Stephen D. Jordan

Disruption of Th1/Th2 cytokine balance by cocaine is mediated by corticosterone

Cocaine has been shown to affect immune function through the release of corticosterone. Acute administration of both cocaine and corticosterone produces an enhancement of the T-dependent antibody response to sheep erythrocytes. The T-independent antibody response to DNP-ficoll is not enhanced under identical conditions, suggesting that the T-cell is involved as a cellular target. We examined T-helper cell cytokine production following in vivo cocaine administration and found an increase in IL-4 and IL-10; while IL-2 and IFN-gamma were unaffected. The rise in Th2 cytokines is consistent with an enhanced T-dependent antibody response, a measure of humoral immunity. Because previous results showed that the enhancement by cocaine is mediated via corticosterone, the direct effects of corticosterone on Th1/Th2 in vitro cytokine production were investigated. Th1 cytokines, IL-2 and IFN-gamma, were dose-dependently suppressed by corticosterone at physiologic concentrations. In contrast Th2 cytokines, IL-4 and IL-10, exhibited a biphasic dose response curve, whereby an enhancement was observed at low doses, followed by suppression at higher doses. In order to determine the consequences of this apparent shift towards a Th2 response on a Th1 response, we looked at the delayed-type hypersensitivity response to sheep erythrocytes. This measure of cell-mediated immunity was not significantly affected by acute cocaine, however, corticosterone administration resulted in a significant suppression. These results indicate that corticosterone can produce a shift towards a Th2 predominate response, possibly at the expense of Th1-mediated responses.

The role of metabolism in carbon tetrachloride-mediated immunosuppression: in vivo studies

The role of metabolic bioactivation for carbon tetrachloride-mediated suppression of humoral responses was investigated in B6C3F1 mice. Subchronic studies with CCl4 demonstrated that this chlorinated hydrocarbon markedly suppressed T-dependent antibody responses following 7 consecutive days of administration at doses between 500 and 5000 mg/kg. No significant difference in the magnitude of suppression was observed between the ip and oral routes of exposure. Thirty-day ip administration of CCl4 at doses as low as 25 mg/kg also resulted in a significant inhibition of T-dependent antibody responses. The results from both the 7-day and the 30-day studies indicate that a greater than 50% suppression of antibody responses could not be achieved even at doses of CCl4 as high as 5000 mg/kg. In vivo studies utilized the cytochrome P450 competitive inhibitor, aminoacetonitrile (AAN), in an effort to block the effects of exposure to CCl4. Both the hepatotoxicity, as measured by serum glutamic pyruvic transaminase levels, and the suppression of the T-dependent antibody response to sRBC were reversed by treatment with AAN. Conversely, induction of cytochrome P450, by pretreatment of mice with ethanol prior to treatment with CCl4, resulted in the potentiation of the immunosuppressive effects of CCl4. AAN and ethanol administered alone had no effect on antibody responses. In order to assess the effect of CCl4 treatment on cytochrome P450 activity at doses which cause immunosuppression, measurements of total microsomal protein and specific substrate activities were determined. Significant decreases were observed in both total hepatic microsomal protein as well as in aminopyrine N-demethylase activity, aniline hydroxylase activity, and aryl hydrocarbon hydroxylase activity following treatment with CCl4 for 7 days at doses ranging from 5 to 1000 mg/kg. All of the cytochrome P450 parameters that were measured, following CCl4 treatment, demonstrated very flat dose-response curves which appeared to parallel the effects of CCl4 on antibody responses.

Discriminative stimulus properties of toluene in the mouse

Little is known about the nature of the acute intoxication produced by exposure to high concentrations of toluene such as that which occurs with spills and in solvent abusers. The intoxication may be similar to that produced by classic central nervous system depressants such as the barbiturates. To investigate this hypothesis, drug discrimination procedures were used to compare the acute effects produced by toluene and pentobarbital (PB). Mice were trained to discriminate toluene (100 mg/kg, ip) from vehicle in a two-lever task in which responding was under the control of a fixed-ratio 20 (FR20) schedule of food presentation. Generalization tests were conducted after 20-min inhalation exposures to toluene (150-3600 ppm) and 20 min after injections with either PB (5-30 mg/kg) or morphine (3-20 mg/kg). Most mice generalized to inhaled toluene and to PB in a concentration- or dose-related fashion, but not to morphine. These results show that the effects of injected toluene can be established as a discriminative stimulus in mice, and that these stimulus effects are independent of route of administration. Shared discriminative stimulus properties with PB suggest that toluene produces an acute intoxication like that of other classic CNS depressants.

Immunosuppression in adult female B6C3F1 mice by chronic exposure to ethanol in a liquid diet.

The overall objective of these studies was to characterize the effects of ethanol on the immunocompetence of adult female B6C3F1 mice. To obtain a significant suppression in the antibody response to SRBC, splenocytes from untreated mice had to be directly exposed to concentrations of ethanol from 0.3% to 3.0%, or to acetaldehyde at concentrations greater than 0.03%. We do not believe that these results are consistent with a role by a direct effect by either ethanol or its primary metabolite because these concentrations are higher than what could be obtained as reasonable blood levels. For in vivo exposure, we employed a pair-feeding regimen which was based on a liquid diet containing 5% ethanol (v/v) that provided 36% of the caloric intake as ethanol. Our results indicated that there was a definite temporal relationship to the consequent suppression of the antibody response to SRBC in that no effect was observed after 14 days exposure, and that the magnitude of the suppression increased from 18% after 21 days to 70% after 42 days. We also monitored the liver for histopathology and observed that the ethanol-induced liver damage was restricted to steatosis (fatty liver), which was also manifested with time and which was most pronounced after 42 days exposure. In contrast to our results with the in vivo antibody response, we saw no effect on mitogen-induced proliferation by splenocytes from ethanol-treated mice. These results prompted us to measure in vitro antibody responses by splenocytes from ethanol-treated mice. We saw no suppression of the in vitro antibody responses to SRBC, DNP-Ficoll or LPS after any length of exposure to ethanol, and speculated that the basis for the suppression of the in vivo antibody response was an indirect consequence of exposure. We subsequently determined that when normal splenocytes were cultured in 5% serum from ethanol-exposed mice (42-day group), there was a > 80% suppression relative to the serum from the pair-fed controls. As important controls for these studies, we have demonstrated that there was no difference between the responses of normal lymphocytes cultured in 5% normal mouse serum and in 5% serum taken from the pair-fed restricted controls. A determination of the ethanol content in the serum from ethanol-exposed mice (42-day group) indicated that the amount of ethanol present in these cultures was < 0.003%.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in B6C3F1 and DBA/2 mice following single and repeated exposures

Abstract Previous studies have demonstrated that repeated (14 day) administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances the suppression of humoral immunity in DBA/2 (Ah-low responder) mice relative to the effect seen with identical cumulative doses after a single treatment (cumulative doses of 4.2, 14.0, and 42 mg/kg). In the present studies, we have explored this phenomenon further by determining the status of several specific parameters, which might account for the increase in antibody suppression in the DBA/2 strain following repeated TCDD exposures. Included in these studies was the induction of hepatic and splenic microsomal 7-ethoxyresorufin-o-deethylase (EROD; P4501A1) activity and biodistribution of the administered TCDD into various target organs and tissues. Changes in lymphocyte subpopulations within the spleen were also assessed by flow cytometry following both single and repeated dosing. All studies made use of direct comparisons between DBA/2 (Ah-low responder) and B6C3F1 (Ah-high responder) female mice. Results of these studies demonstrate that the enhanced suppression of humoral immunity in DBA/2 mice following repeated exposure to TCDD is not directly associated with increases in liver microsomal EROD activity and does not appear to be correlated with changes in the pattern of biodistribution or amount of TCDD within the liver or spleen of these animals. In contrast, the most significant changes that occurred following repeated dosing in either strain were observed in the levels of microsomal EROD activity and immune cell ratios within the spleen. This effect was characterized as an increase in microsomal EROD activity, and a corresponding reduction in the numbers of a non-B/non-T cell population in the spleen.

Role of metabolism in cocaine-induced immunosuppression in splenocyte cultures from B6C3F1 female mice.

Cocaine has been reported to directly suppress the in vitro immune responses at very high concentrations. In the present study, the possible role of metabolism in cocaine-induced immunosuppression was investigated in splenocyte cultures isolated from B6C3F1 female mice. Since cocaine can be metabolized by both esterase and P-450 monooxygenase, we studied the direct effects of cocaine, benzoylecgonine and norcocaine on the in vitro T-dependent antibody response to SRBC. Direct exposure to cocaine only produced a modest (30%) but nonsignificant suppression of the antibody response, while benzoylecgonine, a primary product of metabolism by the esterase pathway, was devoid of activity. In contrast, direct exposure to norcocaine, the initial product of N-demethylation by the P-450 pathway, produced significant suppression at concentrations greater than or equal to 10 microM. Similar results were observed in studies measuring LPS and Con A mitogenicity. Furthermore, a significant suppression was observed when splenocytes were preincubated for 1 h with 1 mM cocaine in the presence of liver S-9 fractions isolated from phenobarbital-induced mice. Meanwhile, no suppression was obtained when splenocytes were preincubated in the presence of untreated S-9 fractions. To characterize the mechanism of our results, the capacity of both untreated and phenobarbital-induced microsomes to produce formaldehyde from cocaine was compared. The N-demethylation of cocaine was NADPH-dependent and phenobarbital-induced microsomes produced approx. 6-times higher amounts of formaldehyde, indicating a greater portion of cocaine could be metabolized through the P-450 pathway to its toxic metabolites. Finally, because benzoylecgonine shares with cocaine the presence of a methyl group on the tropane nitrogen, we also compared the ability of N-demethylation from cocaine and benzoylecgonine in mouse liver microsomes. Our results indicated that benzoylecgonine could not be demethylated as determined by a failure to generate any formaldehyde. These results offer further support that the N-demethylation pathway is a critical step to cause its immunotoxicity.

Cocaine and Immunocompetence: Possible Role of Reactive Metabolites

Although drug abuse has been widely discussed as a possible co-factor in the onset and/or progression of AIDS, available literature is not consistent with cocaine use being associated with marked effects on immunocompetence. The initial objective of the present investigation was to determine the effects of cocaine on immunocompetence following direct addition to cultured splenocytes from female B6C3F1 mice. Because cocaine is a potent local anesthetic, we used procaine as a comparative control in these studies. As discussed below, the results from these studies were not consistent with a role by direct immunomodulatory actions of cocaine. We speculated that the in vivo effects of cocaine on the immune system may be an indirect consequence of exposure. The overall purpose of this study was to begin to examine possible indirect mechanism(s) for the effects of cocaine on the immune system, with an emphasis on a role by reactive metabolites. Cocaine is metabolized to norcocaine and, ultimately to more reactive intermediates, via a minor metabolic pathway mediated by the cytochrome P-450 system. The generation of these metabolites is associated with hepatotoxicity, which is both sex- and strain-dependent (i.e., dependent on the relative activity of the P-450 system). To provide evidence for a role by P-450-dependent metabolites in cocaine’s actions on the immune system, we have compared the effects of subchronic (14 day) exposure to cocaine on the T-dependent antibody response in female B6C3F1 mice, female DBA/2 mice and male B6C3F1 mice.

Role of hydrocortisone in dimethylnitrosamine-induced suppression of antibody response in the mixed culture of murine hepatocytes and splenocytes.

We have previously reported that the hormone-supplemented culture condition for primary hepatocytes is required in dimethylnitrosamine (DMN)-induced suppression of antibody response to sheep erythrocytes in the mixed cultures of murine hepatocytes and splenocytes. In the present investigation, the components of the hormone supplement were screened to identify the component(s) responsible for the increased ability of hepatocytes to activate DMN to its immunosuppressive form. The presence of hydrocortisone in the hepatocyte culture media had the primary role in DMN activation in the co-culture system. Other components of the hormone supplement showed slight or no effects. The effects of hydrocortisone were clearly confirmed through the dose-response study of both DMN and hydrocortisone. To characterize whether the effect of hydrocortisone is glucocorticoid-dependent we tested another potent glucocorticoid, dexamethasone (DEX), and determined if the activity by hydrocortisone could be reversed by RU 486. It was found that hepatocytes cultured in DEX-containing media could also activate DMN to its immunosuppressive form. However, the activity by hydrocortisone to increase DMN-induced immunosuppression was not reversed by RU 486. Furthermore, a possible direct interaction between DMN and hydrocortisone was ruled out. Finally, we transferred DMN-pre-treated culture supernatant from hepatocytes to spleen cell cultures, and found that the metabolite of DMN was very unstable, and that DMN-induced suppression of T-dependent antibody response was hepatocyte-dependent. The present results suggest that glucocorticoids, including hydrocortisone and DEX, in hepatocyte culture media can affect DMN-induced immunosuppression in the hepatocyte/splenocyte co-culture system via a pathway which does not appear to be related to the glucocorticoid receptor.

Color morphs of the crayfish Orconectes propinquus (Girard) (Decapoda, Cambaridae); a preliminary study of agonistic interactions

Interspecific aggressive competition in crayfish has received some attention, since it apparently affects the dispersal of crayfish species (Eberly, 1960; Penn & Fitzpatrick, 1962, 1963; Bovbjerg, 1970; Berrill, 1978; Jezerinac, unpubl.; Momot et al., 1978). It may also underlie the displacement of one species by another (Berrill, 1978; Capelli, 1982; Capelli & Magnuson, 1983). There is no comparable literature on intraspecific competition in crayfish. The predominance of one of three color morphs of Orconectes propinquus in Lake Sim coe (Ontario, Canada), which has apparently appeared rather recently (Dunham et al., 1979; Jordan, unpubl.; Jordan & Dunham, 1981; Jordan et al., 1986), raises the question of whether aggressive competition might have been a factor in its success. In this study we examine intermorph differences in aggression and competitive success in food-motivated agonistic encounters.