TCDD-Mediated Suppression of the In Vitro Anti ... - Oxford Journals

Download PDF
3 downloads 0 Views 174KB Size Report
Comment
light chain, immunoglobulin m heavy chain, and immunoglobulin. J-chain were .... with Dunnett's post hoc test to compare each treatment group to the vehicle- treated controls ..... T lymphocytes may be able to rescue the B lymphocytes and.
TOXICOLOGICAL SCIENCES 107(1), 85–92 (2009) doi:10.1093/toxsci/kfn223 Advance Access publication October 22, 2008

TCDD-Mediated Suppression of the In Vitro Anti-Sheep Erythrocyte IgM Antibody Forming Cell Response is Reversed by Interferon-Gamma Colin M. North,*,†,1 Byung-Sam Kim,‡,1 Neil Snyder,§ Robert B. Crawford,*,† Michael P. Holsapple,{ and Norbert E. Kaminski*,†,2 *Department of Pharmacology and Toxicology; †Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824; ‡Department of Biological Sciences and Immunomodulation Research Center, University of Ulsan, Ulsan, South Korea; §Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298; and {ILSI Health and Environmental Sciences Institute, Washington, DC 20005 Received August 1, 2008; accepted October 9, 2008

Suppression of humoral immune responses by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) has been well established to require the aryl hydrocarbon receptor; however, the downstream mechanisms for this immunotoxic response remain poorly understood. Based on evidence demonstrating that primary hepatocytes pretreated with interferon-gamma (IFN-g) exhibited decreased induction of cytochrome P450 1A1 (CYP1A1) by TCDD, and that serum factors alter the sensitivity of the in vitro T-cell–dependent IgM antibody forming cell (AFC) response, it was hypothesized that IFN-g attenuates suppression of humoral immune responses by TCDD. In fact, concomitant addition of IFN-g (100 U/ml) produced a concentration-related attenuation of TCDD-mediated suppression of the anti-sheep erythrocyte (anti-sRBC) IgM AFC response. Time-of-addition studies performed by adding 100 U/ml IFN-g at 0, 1, 2, 4, 12, 24, 48, and 72 h post-TCDD showed that suppression of the AFC response was prevented only when IFN-g was added within 2 h of TCDD treatment. mRNA levels of the IgM components, immunoglobulin k light chain, immunoglobulin m heavy chain, and immunoglobulin J-chain were significantly decreased by TCDD treatment, an effect that was completely reversed by IFN-g (100 U/ml) cotreatment. Further studies showed that IFN-a, IFN-b, and IFN-g significantly attenuate TCDD-induced increases in CYP1A1 mRNA levels to varying degrees, but concentrations as high as 1000 U/ml of type I IFNs did not reverse the effect of TCDD on the anti-sRBC IgM AFC response. In summary, IFN-g prevents TCDD-mediated suppression of the IgM AFC response in a concentration- and time-related manner by altering transcriptional effects associated with TCDD treatment. Key Words: TCDD; immunotoxicology; IgM, in vitro; IFN-c.

Halogenated aromatic hydrocarbons, which include polychlorinated biphenyls, polychlorinated dibenzofurans, and polychlorinated dibenzo-p-dioxins, are ubiquitous organic environmental contaminants that are resistant to degradation, 1

These authors contributed equally to this work. To whom correspondence should be addressed at 315 National Food Safety & Toxicology Center, Michigan State University, East Lansing, MI 48824. Fax: 517-432-3218. E-mail: [email protected] 2

difficult to remediate in the environment, and bioaccumulate in the food web (Kelly et al., 2007). In animal studies the 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), has been shown to be a carcinogen, endocrine disrupter, teratogen, and immunotoxic (Anonymous, 1997). Extensive investigation by numerous laboratories has widely established that the immune system in rodent models is highly sensitive to suppression by TCDD (reviewed in Kerkvliet, 2002). Among the most sensitive immune responses to suppression by TCDD are primary humoral immune responses, as observed in vivo and in vitro (reviewed in Holsapple et al., 1991). Immune suppression by TCDD, and other structurally similar halogenated aromatic hydrocarbons, is critically dependent on the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that heterodimerizes with the AHR nuclear translocator (ARNT), which is capable of altering the expression of a battery of genes, including cytochrome P450 1A1 (CYP1A1) and components of IgM antibodies including immunoglobulin j light chain (Igj), immunoglobulin l heavy chain (Igl), and immunoglobulin J-chain (IgJ) (Yoo et al., 2004). Types of interferon (IFN) can be broadly classified into type I IFN, such as IFN-a and IFN-b, and type II IFN, specifically IFN-c. IFN-a and IFN-b act through the IFN-a/b receptor, whereas IFN-c signaling begins with activation of the IFN-c receptor. Both types of IFN receptors induce phosphorylation of Janus-activated kinases, and subsequent phosphorylation of family of transcription factors known as signal transducer and transactivator of transcription (STAT) to alter the cellular gene expression program. Differential responses to IFN are partially explained by differences in STAT isoforms generated following receptor activation, with STAT1/2 heterodimers resulting from type I IFNs and STAT1 homodimers resulting from type II IFN. IFNs play a key role in host resistance to viral pathogens, acting in both a cell type and stimulus specific manner for lymphocytes. IFN-c is classically considered a Th1 type cytokine, promoting the T-lymphocyte response toward a cell-mediated response. However, B lymphocytes also respond to IFN-c, albeit in a complex fashion, demonstrating

Ó The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]

86

NORTH ET AL.

inhibition or enhancement of the IgM response depending on the activation stimulus. In vitro sheep erythrocyte (sRBC)– activated B lymphocytes, unlike anti-Ig stimulated B lymphocytes, require the presence of both interleukin (IL)-2 and IFN-c for a robust antibody forming cell (AFC) response (Jelinek et al., 1986), but IFN-c antagonizes the enhancing action of IL-4 on humoral responses (Vitetta et al., 1989). IFN-c can enhance IgM production in CpG-activated B lymphocytes, but inhibits IgM secretion in LPS-activated B lymphocytes (Yi et al., 1996). The complexity of B lymphocyte responses is further demonstrated by IFN-c promotion of class switch recombination to IgG2a (Stevens et al., 1988). Studies utilizing primary hepatocytes have demonstrated that IFN-c can reduce TCDD-mediated increases in CYP1 activity (Jeong et al., 1993), and taken in conjunction with the knowledge that serum factors can prevent TCDD-induced disruption of in vitro IgM AFC responses (Morris et al., 1994), the possibility that IFN-c may prevent effects of TCDD in other cell types such as splenocytes was apparent. The objective of the present investigation was to test the hypothesis that IFN-c treatment attenuates TCDD-mediated suppression of the primary IgM response in splenocytes. Our findings demonstrate that IFN-c, but not IFN-a or IFN-b, reversed TCDD-mediated suppression of the anti-sRBC IgM AFC response in a concentration- and time-related manner by partially or completely preventing TCDD-induced alterations in mRNA levels of CYP1A1, Igj, IgH, and IgJ.

air incubator for the first 4 h, then placed in a stainless steel box that was flushed with a custom blood gas mixture (10% carbon dioxide, 7% oxygen, 83% nitrogen) and pressurized to 5.5 pounds per square inch. For IFN-c additions that occurred at 12, 24, 48, and 72 h the box was slowly pressurized before addition of IFN-c, then returned to the stainless steel box, slowly repressurized, and returned to the incubator. RNA isolation and cDNA generation. Total RNA was isolated using SV Total RNA Isolation kits (Promega, Madison, WI) according to manufacturer’s protocol. Total RNA was quantified using a Nanodrop ND-1000 spectrophotometer and concentrations adjusted by dilution so all reverse transcription reactions contained equal amounts of total RNA. Reverse transcription was performed using Invitrogen Superscript RT (Carlsbad, CA) using anchored oligo dT primers according to manufacturer’s protocol. Quantitative real-time RT-PCR. Detailed description of real-time RTPCR procedures and conditions used have been previously published (Boverhof et al., 2004). All real-time RT-PCR was performed on an Applied Biosystems 7000 PRISM Thermocycler (ABI, Foster City, CA) using SYBR Green as the detection reagent. Primers were designed using Primer3 software (Rozen and Skaletsky, 2000). Conditions used were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 65°C for 1 min. Individual genes were quantified by comparison with a standard curve generated from serial dilution of known quantities of PCR product and normalized to the expression of b-actin. Sequences for PCR primers used to detect CYP1A1 and b-actin can be found in (Boverhof et al., 2004). Igl, Igj, and IgJ PCR primer sequence information was previously published in (Yoo et al., 2004). Statistical analysis. Graphpad Prism 4.00 (Graphpad Software, San Diego, CA) was used for all statistical analysis. One-way ANOVA was used with Dunnett’s post hoc test to compare each treatment group to the vehicletreated controls, with the exception of the CYP1A1 time course in which Tukey’s post hoc test was used to further compare the TCDD-treated group to the TCDD and IFN-c cotreated group. Grubb’s outlier test was used to test for significant outliers, which were then excluded from analysis. No more than one outlier was removed from any single experiment.

MATERIALS AND METHODS Animals. Virus-free, female B6C3F1 mice of at least 12 weeks of age (National Cancer Institute) were housed under pathogen free conditions and provided ad libitum access to water and food (Purina Certified Laboratory Chow, Ralston Purina, St Louis, MO). Animal holding rooms were maintained at 20°C–25°C and 40–60% humidity with a 12-h light/dark cycle. Mice were used in accordance with guidelines set forth by the Michigan State University Institutional Animal Care and Use Committee. Chemicals and culture reagents. Unless otherwise stated all chemicals and reagents were purchased from Sigma-Aldrich (St Louis, MO). TCDD was purchased from Accustandard (New Haven, CT). RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated bovine calf serum (Hyclone, Logan, UT), 100 U/ml Penicillin/Streptomycin (Invitrogen), and 50lM 2-mercaptoethanol were used for all cell culture. Sheep erythrocytes were purchased from Colorado Serum (Denver, CO). IFN-a, IFN-b, and IFN-c were purchased from Roche Applied Science (Indianapolis, IN). Guinea pig complement was purchased from Cedarlane Laboratories (Burlington, NC). Bactoagar was obtained from Difco (Detroit, MI). Custom blood gas mixture was purchased from Airgas Great Lakes (Lansing, MI). In vitro T-dependent AFC response. Methods used for the preparation and culture of splenocytes, as well as enumeration of IgM secreting plasma cells, have been previously reported (Holsapple et al., 1984), with the following modifications. Spleens were processed by mechanical disruption into a single cell suspension and 0.5 ml of cells were seeded in 48-well Costar cell culture plates (Corning, Corning, NY) at a density of 1 3 107 cells/ml and sheep erythrocytes added at a concentration of 1 3 109 cells/ml. For time-of-addition studies, cells were incubated at 37°C in a humidified 5% carbon dioxide/95%

RESULTS

A Concentration- and Time-Related Attenuation of TCDDMediated Suppression of the Anti-sRBC IgM AFC Response by IFN-c TCDD at concentrations of 10nM or greater produced a significant impairment (p < 0.01) of the anti-sRBC IgM AFC response in a concentration-related manner (Fig. 1A). AFC responses in splenocytes that received both TCDD and 100 U/ml IFN-c (approximately equivalent to 20 ng/ml) did not differ significantly from vehicle-treated splenocytes, indicating an ablation of the TCDD-mediated suppression. Notably, direct addition of 100 U/ml IFN-c alone, did not significantly alter the AFC response. As demonstrated in Figure 1A, 30nM TCDD significantly suppressed the AFC response (42% of vehicle-treated splenocytes, p < 0.01), a concentration that causes a near maximal suppression of the anti-sRBC IgM AFC response. Based on the strong level of suppression observed with 30nM TCDD, this concentration was deliberately selected to rigorously assess the ability of IFN-c to block the TCDDmediated suppression of the IgM AFC. Concentration response studies showed that 1 and 10 U/ml IFN-c did not abolish the

IFNc REVERSAL OF IGM SUPPRESSION BY TCDD

87

FIG. 2. IFN-c reversal of TCDD-mediated AFC response suppression is time-dependent. Splenocytes cultures were stimulated with sheep erythrocytes then treated with 0.015% DMSO or 30nM TCDD. RPMI-1640 vehicle (dotted line) or 100 U/ml IFN-c (solid line —) were added at 0, 1, 2, 4, 8, 24, 48, and 72 h post-TCDD. Five days following the initial activation with sheep erythrocytes antibody secreting plasma cells were enumerated by AFC response. Data depicted are mean ± SE AFC response. *p < 0.05 or **p < 0.01 as determined by one-way ANOVA using Dunnett’s post hoc test comparing to vehicle treatment at 0 h post-TCDD.

FIG. 1. IFN-c reversal of TCDD-mediated AFC response suppression is concentration-dependent. (A) Splenocytes cultures were stimulated with sheep erythrocytes then treated with 0.015% DMSO vehicle, 3, 10, or 30nM TCDD in combination with RPMI-1640 vehicle (open bars) or 100 U/ml IFN-c (filled bars). Following 5 days of incubation the number of antibody-secreting plasma cells was determined by AFC response. Data depicted are mean ± SE AFC response. **p < 0.01 as determined by one-way ANOVA using Dunnett’s post hoc test comparing to vehicle-treatment alone. (B) Splenocytes cultures from mice were stimulated with sheep erythrocytes then treated with 0.015% DMSO vehicle (open bars) or 30nM TCDD (filled bars) in combination with RPMI1640 vehicle, 1, 10, or 100 U/ml IFN-c. Following 5 days of incubation the number of antibody-secreting plasma cells was determined by AFC response. Data depicted are mean ± SE AFC response. *p < 0.05 or **p < 0.01 as determined by one-way ANOVA using Dunnett’s post hoc test comparing to vehicle treatment alone.

TCDD-mediated suppression of the AFC response, whereas splenocytes cotreated with TCDD and 100 U/ml IFN-c exhibited complete attenuation of the TCDD-mediated effect (Fig. 1B). Interestingly, IFN-c attenuation of TCDD-mediated suppression of the anti-sRBC AFC response was consistently observed using splenocytes from mice of at least 12 weeks of age or older, whereas mice younger than 12 weeks of age exhibit some variability with respect to their response to IFN-c

(Data not shown). The reason for the variability in mice under 12 weeks is not altogether clear but may reflect modest changes in the maturing murine immune system. To further characterize the ability of IFN-c to reverse TCDD-mediated suppression of the AFC response, timeof-addition studies were conducted. Figure 2 shows that TCDD-mediated suppression of the IgM AFC by IFN-c was time-related. After addition of 30nM TCDD to splenocyte cultures at time 0, 100 U/ml IFN-c was added at 0, 1, 2, 4, 12, 24, 48, and 72 h post addition of TCDD and antigen (sRBC). AFC responses for splenocyte cultures that received IFN-c 2 h or later after TCDD and antigen showed significant suppression of the AFC response when compared with vehicle-treated controls, indicating that the window of sensitivity for reversal of TCDD-mediated suppression of the anti-sRBC IgM AFC response by IFN-c is less than 2 h. Effects of TCDD on Steady State mRNA Abundance were Partially or Completely Attenuated with Simultaneous IFN Treatment TCDD alters the mRNA levels of many genes, including CYP1A1 and components of IgM. On the final day of culture sRBC-stimulated splenocytes were collected and RNA analyzed by quantitative real-time RT-PCR to assess gene expression. TCDD treatment decreased the amount of Igj (Fig. 3A, 63% of vehicle-treated splenocytes, p < 0.01), Igl (Fig. 3B, 63% of vehicle-treated splenocytes, p < 0.05), and

88

NORTH ET AL.

FIG. 3. IFN-c treatment reverses TCDD-mediated reductions in gene expression for components of the IgM antibody. Splenocytes cultures were stimulated with sheep erythrocytes then treated with 0.015% DMSO or 30nM TCDD in combination with RPMI-1640 vehicle (open bars) or 100 U/ml IFN-c (filled bars). Following 5 days of incubation splenocytes were collected and RNA isolated for analysis of (A) Igj, (B) Igl, or (C) IgJ steady state mRNA levels by quantitative real-time RT-PCR. cDNA copy number was determined by comparison with a standard curve generated from known quantities of PCR product specific for each gene assessed, then normalized to b-actin cDNA copy number. Data depicted as mean ± SE fold change in expression relative to vehicle-treated splenocytes. *p < 0.05 or **p < 0.01 as determined by one-way ANOVA using Dunnett’s post hoc test comparing to vehicle treatment.

IgJ (Fig. 3C, 21.5% of vehicle-treated splenocytes, p < 0.01). Reductions in the mRNA abundance of the IgM components caused by TCDD were completely attenuated by the direct addition of 100 U/ml of IFN-c to the splenocyte culture. Induction of CYP1A1 gene and protein expression by TCDD and other dioxin-like chemicals has been a well characterized and commonly used indicator of AHR activation. In order to evaluate the possibility that AHR activation by TCDD is altered by IFN-c, the time course for TCDD-induced CYP1A1 mRNA levels was evaluated at 4, 8, and 24 h postTCDD (Fig. 4). IFN-c treatment alone did not significantly alter CYP1A1 mRNA levels compared with vehicle-treated splenocytes, but 30nM TCDD treatment induced a significant increase (p < 0.01) in CYP1A1 mRNA levels by 4 h, an increase that peaked at 8 h and remained sustained throughout the 24-h time period. Splenocytes treated with both IFN-c and TCDD showed a 5.2-fold increase in CYP1A1 mRNA levels compared with vehicle-treated cells at 4 h post-TCDD (p < 0.01), 37% less than the induction observed in cells treated with TCDD alone (p < 0.01). Cotreatment of splenocytes with IFN-c and TCDD produced a consistent and significant attenuation of CYP1A1 mRNA levels compared with TCDD treatment alone at 8 and 24 h post-TCDD treatment. Based on the observation that IFN-c, which signals through type II IFN receptors, attenuated TCDD-induced increases in CYP1A1 mRNA levels, we further evaluated whether signaling through type I IFN receptors was equally capable of interfering with TCDD-mediated CYP1A1 induction in splenocytes. Importantly, published studies have provided evidence suggesting that type I IFN can modulate CYP1A1 expression. In addition, type I and II IFNs can be induced under similar conditions in vivo. In light of the above, the potential effects of type I IFNs, IFN-a and IFN-b, were assessed at 8 h post-TCDD addition, the time of maximal CYP1A1 mRNA abundance previously observed (Fig. 4). To normalize for potential effects of IFNs on the background level of CYP1A1 mRNA, Table 1 presents mRNA levels as both fold change compared with vehicle treatment and as a stimulation index, in which each treatment group that received IFN and TCDD cotreatment is normalized to the level of mRNA present in the respective IFN control treatment. Table 1 shows that IFN-c treatment decreased the abundance of TCDDinduced CYP1A1 mRNA levels (28.9-fold increase relative to vehicle-treated splenocytes cotreated with TCDD and IFN-c compared 58.5-fold increase relative to vehicle for TCDDtreated splenocytes, a 50.5% reduction in CYP1A1 mRNA levels). Both type I IFNs also caused statistically significant (p < 0.01) decreases in TCDD-induced CYP1A1 mRNA levels, albeit more modest in magnitude than that observed with IFN-c treatment (39.1% reduction in TCDD-induced CYP1A1 mRNA levels for IFN-a, and 46.0% reduction in TCDD-induced CYP1A1 mRNA levels for IFN-b). Type I IFNs, unlike IFN-c, also produced decreases in the basal levels of CYP1A1 mRNA levels, and while not statistically

89

IFNc REVERSAL OF IGM SUPPRESSION BY TCDD

TABLE 1 IFN Effects on TCDD-Induced CYP1A1 mRNA Abundance

Treatment

FIG. 4. TCDD-induced CYP1A1 gene expression is partially reduced by IFN-c. Splenocytes cultures were stimulated with sheep erythrocytes then treated with 0.015% DMSO (open squares), 30nM TCDD (open circles), 100 U/ml IFN-c (filled squares), or the combination of 30nM TCDD and 100 U/ml IFN-c (filled circles). Splenocytes were collected at 4, 8, and 24 h post-TCDD addition and RNA isolated for gene expression analysis by quantitative real-time RTPCR. cDNA copy number was determined by comparison with a standard curve generated from known quantities of PCR product specific for CYP1A1 then normalized to b-actin cDNA copy number. Data depicted as mean ± SE fold change in expression relative to vehicle-treated splenocytes. a ¼ p < 0.01 as determined by One-way ANOVA using Tukey’s post hoc test comparing to vehicle-treatment. b ¼ p < 0.01 as determined by one-way ANOVA using Tukey’s post hoc test comparing the combination of 30nM TCDD and 100 U/ml IFN-c to 30nM TCDD treatment alone.

significant, may reflect inhibition of basal CYP1A1 mRNA that replicate similar findings in liver (Calleja et al., 1997; Donato et al., 1997; Funseth et al., 2002; Jeong et al., 1993). Tissues such as liver that contain higher basal levels of drug metabolizing enzymes may be more sensitive to IFNmediated alteration in CYP1A1 expression, but because the levels of CYP1A1 are comparatively low in splenocytes, IFN alteration of TCDD-induced CYP1A1 mRNA levels appears more subtly affected. IFN-a, IFN-b, and IFN-c all caused significant (p < 0.01) decreases in CYP1A1 mRNA levels induced by TCDD treatment, though reversal of CYP1A1 mRNA induction by IFN-a and IFN-b following TCDD treatment was not as large in magnitude as the reversal observed with IFN-c treatment, particularly in the context of the stimulation index. Reversal of TCDD-Mediated Suppression of the AFC Response was Specific to IFN-c, not IFN-a or IFN-b To appraise the possibility that both type I and type II IFNs are capable of reversing TCDD-mediated suppression of the anti-sRBC IgM AFC response, both type I IFN, IFN-a, and IFN-b, were evaluated in a concentration response study. IFN-a alone at 10 U/ml or higher produced a significant increase (p < 0.01) in the IgM AFC response, but did not alter the suppressive effect of TCDD on the AFC response, even at 1000 U/ml (Fig. 5A). Similarly, IFN-b at concentrations of 100 U/ml or higher produced a significant increase in the AFC

Naive Vehicle Vehicle þ IFN-a Vehicle þ IFN-b Vehicle þ IFN-c TCDD TCDD þ IFN-a TCDD þ IFN-b TCDD þ IFN-c

Fold change (relative to naive splenocytes) 1 0.965 0.601 0.631 1.015 58.549 35.636 31.621 28.882

± ± ± ± ± ± ± ± ±

0.07c 0.04c 0.01c 0.03c 0.12c 0.67a,b 1.15a,b,c,d 2.08a,b,c,d 0.24a,b,c,d

Stimulation index (relative to respective interferon treated splenocytes)

1.035 0.968 1.013 1.121 62.795 54.110 50.908 31.958

± ± ± ± ± ± ± ±

0.04c 0.01c 0.01c 0.13c 0.72b,d 1.26b,c,d 3.35b,c,d 0.27b,c,d

Note. Splenocytes cultures treated with 0.015% DMSO vehicle or 30nM TCDD in combination with RPMI-1640 vehicle, 100 U/ml IFN-a, 100 U/ml IFN-b, or 100 U/ml IFN-c. Following 8 h incubation, samples were collected for RNA isolation and analyzed for CYP1A1 mRNA quantity by real-time PCR. Data depicted are mean ± SE fold change or mean ± SE stimulation index (fold change in TCDD þ IFN treatment divided by fold change in Vehicle þ IFN treatment). Statistically significant differences as determined by one-way ANOVA using Tukey’s post hoc test comparing treatment groups are a significantly different from Naive, bsignificantly different from Vehicle, c significantly different from TCDD, and dsignificantly different from Vehicle þ Respective IFN.

response (p < 0.01), but also did not alter the effect of TCDD (Fig. 5B). These results suggest that reversal of TCDDmediated suppression of the IgM AFC response is specific to type II IFN, and cannot be mediated by type I IFNs.

DISCUSSION

Evidence presented in this series of studies shows that IFN-c attenuated several effects produced by TCDD, including suppression of the in vitro T-dependent anti-sRBC IgM AFC response and induction of CYP1A1 mRNA levels, a hallmark of AHR activation. The observation that the IFN-c–induced reversal of TCDD-associated effects is time and concentration related, at levels that do not increase the AFC response, suggests the engagement of a mechanism that impairs downstream events required for TCDD-mediated suppression of the IgM AFC response. Ligands with lower affinity than TCDD for AHR can antagonize the actions of TCDD by competition for binding to AHR (Suh et al., 2003), but the IFN-c–signaling cascade, which involves activation of the JAK-STAT pathway, is not known to directly interact with the AHR signaling pathway. The recent work of Kimura et al., which demonstrated that treatment of T lymphocytes with TGFb and IL-6, alone or in combination, could induce a physical association between the AHR and STAT1 (Kimura et al., 2008), provides potentially important new insights into the putative mechanism by which IFN-c may attenuate TCDDmediated effects described in this report. However, presently, it

90

NORTH ET AL.

FIG. 5. Type I IFN does not reverse TCDD-mediated suppression of the AFC response. Splenocytes cultures from mice were stimulated with sheep erythrocytes then treated with 0.015% DMSO (open bars) or 30nM TCDD (filled bars) in combination with (A) RPMI-1640 vehicle, 1, 10, 100, or 1000 U/ml IFN-a or (B) RPMI-1640 vehicle, 1, 10, 100, or 1000 U/ml IFN-b. 100 U/ml IFN-c was used as a positive control to demonstrate that splenocytes were competent to reverse TCDD-mediated suppression of the AFC response. Following 5 days of incubation antibody-secreting plasma cells were enumerated by AFC response. *p < 0.05 or **p < 0.01 as determined by one-way ANOVA using Dunnett’s post hoc test comparing to vehicle treatment.

is not known whether IFN-c treatment induces AHR and STAT1 interactions in B lymphocytes. Published results from several research groups have demonstrated inflammation in general (Bleau et al., 2003), and cytokines specifically (Calleja et al., 1997, 1998), reduce the expression of drug metabolizing enzymes such as CYP1A1 in the liver (Donato et al., 1997), bone marrow (Hrelia et al., 1994), and lymphocytes (Delaporte et al., 1995). The work of Jeong et al. (1993) demonstrated that IFN-c treatment of mouse primary hepatocytes prevented TCDD-induced increases in CYP1 family enzyme activity, but to our knowledge no one has demonstrated prevention of TCDD effects on the IgM AFC response or diminished CYP1A1 mRNA expression induced by TCDD in primary splenocytes. IFN-c–mediated suppression of CYP1A1 has been reported to occur in hepatocytes from rats (Stadler et al., 1994), mice (Jeong et al., 1993), and humans (Abdel-Razzak et al., 1993; Donato et al., 1997). Conservation of IFN-c effects on CYP1A1 expression across species suggests that IFN-c may also prevent TCDD suppression of the primary IgM response in the rat and human. Although the observation that IFN-c lowers CYP1A1 gene and protein expression in hepatocytes has been demonstrated in several species, it is also noteworthy that the effects of IFN on CYP1A1 extend to other cell types as well. Treatment of mice with benzo[a]pyrene, a well-known AHR ligand, causes an increase in the number of chromosome aberrations in bone marrow cells as a result of CYP1A1 metabolism of benzo[a]pyrene into DNA reactive intermediate. Pretreatment of mice with IFN-a/b reduced the number of chromosomal aberrations caused by benzo[a]pyrene, an observation that correlated with depression of CYP1A1 activity (Hrelia et al., 1994). Our results demonstrate that type I IFN such as IFN-a or IFN-b do not attenuate the suppressive action of TCDD on the IgM AFC response, but type I IFN does reduce CYP1A1 expression in splenocytes, which is consistent with published reports of type I IFN inhibition of CYP1A1 in human peripheral lymphocytes (Moochhala and Lee, 1991) and the human B lymphoblastoid cell line, AHH-1 TKþ/ (Delaporte et al., 1995). The findings of Delaporte and coworkers show IFN-a inhibits AHR ligand dibenz[a,h]anthracene-induced CYP1A1 expression by a pretranscriptional mechanism, because AHH-1 TKþ/ cells, which have been transfected with human CYP1A1 cDNA under the control of a constitutively active herpes simplex virus promoter, do not downregulate the expression of CYP1A1 in response to IFN-a treatment, when compared with control AHH-1 TKþ/ cells which do downregulate endogenously controlled CYP1A1 in response to IFN-a. The demonstration that IFN-c attenuates TCDD-mediated suppression of the IgM AFC response strongly suggests an interaction or convergence between signaling pathways downstream of the type II IFN receptor and signaling resulting from AHR activation by TCDD. Moreover, it is tempting to speculate that the ability of IFN-c

91

IFNc REVERSAL OF IGM SUPPRESSION BY TCDD

to attenuate suppression of the anti-sRBC IgM AFC response by TCDD can be differentiated from IFN-a and IFN-b due to differences in the types of STAT dimers formed following IFN receptor engagement, with STAT1 homodimers being the result of IFN-c–mediated type II receptor activation and STAT1/STAT2 heterodimers resulting from IFN-a/b–mediated type I receptor activation. In addition, evidence does exist that IFN-a and IFN-b, despite signaling through the same receptor, can also induce differential activities (Lewerenz et al., 1998; van Boxel-Dezaire et al., 2006), an observation that may, in part, explain the apparent differences in the magnitude of IFN-a and IFN-b reversal for TCDD-induced CYP1A1 mRNA abundance. Collectively, the differential activities observed by the various forms of IFNs on TCDD-induced biological responses is likely largely due to the differential STAT family members induced by the respective IFNs. Antibody secretion is a defining characteristic of B lymphocyte differentiation into antibody secreting plasma cells, and in the case of the response against sRBC, the triad of immune cells (antigen-presenting cells, T lymphocytes, and B lymphocytes) normally associated with adaptive immunity are required for mounting a robust response. Although TCDD has been demonstrated to act directly on the B lymphocyte (Dooley and Holsapple, 1988) using the in vitro AFC response, it is possible that the effect of IFN-c to reverse suppression of the IgM AFC response is mediated by direct effects on either B lymphocytes or accessory cells, or both simultaneously. Alteration or augmentation of the action of T lymphocytes may be able to rescue the B lymphocytes and return them to the path of normal differentiation. The relatively brief window of sensitivity (i.e., less than 2 h) during which IFN-c can reverse TCDD-mediated suppression of the IgM AFC response suggests that the effect of IFN-c is directly on the B lymphocyte, or accessory cells that are intermediaries of the IFN-c effect must respond rapidly to signal the B lymphocyte for differentiation to occur unperturbed. In revisiting the findings of Jeong et al., IFN-c did not prevent TCDD-induced increases in CYP1 activity in the Hepa1c1c7 hepatoma line. On the other hand, conditioned media from IFN-c–treated primary hepatocytes did prevent the TCDD-mediated increases in CYP1 activity for Hepa1c1c7, a result that led Jeong and coworkers to suggest that a soluble factor may be responsible for the effects of IFN-c after finding that trypsinized or heat-conditioned media was not effective in preventing TCDD-induced CYP1A1 expression (Jeong et al., 1993). Unless measures are taken to exclude them from culture, Kupffer cells of the liver will often be present in hepatocyte cultures. If macrophages such as Kupffer cells are responsible for production of a soluble factor that prevents CYP1A1 induction by TCDD in Hepa1c1c7 cells cultured in conditioned media, it is similarly possible that macrophages present in splenocyte cultures may produce the same factor. It is notable that our results do not exclude the possibility that other cytokines that were not evaluated in

this study, such as IL-1, IL-6, or TNF-a may also reverse TCDD-mediated suppression of the in vitro anti-sRBC AFC response. Future studies will address whether IFN-c acts directly on B lymphocytes to prevent TCDD-mediated suppression of the AFC response, or whether it is acting through an intermediate factor. In order to address the underlying mechanism(s) responsible for the effect of IFN-c there are several distinct possibilities that could be tested, including alteration in AHR protein expression, reduced nuclear translocation of ligand-activated AHR, increased proteolytic degradation of ligand-activated AHR, induction of the AHR repressor protein, increased production of a lower affinity endogenous AHR ligand that competes with TCDD for AHR binding, alteration in coregulator availability for AHR, or direct competition for genomic DNA binding sites between AHR/ ARNT and STAT dimers. Further broadening the possibilities by which IFN-c may be acting, those mechanisms postulated above may also apply to ARNT. Through the systematic evaluation of these possibilities the mechanism responsible for IFN-c–induced attenuation of the TCDD-mediated suppression of the anti-sRBC IgM AFC response and CYP1A1 induction can be clarified. In summary, IFN-c, but not IFN-a or IFN-b, reverses the suppressive effect of the potent AHR ligand TCDD on the murine in vitro anti-sRBC IgM AFC response in a time- and concentration-related manner. The findings demonstrating IFN-c attenuation of the suppressed AFC response by TCDD presented here raise the possibility that IFN-c may prove to be a useful and specific biological probe to further extend mechanistic understanding of how TCDD acts to impair the primary IgM AFC response.

FUNDING

National Institutes of Health grants (P42 ES04911) to N.E.K. and (T32 ES07255) to C.M.N. ACKNOWLEDGMENTS

Norbert E. Kaminski has grant support from the Dow Chemical Company for research on dioxin immunotoxicity; however, these funds were not used in support of research described in this manuscript. REFERENCES Abdel-Razzak, Z., Loyer, P., Fautrel, A., Gautier, J. C., Corcos, L., Turlin, B., Beaune, P., and Guillouzo, A. (1993). Cytokines down-regulate expression of major cytochrome P-450 enzymes in adult human hepatocytes in primary culture. Mol Pharmacol 44, 707–15. Anonymous. (1997). Polychlorinated dibenzo-para-dioxins. IARC Monogr Eval Carcinog Risks Hum 69, 33–343.

92

NORTH ET AL.

Bleau, A.-M., Maurel, P., Pichette, V., Leblond, F., and du Souich, P. (2003). Interleukin-1beta, interleukin-6, tumour necrosis factor-alpha and interferongamma released by a viral infection and an aseptic inflammation reduce CYP1A1, 1A2 and 3A6 expression in rabbit hepatocytes. Eur J Pharmacol 473, 197–206. Boverhof, D. R., Tam, E., Harney, A. S., Crawford, R. B., Kaminski, N. E., and Zacharewski, T. R. (2004). 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces suppressor of cytokine signaling 2 in murine B cells. Mol Pharmacol 66, 1662–70. Calleja, C., Eeckhoutte, C., Dacasto, M., Larrieu, G., Dupuy, J., Pineau, T., and Galtier, P. (1998). Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6[beta]-hydroxylation. Biochemical Pharmacology 56, 1279–1285. Calleja, C., Eeckhoutte, C., Larrieu, G., Dupuy, J., Pineau, T., and Galtier, P. (1997). Differential effects of interleukin-1 beta, interleukin-2, and interferon-gamma on the inducible expression of CYP 1A1 and CYP 1A2 in cultured rabbit hepatocytes. Biochem Biophys Res Commun 239, 273–8. Delaporte, E., Cribb, A. E., and Renton, K. W. (1995). Interferon-mediated changes in the expression of CYP1A1 in human B lymphoblastoid (AHH-1 TK) cells. Can J Physiol Pharmacol 73, 1692–7. Donato, M. T., Guillen, M. I., Jover, R., Castell, J. V., and Gomez-Lechon, M. J. (1997). Nitric oxide-mediated inhibition of cytochrome P450 by interferongamma in human hepatocytes. J Pharmacol Exp Ther 281, 484–90. Dooley, R. K., and Holsapple, M. P. (1988). Elucidation of cellular targets responsible for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of antibody responses: I. The role of the B lymphocyte. Immunopharmacology 16, 167–80. Funseth, E., Pahlman, M., Eloranta, M.-L., Friman, G., and Ilback, N.-G. (2002). Effects of coxsackievirus B3 infection on the acute-phase protein metallothionein and on cytochrome P-4501A1 involved in the detoxification processes of TCDD in the mouse. The Science of The Total Environment 284, 37–47. Holsapple, M. P., Snyder, N. K., Wood, S. C., and Morris, D. L. (1991). A review of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence: 1991 update. Toxicology 69, 219–55. Holsapple, M. P., Tucker, A. N., McNerney, P. J., and White, K. L., Jr. (1984). Effects of N-nitrosodimethylamine on humoral immunity. J Pharmacol Exp Ther 229, 493–500. Hrelia, P., Morotti, M., Vigagni, F., Maffei, F., Paolini, M., Mesirca, R., and Cantelli-Forti, G. (1994). The modulating activity of interferon on benzo(a)pyrene bioactivation and clastogenesis in mice. Pharmacol Toxicol 74, 249–54. Jelinek, D. F., Splawski, J. B., and Lipsky, P. E. (1986). The roles of interleukin 2 and interferon-gamma in human B cell activation, growth and differentiation. Eur J Immunol 16, 925–32. Jeong, H. G., Jeong, T. C., and Yang, K. H. (1993). Mouse interferon gamma pretreated hepatocytes conditioned media suppress cytochrome P-450

induction by TCDD in mouse hepatoma cells. Biochem Mol Biol Int 29, 197–202. Kelly, B. C., Ikonomou, M. G., Blair, J. D., Morin, A. E., and Gobas, F. A. P. C. (2007). Food Web-Specific Biomagnification of Persistent Organic Pollutants. Science 317, 236– 239; 10.1126/science.1138275 Kerkvliet, N. I. (2002). Recent advances in understanding the mechanisms of TCDD immunotoxicity. Int Immunopharmacol 2, 277–91. Kimura, A., Naka, T., Nohara, K., Fujii-Kuriyama, Y., and Kishimoto, T. (2008). Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells. Proc Natl Acad Sci US A 105, 9721–6. Lewerenz, M., Mogensen, K. E., and Uze, G. (1998). Shared receptor components but distinct complexes for alpha and beta interferons. J Mol Biol 282, 585–99. Moochhala, S. M., and Lee, E. J. (1991). Effects of recombinant human interferon alpha on aryl hydrocarbon hydroxylase activity in cultured human peripheral lymphocytes. Life Sci 48, 1715–9. Morris, D. L., Jeong, H. G., Stevens, W. D., Chun, Y. J., Karras, J. G., and Holsapple, M. P. (1994). Serum modulation of the effects of TCDD on the in vitro antibody response and on enzyme induction in primary hepatocytes. Immunopharmacology 27, 93–105. Rozen, S., and Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365–86. Stadler, J., Trockfeld, J., Schmalix, W. A., Brill, T., Siewert, J. R., Greim, H., and Doehmer, J. (1994). Inhibition of cytochromes P4501A by nitric oxide. Proc Natl Acad Sci U S A 91, 3559–63. Stevens, T. L., Bossie, A., Sanders, V. M., Fernandez-Botran, R., Coffman, R. L., Mosmann, T. R., and Vitetta, E. S. (1988). Regulation of antibody isotype secretion by subsets of antigen-specific helper T cells. Nature 334, 255–258. Suh, J., Kang, J. S., Yang, K.-H., and Kaminski, N. E. (2003). Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di-ortho-substituted polychlorinated biphenyls. Toxicol Appl Pharmacol 187, 11–21. van Boxel-Dezaire, A. H., Rani, M. R., and Stark, G. R. (2006). Complex modulation of cell type-specific signaling in response to type I interferons. Immunity 25, 361–72. Vitetta, E. S., Fernandez-Botran, R., Myers, C. D., and Sanders, V. M. (1989). Cellular interactions in the humoral immune response. Adv Immunol 45, 1–105. Yi, A. K., Chace, J. H., Cowdery, J. S., and Krieg, A. M. (1996). IFN-gamma promotes IL-6 and IgM secretion in response to CpG motifs in bacterial DNA and oligodeoxynucleotides. J Immunol 156, 558–564. Yoo, B. S., Boverhof, D. R., Shnaider, D., Crawford, R. B., Zacharewski, T. R., and Kaminski, N. E. (2004). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters the Regulation of Pax5 in Lipopolysaccharide-Activated B Cells. Toxicol. Sci. 77, 272– 279; 10.1093/toxsci/kfh013