CD3 was not sufficient to induce IL-2 or LT release from the II-23 hybrid; however, soluble anti-CD3 combined with PMA was a strong stimulus for lymphotoxin and IL-2 release. The II-23 hybrid also functioned as a cytotoxic T cell line in a 51Chromium release assay. Induction of killer cell function required similar perturbation of Ti/CD3 complex. This human T cell hybrid line offers an inducible model system for the simultaneous study of the molecular events regulating the production of growth inhibitory/cytotoxic (LT) and growth promoting (IL-2) lymphokines and cytotoxic T cell function.

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxins produced by antigen-specific and natural killer-like CTL are dissimilar to classical lymphotoxins.

Several cloned lines of IL 2-dependent human T cells derived from alloantigen, mitogen, or IL 2-stimulated peripheral blood lymphocytes were examined for their surface marker expression, cytolytic activity in a 51Cr-release assay, and capacity to release cytotoxic lymphokines. Thirty cell lines exhibiting either antigen-specific natural killer cell activity or lectin-dependent killer cell function, which expressed either the CD4 or CD8 surface differentiation markers, were capable of producing cytotoxin(s) in response to the lectins phytohemagglutinin and concanavalin A. Cytotoxin activity was detected on the murine L929 target cell in a 16-hr cytotoxicity assay. In contrast, several nonlytic T cell tumor lines failed to produce a soluble cytotoxin. Antibodies capable of neutralizing human alpha-lymphotoxin were completely ineffective in inhibiting the cytotoxin(s) produced by any of the cytotoxic T lymphocytes (CTL) cell lines. Comparative gel filtration and HPLC hydrophobic chromatography of alpha-lymphotoxin and CTL toxin produced by the CTL-830.B2 clone revealed significant differences in their elution profiles. The CTL-produced toxin and alpha-lymphotoxin exhibited similar kinetics of lysis of the L929 target cells, with 50% target cell lysis occurring at 10 hr. These data indicate human CTL produce a cytotoxin(s) antigenically distinct from alpha-lymphotoxin and imply that human cytolytic effector T cells are not the cellular source for the production of human alpha-lymphotoxin. The relationship of alpha-lymphotoxin and CTL toxin production was investigated in unseparated peripheral blood mononuclear cells stimulated with lectins or IL 2 for 1 and 5 days. Anti-alpha-lymphotoxin antibodies were capable of neutralizing only 30 to 50% of the cytotoxic activity in 24-hr supernatants. Cytotoxic activity in supernatants harvested after 120 hr stimulation with PHA or Con A was neutralized 70 to 100%, whereas the toxin(s) released from IL 2-stimulated lymphocytes was only neutralized 30%. These data suggest the observed heterogeneity of cytotoxic lymphokines produced by unseparated mononuclear cells depends in part on the subpopulations of effector cells responding to a given stimulus and the capacity of different subpopulations to produce distinct cytotoxins.

Physical properties of L-asparaginase from Serratia marcescens.

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.