Cytokine mRNA profiles for isocyanates with known and unknown potential to induce respiratory sensitization.

Isocyanates are low-molecular-weight chemicals implicated in allergic asthmatic-type reactions. Identification of chemicals likely to cause asthma is difficult due to the lack of a validated test method. One hypothesis is that differential cytokine induction (Th1 versus Th2 profiles) in the draining lymph node following dermal application can be used to identify asthmagens and distinguish them from contact allergens. In this study, we compared the cytokine mRNA profiles of six chemicals: toluene diisocyanate (TDI), diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), p-tolyl(mono)isocyanate (TMI), and meta-tetramethylene xylene diisocyanate (TMXDI). Whereas TDI and MDI are well-known respiratory sensitizers, documentation for HMDI, IPDI, TMI, and TMXDI is limited, but suggests that HMDI and IPDI may have respiratory sensitization potential in humans and TMI and TMXDI do not. Following dermal exposure of BALB/c mice, all six isocyanates induced cytokines characteristic of a Th2 response. Although LLNAs suggested that the doses chosen for the RPA were immunologically equivalent, the isocyanates tested differentiated into two groups, high responders and low responders. However, two of the low responders (TMI and TMXDI) were further tested and induced higher levels of Th2 cytokine message than dinitrochlorobenzene (not an asthmagen). Further study of these chemicals is needed to determine whether the Th2 cytokine responses observed for these low responders is predictive of asthmagenic potential or represents an insufficient signal.

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides.

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells-interleukin (IL)-4, 10, and 13-respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-gamma). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification.

Oral bioaccessibility of dioxins/furans at low concentrations (50-350 ppt toxicity equivalent) in soil.

Animal studies have indicated that the oral bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in environmentally contaminated soil could range from 0.5 to 60%. To estimate the oral bioavailability of TCDD, and the 16 other 2,3,7,8-substituted dioxin/furan congeners, this study used a physiologically based extraction test, designed around the anatomic and physiologic characteristics of the human digestive tract. This test measures the fraction of dioxins/furans in soil that would be solubilized in the gastrointestinal tract (i.e., that would be bioaccessible) and therefore available for absorption. Eight soils from Midland, MI, were evaluated in this study and exhibited TCDD concentrations of 1.7-139 pg/g (ppt) and total TEQ concentrations of 6-340 ppt. Bioaccessibility of dioxins/furans from these soils ranged from 19 to 34% averaged across the 17 2,3,7,8-substituted dioxin/furan congeners), with an average of 25%. The total organic carbon in these soils was low--ranging from 1 to 4%--particularly for the soil series from which they were collected. Bioaccessibility of individual congeners did not appear to be correlated with degree of chlorination. Even though these dioxin/furan concentrations are much less than studied previously, these results are consistent with those from animal studies at other sites, which have generally yielded values of 20-60% relative bioavailability for TCDD in soil.

Developmental immunotoxicology and risk assessment: a workshop summary.

A workshop entitled 'Developmental Immunotoxicology and Risk Assessment' was held on 12-13 June 2001, in Washington, DC. The workshop was organized jointly by the Immunotoxicology Technical Committee (ITC) of the International Life Sciences Institute's (ILSI) Health and Environmental Sciences Institute (HESI) with input from the U.S. Environmental Protection Agency (EPA). Growing public concern that early exposure of the developing immune system to immunotoxic compounds may cause significant or persistent postnatal immunosuppression prompted the workshop. The main goal of the workshop was to examine scientific questions that underlie developmental immunotoxicity tests and the interpretation of the results as they relate to human risk assessment. A second goal was to provide a framework, based on current scientific knowledge, for the development of meaningful testing guidelines. The workshop focused on a series of questions that included how to address critical windows of exposure, how to develop and apply more predictive endpoints, does early chemical exposure cause transient or permanent effects on the immune system, as well as other related questions. On the first day, experts were invited to give scientific presentations relating to comparative developmental immunology, models of immunosuppression, and the regulatory aspects of developmental immunotoxicology. The second day was devoted to a panel discussion that included all the speakers as well as meeting participants, which attempted to answer each of the specific questions raised at the workshop. In general, it was acknowledged that there are a variety of techniques available for assessing immunosuppression in adult animal models, but there is uncertainty about how to apply these to a developing animal, especially if the goal is to have some standard procedure that can be applied for regulatory risk assessment. It was pointed out that although we know a lot about the developing immune system of individual species, we do not know how to relate the significance of drug or chemical effects on these systems in terms of human hazard. Overall, the panel deemed the area of developmental immunotoxicity to be still in its infancy and outlined strategies that could lead to the development of standard practices.

Cytokine profiling for chemical sensitizers: application of the ribonuclease protection assay and effect of dose.

Exposure to chemicals in domestic and occupational settings may contribute to increases in asthma and allergy. Airway hypersensitivity (AHS) is T helper-2 (Th2) cell associated, whereas contact hypersensitivity (CHS) is T helper-1 (Th1) cell associated. The distinct cytokine profiles produced by these cells may provide a means of distinguishing respiratory sensitizers from contact sensitizers. In this study, female BALB/c mice were exposed twice on the flanks and three times on the ears using the airway sensitizer trimellitic anhydride (TMA) or the contact sensitizer dinitrochlorobenzene (DNCB). At various times following exposure, total mRNA was extracted from draining lymph node cells and cytokine mRNA profiles analyzed using a multiprobe ribonuclease protection assay (RPA). The Th2 cytokines IL4, IL10, and IL13 were significantly increased in response to TMA compared to DNCB, with optimal detection occurring 14 days following initial exposure. To determine its effect, dose was varied in flank exposures, ear exposures, or both simultaneously. When dose was varied during flank exposures only, TMA induced higher levels of Th2 cytokines than DNCB at all doses tested. DNCB did not induce Th1 cytokines at any dose tested. Variation of TMA dose during both exposures similarly induced Th2 cytokines. Dose only appeared to be a factor when TMA concentration was varied during the ear exposures alone. Thus, these studies suggest that quantitative differences in Th2 responses between TMA and DNCB may be demonstrated over a wide range of doses and these differences may be detected by RPA following dermal exposure to these sensitizers.

Putative link between transcriptional regulation of IgM expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the aryl hydrocarbon receptor/dioxin-responsive enhancer signaling pathway.

The B-cell, a major cellular component of humoral immunity, has been identified as a sensitive target of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The actual molecular mechanism responsible for the immunotoxic effects produced by TCDD is unclear; however, many of the biological effects produced by TCDD are thought to be mediated by the aryl hydrocarbon receptor (AhR). Using the CH12.LX B-cell line, the present studies show that inhibition of mu gene expression and IgM protein secretion by polychlorinated dibenzo-p-dioxin congeners follow a structure-activity relationship for AhR binding. Furthermore, these effects may be mediated by the two dioxin-responsive enhancer (DRE)-like sites that were identified within the Ig heavy chain 3'alpha-enhancer. Electrophoretic mobility shift assay-Western analysis demonstrated TCDD-induced binding of the AhR nuclear complex to both DRE-like sites as well as TCDD-induced binding of several nuclear factor-kappa B/Rel proteins to a kappa B site, which overlaps one of the DRE-like sites. Interestingly, kappa B binding in the AhR-deficient BCL-1 B-cells was also induced by TCDD, demonstrating an AhR-independent effect of TCDD on kappa B binding. Taken together, these results support an AhR/DRE-mediated mechanism for TCDD-induced inhibition of IgM expression.

Influence of p53 zygosity on select sperm parameters of the mouse.

The influence of p53 gene zygosity on select parameters of mouse sperm was investigated by employing knock-out animal models. The background incidence of sperm shape abnormalities, total sperm count, and DNA double strand breaks were determined in p53 nullizygous (-/-) and heterozygous (+/-) mice and these estimates were compared to the corresponding measures in p53 wild-type (+/+) and the inbred C57Bl6 mouse strains. There were no qualitative differences in the incidence of sperm shape abnormalities and sperm counts regardless of p53 zygosity. However, the number of DNA double strand breaks, as measured by the comet assay, were significantly lower in the p53 knock-out mice. This apparent decrease was interpreted to be the result of a possible change in DNA-protein and/or DNA-DNA cross-linking in the germ cells of the knock-out mice. These data show that there is no evidence of increased incidence of gross alterations in spermatogenesis (no significant loss in sperm production nor any increase in the proportion of abnormal sperm produced) in knock-out mice deficient or absent in p53 protein; however, there appear to be changes at the genomic level where the degree of cross-linking was apparently elevated in DNA from p53 nullizygous and heterozygous mice.

Lipopolysaccharide activation of murine splenocytes and splenic B cells increased the expression of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator.

These studies characterized the profile of AhR and ARNT expression in primary splenocytes and purified splenic B cells after cellular activation with lipopolysaccharide (LPS). LPS treatment of mouse splenocytes markedly increased the magnitude of both AhR and ARNT steady state mRNA expression. AhR mRNA expression peaked at 8 hr post-LPS activation and was increased by approximately 5-fold compared with freshly isolated splenocytes (i.e., time 0). ARNT mRNA expression began to increase at 8 hr postactivation, peaked at approximately 48 hr and was increased by approximately 4-fold when compared with nonactivated splenocytes at time 0. Western blotting also demonstrated an increase in the relative magnitude of both the AhR and ARNT proteins in LPS activated splenocytes. Likewise, the presence of the AhR, ARNT and cytochrome P450IA1 (CYP1A1) proteins were also detected in purified primary splenic B cells, and the magnitude of protein expression was enhanced in LPS activated splenic B cells at 12 and 24 hr relative to time matched controls for each of these proteins. In summary, these findings suggest that on LPS activation the magnitude of AhR and ARNT mRNA and protein increases in both splenocytes and purified primary splenic B cells. Moreover, because the increase in the relative magnitude of CYP1A1 protein in response to LPS occurred in the absence of exogenous AhR ligand, these results suggest that B-cell activation is sufficient to induce AhR nuclear translocation and binding to dioxin-responsive elements in the promoter region of AhR-responsive genes.

Aryl hydrocarbon receptor-dependent suppression by 2,3,7, 8-tetrachlorodibenzo-p-dioxin of IgM secretion in activated B cells.

The immune system has been identified as a sensitive target for the toxic effects produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Furthermore, the B cell has been identified as a sensitive cellular target of TCDD by previous cell-type fractionation studies from this laboratory. The mechanism responsible for the immunotoxic effects produced by TCDD is unclear; however, many of the biological effects of TCDD are thought to be mediated by the aryl hydrocarbon receptor (AhR). Here, we describe two B cell lines that differ considerably in their expression of the AhR and in their sensitivity to TCDD. Our results demonstrated a marked expression of the AhR protein in the CH12.LX B cell line but not in the BCL-1 B cell line. Transcripts for the AhR were not detected by reverse transcriptase-polymerase chain reaction in the BCL-1 cells. The AhR nuclear translocator (ARNT) protein was highly expressed in both cell lines. In addition, the AhR and ARNT are functional in CH12.LX cells as demonstrated by TCDD-induced CYP1A1 induction. TCDD did not induce CYP1A1 in BCL-1 cells. Furthermore, TCDD treatment resulted in suppression of lipopolysaccharide (LPS)-induced IgM secretion in CH12.LX cells. Conversely, TCDD-induced inhibition of IgM secretion was not demonstrated in LPS-stimulated BCL-1 cells, implicating a role for the AhR in the inhibition of B cell effector function. LPS-induced differentiation of the CH12.LX cells also resulted in a marked induction of Ahr expression which was not induced in LPS-stimulated BCL-1 cells. These studies have implicated the AhR as a critical factor in TCDD-induced inhibition of IgM secretion and have demonstrated an induction of AhR gene and protein expression after B cell activation.

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

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.

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.

Role of corticosterone in the enhancement of the antibody response after acute cocaine administration.

A model has been developed in which acute cocaine administration results in an enhanced T-dependent antibody response to sheep erythrocytes. This enhancement occurs when cocaine (30 mg/kg, twice in 1 day) is administered 1 or 2 days before sensitization with antigen, in mice older than 16 wk. Acute cocaine has been shown to elicit a rise in serum corticosterone, and the administration of exogenous corticosterone, under similar conditions as cocaine, also results in a similar immunoenhancement. Further evidence in support of a role by corticosterone is the lack of an enhancement in adrenalectomized mice and the ability of alpha-helical corticotropin releasing factor to block the enhancement by cocaine. The role of concomitant epinephrine release from the adrenal was addressed by adrenal demedullation. Eliminating epinephrine, but not corticosterone release, had no effect on the cocaine-induced immunoenhancement. The evidence presented provides support for a major role by corticosterone in mediating cocaine's effects on at least one measure of immune function, the T-dependent antibody response.

Characterization of the role played by antigen challenge in the suppression of in vivo humoral immunity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The goal of these studies was to characterize the role played by antigen challenge in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunosuppression. The effects of single exposure to TCDD (4.2, 14, and 42 microg/kg) in B6C3F1 mice on the reverse plaque assay (RPA; no sensitization) and the sheep red blood cell (SRBC) antibody response were compared. While the RPA was suppressed in a dose-dependent fashion with significance at the two highest doses, a much more dramatic effect was noted with the primary anti-SRBC response: a suppression was noted at the lowest dose, which was comparable to that observed with the highest dose in the RPA. Subsequent studies compared the RPA in B6C3F1 (Ah-high responder) and DBA/2 (Ah-low responder) mice after both single and repeated exposure to identical cumulative doses of TCDD (4.2, 14, and 42 microg/kg). The repeated exposures consisted of 14 consecutive daily treatments of 0.3, 1.0, and 3.0 microg/kg. The results indicated only a slight difference in the effects of TCDD in the two strains after single exposure, and even less difference after repeated exposure. Moreover, administering TCDD on different days relative to the SRBC challenge indicated a suppression in both strains when given 1, 2, or 3 days before antigen challenge, on the day of antigen challenge, or 1 or 2 days after antigen challenge. The only day of administration where the suppression was attenuated was 3 days after antigen challenge. These results confirm an important relationship between antigen challenge and TCDD exposure on immunosuppression.

Leukocyte activation induces aryl hydrocarbon receptor up-regulation, DNA binding, and increased Cyp1a1 expression in the absence of exogenous ligand.

The aryl hydrocarbon receptor (AhR) functions as a transcription factor after ligand binding by halogenated aromatic hydrocarbons. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic halogenated aromatic hydrocarbon, is dependent on binding to the AhR to mediate a broad range of toxic effects. Immune suppression is one of the most sensitive sequela associated with TCDD exposure, yet, paradoxically, resting leukocytes express a relatively low amount of AhR. Here we report that activation of leukocytes produced a 6-fold increase in AhR steady state mRNA levels and a concordant increase in AhR protein expression. Furthermore, leukocyte activation induced AhR translocation, DNA binding to a dioxin response element, and CYP1A1 transcription in the absence of TCDD. Activated leukocytes exhibited an even greater enhancement of dioxin response element binding by the AhR in the presence of TCDD than in the absence of TCDD. These studies suggest that the mechanism responsible for the sensitivity of immunocompetent cells to TCDD may be directly associated with a marked increase in AhR expression, which accompanies leukocyte activation.

Evaluation of sex- and strain-dependency of cocaine-induced immunosuppression in B6C3F1 and DBA/2 mice.

The objective of these studies was to determine if the immunotoxic effects of cocaine in mice are sex- and strain-dependent, a profile of activity previously described for cocaine-induced hepatotoxicity. The latter effect has been attributed to differences in the metabolism of cocaine by the cytochrome P-450 system. Subchronic, (14-day) in vivo administration of cocaine to female B6C3F1 mice showed a significant decrease (80%) in the T-dependent primary antibody response only at 80 mg/kg, although exposure to 60 mg/kg produced only a 20% decrease. In contrast, exposure to 60 mg/kg cocaine in female DBA/2 mice produced a significant decrease of 50%. An even greater effect was observed in male mice where exposure to 40 mg/kg cocaine produced > 50% decreases in both B6C3F1 and DBA/2 mice. Similar results were obtained when male mice were only exposed for 7 days. Confirmation that hepatotoxicity occurred with a similar profile of sex- and strain-dependency was obtained in parallel studies when serum chemistries were measured. The immunosuppressive activity of cocaine in female B6C3F1 mice was markedly increased when mice were pretreated with phenobarbital, a cytochrome P-450 inducer. These results extend our previous studies that indicated that cocaine-induced immunosuppression occurs under conditions that are consistent with a mechanism mediated through metabolism by the cytochrome P-450 pathway.

Identification of functional aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in murine splenocytes.

The objective of the present studies was to determine whether the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein are present and functional in B6C3F1 (C57BL/6 x C3H) mouse splenocytes. Northern analysis of poly(A) RNA isolated from splenocytes revealed transcripts of approximately 6.6 kb which hybridized to the AhR complementary DNA (cDNA) probe. Anti-AhR antibodies identified two major cytosolic forms of the AhR in splenocytes, approximately 95 and 104 kDa, corresponding to the codominately expressed Ahrb alleles in the B6C3F1 mice. Northern analysis utilizing an oligomer probe for ARNT identified three messenger RNA (mRNA) transcripts, approximately 5.6, 2.0, and 1.1 kb, in spleen which was consistent with the banding pattern observed in the B6C3F1 mouse liver. Western blotting confirmed the presence of the approximately 87 kDa ARNT protein in splenocytes. Protein quantitation by slot blot analysis demonstrated approximately 2.0-fold more AhR in liver than in splenocytes. Interestingly, ARNT was approximately 2.4-fold more abundant in splenocytes than in liver. Consistent with these results, comparison by quantitative reverse transcriptase-polymerase chain reaction analysis of AhR and ARNT transcripts in liver and splenocytes demonstrated approximately 2.3-fold more AhR transcripts in liver than in splenocytes and approximately 3.2-fold more ARNT transcripts in splenocytes than in liver. In addition, comparisons between AhR and ARNT transcripts isolated from the liver and splenocytes indicated a greater number of ARNT transcripts as compared with AhR in both preparations. TCDD treatment of splenocytes induced binding of the AhR nuclear complex to the dioxin-responsive enhancer (DRE) as detected by the electrophoretic mobility shift assay. These findings confirm that the AhR and ARNT are present in mouse splenocytes and are capable of binding to the DRE.

Alternations in splenocyte and thymocyte subpopulations in B6C3F1 mice exposed to cocaine plus diazinon.

Our laboratory has proposed a working model which asserts that cocaine's effects on immunity are mediated by reactive metabolites generated by the cytochrome P-450 system. This metabolic pathway is normally a minor one in humans, but takes on significance when metabolism of cocaine by the P-450 system is increased, as may occur with excessive alcohol consumption (enzyme induction) or after exposure to organophosphate pesticides (esterase inhibition). Results from our laboratory demonstrate that cocaine exerts its most dramatic effects on immunocompetence when administered to mice that have been pretreated with diazinon, an organophosphate esterase inhibitor. Most notably, we observed decreases in both the splenic T-dependent antibody response to sheep erythrocytes and the splenic T-independent antibody response to DNP-ficoll and a dramatic thymic atrophy in mice exposed to cocaine + diazinon, which were not seen in mice exposed to cocaine alone. The primary objective of the present investigation was to determine whether the exposure conditions used to produce the changes noted above are also capable of causing changes in lymphocyte cell types by use of flow cytometric analysis. Administration of cocaine after pretreatment with diazinon only modestly affected splenic lymphocyte subsets, which caused a slight decrease in the number of B cells. No effect was observed in the macrophage, T-helper or T-suppressor subpopulations in the spleen. These results suggest that changes in splenocyte subpopulations induced by cocaine + diazinon cannot account for the suppression of the antibody response. In contrast, all T-cell subsets in the thymus were decreased significantly, with immature double-positive thymocytes suffering the greatest loss in cell number. These results indicate that T cells, especially immature thymocytes located in the thymus, are sensitive to effects associated with the combined treatment of cocaine + diazinon.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elevates basal B-cell intracellular calcium concentration and suppresses surface Ig- but not CD40-induced antibody secretion.

Humoral immune responses to either T-independent or T-dependent antigens have previously been shown to be suppressed by the halogenated aromatic hydrocarbon environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through direct action on B-lymphocytes. To better understand the molecular nature of the TCDD-induced suppression of B-cell differentiation, we studied the effects of TCDD using in vitro models of T-independent (antibody directed against surface IgM) and T-dependent [activated T-helper (TH) cells bearing CD40 ligand] B-cell maturation. We report here that TCDD suppresses murine B-cell IgM secretion induced by either soluble or insolubilized anti-IgM plus lymphokines but does not affect IgM secretion stimulated by activated T(H)-cells and lymphokines. Because soluble or insolubilized anti-IgM but not fixed, activated TH-cells was found to trigger increases in intracellular ionized calcium in isolated B-cells, the effect of TCDD exposure on B-cell intracellular calcium concentration and mobilization was examined. In comparison to the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, which induces an immediate rise in resting [Ca2+]i of up to four- to fivefold, TCDD treatment did not produce a rapid increase in [Ca2+]i but did result in an elevation of basal levels of nearly the same magnitude 18 hr postexposure. However, anti-IgM-induced calcium transients were similar in the presence or absence of TCDD. TCDD exposure also produced instability of the calcium concentration curve, with the observed elevation of basal intracellular calcium occurring after both in vitro and in vivo treatment paradigms. The immunomodulatory profiles of activity of TCDD and thapsigargin on the B-cell proliferative response to PMA plus ionomycin differ, suggesting that the kinetics of calcium release by these compounds dictates the overall effect on the responding B-cell. Taken together, the data indicate that TCDD elevates resting intracellular calcium levels in murine B-cells and may selectively inhibit calcium-dependent signaling pathways linked to surface Ig.

Immunosuppression induced by acute exposure to cocaine is dependent on metabolism by cytochrome P-450.

The primary objective of this paper was to characterize the role of metabolism in immunosuppression by acute exposure to cocaine. beta-Ionone has been used to study the role of metabolism in hepatotoxicity associated with acute exposure to cocaine, and was shown to produce a greater effect than other cytochrome P-450 (P-450) inducers. When beta-ionone (600 mg/kg s.c.) was pretreated 72 and 48 hr before the acute administration of cocaine (30 mg/kg i.p.) in B6C3F1 female mice, the antibody response to sheep red blood cells was significantly suppressed. Exposure to cocaine alone produced little or no suppression. The immunosuppression in cocaine + beta-ionone-treated mice was accompanied by a decrease in thymus weight and an increase in liver weight. Administration of metyrapone (40 mg/kg i.p.) 30 min before cocaine administration (40 mg/kg) blocked completely the suppression of the antibody response by cocaine in beta-ionone-pretreated mice. The reversal by metyrapone was additional evidence that a P-450 pathway was the critical metabolic pathway of cocaine to be immunosuppressive, and the inhibitory effect of metyrapone on cocaine N-demethylase was confirmed in liver microsomes. The inductive effects of beta-ionone were also characterized further. Cocaine N-demethylase activity was significantly induced by beta-ionone. The induction of P-450IIB1/2, the only isozyme shown previously to be associated with the hepatotoxicity by cocaine, was demonstrated by Western immunoblotting to be induced by beta-ionone at doses as low as 300 mg/kg; but was less than the induction associated with phenobarbital. Studies confirmed that acute exposure to cocaine also was immunosuppressive in phenobarbital-pretreated mice. Taken together, our present results suggest that the immunosuppression by acute exposure to cocaine is associated with the increased metabolism of cocaine to toxic metabolites by P-450, probably P-450IIB1/2, as demonstrated previously for its hepatotoxicity.