Aryl hydrocarbon receptor (AhR) agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study
© Jensen et al; licensee BioMed Central Ltd. 2003
Received: 01 October 2003
Accepted: 16 December 2003
Published: 16 December 2003
Bone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR) agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to alter stromal cell cytokine responses.
Bone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS) to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA) and quantified by real-time PCR. Cytokine (IL-6) protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation.
RPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in the LPS-mediated induction of DNA-binding RelA/p50 and c-Rel/p50 heterodimers in the presence of DMBA.
Common environmental AhR agonists can suppress the response to bacterial lipopolysaccharide, a model for innate inflammatory responses, through down-regulation of IL-6, a cytokine critical to the growth of several hematopoietic cell subsets, including early B cells. This suppression occurs at least at the level of IL-6 gene transcription and may be regulated by NF-κB.
Bone marrow stromal cells support the growth and development of all eight hematopoietic cell lineages through cell-cell contact and the production of soluble cytokines [1, 2]. Although this process generally is well-regulated by both adhesion molecules and receptor/ligand pairs, aberrant stromal cell-blood cell interactions have been documented and are associated with a variety of diseases that involve abnormal growth and development of blood cells [3–5].
Perhaps the best studied stromal cell-blood cell interaction is that which occurs between bone marrow stroma and developing B lymphocytes [6–9]. Of particular interest in these studies is the contribution of stromal cell-derived IL-6 to B lymphopoiesis. Originally described as a tumor-derived growth factor , IL-6 now is known to be required for the growth of normal bone marrow progenitor B cells , for terminal differentiation of surface immunoglobulin-bearing B cells [12, 13], and for the long-term survival of bone marrow plasma cells [13–15].
Aberrant IL-6 expression has been associated with autoimmune diseases, including, but not limited to vitiligo , systemic lupus erythematosus (SLE) [17, 18], rheumatoid arthritis , and multiple sclerosis . Moreover, IL-6 has been recognized as an important growth and survival factor for neoplastic bone marrow plasma cells in multiple myeloma [5, 14, 21] and has been targeted with specific antibodies for myeloma therapy . In addition, IL-6 is emerging as an important survival and angiogenesis factor in other cancers, including basal cell carcinoma, prostate cancer, and Kaposi's carcinoma [23–25]. These studies illustrate the importance of IL-6 regulation in normal cell function and suggest that any modulation of its expression could have important pathologic consequences.
We and others demonstrated that exposure of bone marrow stromal cells to common environmental contaminants, such as polycyclic aromatic hydrocarbons (PAHs), adversely affect their function [26–34]. Specifically, the prototypic PAHs, benzo [a]pyrene (B [a]P) and 7,12-dimethylbenz [a]anthracene (DMBA) induce primary or cloned stromal cells to deliver a death signal to adjacent pre- and pro/pre-B cells [28–34]. Induction of this apoptosis signal in stromal cells is dependent on activation of the aryl hydrocarbon receptor (AhR), a cytosolic receptor that is converted into a transcription factor on binding of any one of a number of PAHs, halogenated aromatic hydrocarbons (HAHs), or planar polychlorinated biphenyls (PCBs) [35–37]. Other laboratories demonstrated that HAH, such as 2,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suppress the production of bone marrow-derived T cell precursors [38, 39], although the role of AhR+ bone marrow stromal cells in this process was not evaluated. Interestingly, exposure to AhR ligands has been associated with increased multiple myeloma risk [40–42], suggesting a possible link between AhR activation, aberrant bone marrow stromal cell cytokine production, and plasma cell dyscrasia.
In light of the ability of AhR ligands to target bone marrow stromal cells and the importance of bone marrow stromal cells to blood cell development, we sought to determine if AhR ligands compromise production of bone marrow stromal cell cytokines such as IL-6. To this end, a well-characterized AhR+ bone marrow stromal cell line (BMS2) was used to evaluate the effects of a PAH (DMBA) and an HAH (TCDD) on cytokine gene expression induced by the Toll-like receptor-4 (TLR-4) ligand, lipopolysaccacharide (LPS). LPS was chosen for these studies since it up-regulates the production of several bone marrow-derived growth factors, including IL-6, in models of inflammatory cytokine responses [8, 11, 43–45]. Particular attention was paid to the potential role of the promiscuous transcription factor NF-κB in AhR ligand effects since we and others demonstrated a physical and functional interaction between the AhR and the p65 (RelA) subunit of NF-κB [46, 47] and since NF-κB plays an important role in regulating IL-6 gene transcription .
TCDD (99.99% pure) was obtained from Ultra Scientific (North Kingstown, RI). DMBA and Staphylococcus lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO).
Cell culture and treatment
BMS2, a bone marrow stromal cell line that supports the growth and differentiation of granulocytes, pro/pre-B, and pre-B cells [8, 34] was a gift from Dr. P. Kincade (Oklahoma Medical Research Center). BMS2 cells were maintained in DMEM (Mediatech, Washington, DC) supplemented with 5% fetal calf serum, L-glutamine (Gibco/BRL, Gaithersburg, MD), 2-mercaptoethanol (Sigma Chemical Co) and 25 μg/ml plasmocin (Invivogen, Carlsbad, CA) at 37°C and 7.5% CO2. Cells were passed twice weekly and determined to be mycoplasma-free by a PCR based protocol (Mycoplasma Detection Kit; ATCC, Manassas, VA).
BMS2 cells were plated in antibiotic-free medium for 24 hours prior to treatment with vehicle (0.01% ethanol final concentration), 1–10 μM DMBA dissolved in ethanol, or 1 nM TCDD dissolved in DMSO (0.01% final concentration). One hour later, LPS was added to a final concentration of 1 μg/ml. Addition of LPS was delayed one hour to maximize AhR ligand uptake prior to LPS challenge . Cells were harvested at various times thereafter for RNA analysis (RNase protection assays; Real-time PCR) or nuclear protein-DNA binding analyses (EMSA), and supernatants were harvested for IL-6 quantitation by ELISA.
Culture well-adherent BMS2 cells were removed with 0.05% trypsin (Sigma), rinsed with PBS, pelleted, and frozen at -80°C until use. Total RNA was isolated using the RNeasy kit (Qiagen; Valencia, CA) or SV Total RNA System (Promega, Madison, WI). RNA was quantitated and samples visualized on an RNA gel to ensure RNA integrity.
Ribonuclease Protection Assay (RPA)
The RiboQuant Multi-probe RNase Protection Assay System (BD PharMingen, San Diego, CA) was used for RPA experiments. Riboprobes were generated with kit components and fresh [α-32P]-UTP 3000 Ci/mmol (Perkin-Elmer, Boston, MA) and used within 2 days. Two templates (mCK4 and mCK3b) were tested to screen for changes in genes encoding multiple cytokines: IL-3, IL-6, IL-7, IL-11, IFNγ, TNFα, TNF-β, LT-β, TGFβ 1, TGFβ 2, TGFβ 3, MIF, GM-CSF, M-CSF, G-SF, LIF, and SCF. L32 and GAPDH were also represented as internal standards. Purified probes were mixed with 7–15 μg of total RNA and hybridized overnight. After cooling, annealed targets were treated with RNase and the resulting fragments were purified, denatured and resolved on a 4.5% sequencing gel. The gels were dried completely, exposed to film and quantified with a Molecular Dynamics Phosphor Imager (Amersham Biosciences, Sunnyvale, CA) using Imagequant software (Amersham).
IL-6 primers were designed to span exon junctions and were tested against genomic templates. PCR products were resolved on agarose gels to show minimal contribution from genomic products or primer dimers. IL-6 primer sequences were: 5-'CAAGAGACTTCCATCCAGTTGCCT-3' and 5'-TTTCTCATTTCCACGATTTCCCAG-3'. β-actin primers were 5'-GTCGTCGACAACGGCTCCGGCATGTG-3' and 5'-CATTGTAGAAGGTGTGGTGCCAGATC-3'. Total RNA was reverse transcribed into cDNA using TaqMan Reverse Transcription Reagents (PE Applied Biosystems, Foster City, CA). cDNA (0.01 μg) was used in the hot start Real-time PCR with SYBR Green PCR Master Mix (PE Applied Biosystems). Real-time PCR was carried out with the ABI Prism 7700 Sequence Detector (PE Applied Biosystems). The PCR conditions were: 95°C for 10 min, 40 cycles of 90°C for 15 s, and 60°C for 1 m, with fluorescence measurements read during each cycle. Fluorescence measurements were used to compare transcripts between samples using the Comparative CT Method, a method for determining relative amounts of transcripts based on an internal control when the absolute number of transcripts represented is unknown (PE Applied Biosystems).
BMS2 cells were plated at 25,000 cells/ml in T75 flasks and cultured for 18–24 hours prior to treatment with vehicle (0.01% ethanol or DMSO), 1 μM DMBA, or 1 nM TCDD. One hour later, cultures were challenged with 1 μg/ml LPS. At each time point indicated, 500 μl aliquots of BMS2 cell culture supernatants were drawn off and frozen at -20°C. ELISAs were carried out with the OptEIA mouse IL-6 ELISA kit (BD Pharmingen) according to the manufacturer's instructions. Supernatant samples were diluted 1:50 and plated in duplicate wells of a 96 well plate. Freshly diluted rIL-6 was used to generate a standard curve on each plate analyzed. The lower limit of detection for this ELISA was 15 pg/ml IL-6. Plates were read at 450 nm using a spectrophotometric plate reader. Raw data were corrected against blank wells and converted to pg/ml using the standard curve.
BMS2 cells were treated in 6-well plates with vehicle or 1 μM DMBA. One hour later, 1 μg/ml LPS or PBS was added to the cultures, and cells were harvested 16 or 24 hours later. Nuclear proteins were extracted as described previously . For determination of NF-κB activation, a double stranded oligonucleotide containing the NF-κB binding site from the upstream regulatory element of c-myc (5'-GATCCAAGTCCGGGTTTTCCCC AACC-3')  was used. The DNA probe was end-labeled using T4 polynucleotide kinase (Promega, Madison, WI) and [γ-32P]-ATP and was purified using a Centrispin-20 column (Princeton Separations, Adelphia, NJ). EMSAs were performed as follows: 32P-labeled DNA (~0.5 ng, 50,000 cpm) and 2 μg of nuclear protein were combined with buffer (final concentrations: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM sodium chloride, 0.5 mM magnesium chloride, 1 mM DTT, 20 μg BSA, 10% glycerol, 0.5% Triton X-100) and poly dI-dC (1 μg) in a final volume of 20 μl. The mixture was incubated at room temperature for 30 min. The gel was run as above, dried and exposed to film. For quantification, gels were analyzed by phosphorimaging on a Molecular Dynamics Phosphor Imager (Amersham Biosciences). The identity of the NF-κB subunits was determined by including antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) specific for p50 (sc-114), p52 (sc-848), Rel A (sc-372) or c-Rel (sc-71).
Statistics were calculated using Prism version 3.0 for Macintosh (GraphPad). Data are presented as means ± SE. Data were analyzed using the Student's T-test or single factor ANOVA's with the Dunnet's multiple comparisons test.
LPS up-regulates cytokine gene induction in bone marrow stromal cells
Bone marrow stromal cells support the growth and differentiation of early B cells . In previous studies, we demonstrated that cloned bone marrow stromal cell lines, including BMS2 cells, support the growth of primary pre-B and cloned pro/pre-B cells and that treatment of the bone marrow stromal cells with AhR agonists alters their function [28–30, 32–34, 52]. As a first step in determining if AhR ligands affect stromal cell cytokine production, baseline levels of cytokine mRNAs in BMS2 cells were determined by RNAse protection assays.
LPS induces GM-SCF, G-CSF, LIF, and IL-6 mRNAs in bone marrow stromal cells
Mean Fold Changea ± SE
1.3 ± 0.2
0.9 ± 0.2
5.7 ± 1.2
7.0 ± 1.5
1.1 ± 0.1
1.0 ± 0.1
The effect of AhR agonists on LPS-mediated IL-6 mRNA induction
To determine the effect of AhR ligands on LPS-induced cytokine induction, BMS2 cells were treated with vehicle (0.01% ethanol), 1 μM DMBA, or 1 nM TCDD one hour prior to addition of 1 μg/ml LPS. Cells were harvested 8 hours later. RNA was extracted and assayed for cytokine mRNA levels by RPA.
DMBA and TCDD significantly abrogate LPS-induced IL-6 production
The results described above indicate consistent suppression of LPS-mediated IL-6 mRNA induction. To determine if this decrease in steady state mRNA is reflected in a proportional decrease in secreted IL-6 protein levels, BMS2 cells were cultured for 18 hrs prior to addition of vehicle, 1 μM DMBA, or 1 nM TCDD. One hour later, cultures were treated with PBS (to assay effects of AhR ligands on background IL-6 levels) or challenged with 1 μg/ml LPS (i.e. time zero). Culture supernatants were harvested at time zero and 6–72 hours thereafter and assayed by ELISA for IL-6 levels.
A significant increase in secreted IL-6 levels was seen as early as 6 hours after LPS treatment (Figure 4B). IL-6 levels continued to rise throughout the 72 hour period after LPS exposure. However, this LPS-dependent increase in secreted IL-6 was suppressed in cultures containing either DMBA or TCDD, with TCDD suppressing IL-6 levels as much as 88% at the 72 hour time point. Neither TCDD nor DMBA affected BMS2 proliferation as all cultures reached confluency at the same time (approximately 96 hrs after plating). These data demonstrate that activation of the AhR has a significant effect on the ability of bone marrow stromal cells to produce IL-6. Furthermore, the profound decrease in IL-6 protein levels in TCDD-exposed cells exhibiting only a 40–60% decrease in IL-6 mRNA levels, suggests that at least this AhR ligand may suppress IL-6 production at more than just the RNA level.
NF-κB activation correlates with IL-6 mRNA levels
LPS-induced IL-6 gene transcription in murine monocytes is controlled primarily by NF-κB, although other transcription factors may play minor roles . Since we and others have demonstrated interactions between the AhR and the NF-κB signaling pathways [46, 47, 53, 54], it was important to determine if activation of the AhR influenced NF-κB activity in bone marrow stromal cells. To this end, BMS2 cells were treated with vehicle or DMBA one hour prior to exposure to LPS as in previous experiments. Cells were harvested 16–24 hours later. Nuclear proteins were extracted and assayed in electromobility shift assays (EMSAs) for binding of nuclear proteins to a radiolabelled NF-κB probe consisting of the NF-κB-binding upstream regulatory element of the c-myc promoter .
The present studies were designed to evaluate the ability of AhR ligands to influence a spectrum of bone marrow stromal cell cytokines. In this system, exposing AhR+ stromal cells to LPS induced an inflammatory response characterized in part by up-regulation of IL-6 Any environmental chemical capable of compromising this response has the potential to disrupt the regulation of many important stromal cell functions, including generation of inflammatory responses in general and the elaboration of several cytokines, including IL-6, to regulate blood cell development in particular.
Previously, we demonstrated that DMBA and TCDD, two prototypic AhR ligands, activate the AhR in BMS2 cells [28, 34]. In studies presented here we demonstrate that both TCDD and DMBA suppress IL-6 production at least through the reduction of steady state IL-6 mRNA levels. The reduction in IL-6 mRNA levels does not exclude the possibility that these AhR ligands also suppress IL-6 protein secretion or stability. The specificity of this toxicity is underscored by the failure of these AhR agonists to affect either the baseline or LPS-induced levels of mRNAs specific for several other cytokines at the time points assayed here, although modulation of these or other cytokines at other time points cannot be excluded. This result also indicates that the observed changes in IL-6 do not result from overt toxicity or suppression of cell growth by AhR agonists.
In our continuing investigations into interactions between AhR and NF-κB signaling pathways , we sought to determine if AhR signaling influences NF-κB activity in bone marrow stromal cells. An added incentive for these studies was provided by the observation that the IL-6 gene promoter contains an NF-κB binding site which plays a major role in regulating LPS-induced IL-6 transcription [55–57]. Indeed, EMSAs indicated a profound increase in NF-κB activity following LPS exposure. This induction was suppressed modestly (33%) but significantly (p < 0.05) by DMBA. These results suggest a possible mechanism by which the AhR may regulate IL-6 transcription, i.e. suppression of NF-κB signaling.
The AhR ligand-mediated decrease in NF-κB activity documented here differs from results obtained with other cell types. For example, coplanar PCBs, which can activate the AhR, increase IL-6 production in endothelial cells . Furthermore, TCDD increases both AP-1 and NF-κB binding to their cognate response elements in hepatoma cells [59, 60]. In the latter example, NF-κB up-regulation involved an increase in binding of p50 homodimers to a consensus NF-κB nucleotide sequence. Unlike the p50/RelA-containing complexes implicated in the present studies (Figure 5B), p50 homodimers may actually block p50/p65-dependent gene activation . Indeed, p50/RelA dimers are well known to activate gene transcription [29, 51, 62, 63]. Therefore, its suppression by AhR ligands is likely to have important biological consequences.
Finally, the results presented here demonstrate the ability of AhR ligands to affect cytokine production in vitro. Several studies indicate cytokine dysregulation in vivo following exposure to TCDD or PAH, although the nature of these changes differs significantly between systems. In vivo exposure to TCDD in and of itself increases serum TNF-α levels  and the administration of a TNF-immunoglobulin fusion protein  or TNF-α-specific antibodies ameliorates TCDD-mediated inflammatory responses . Furthermore, TCDD exacerbates the TNF-mediated inflammatory responses to LPS in vivo . In contrast, TCDD injection suppresses IL-6 responses of splenic T lymphocytes to a protein antigen .
Results of in vivo exposures to AhR ligands may even differ between tissues. For example, TCDD injection results in a significant decrease in IL-18 mRNA in the thymus but a significant increase in the spleen [68, 69]. Similarly, IFN-γ levels in lymph nodes from TCDD-treated and virus-infected mice decrease while levels in the lung increase 10 fold . Clearly, the effects of AhR ligands in vivo are varied and complex, likely involving interactions between many cell types and cytokine feedback loops. Significantly, none of these studies have evaluated inflammatory cytokine production in the bone marrow. Future experiments from our laboratory will address this gap in knowledge about AhR ligand toxicity.
The studies presented here demonstrate that prototypic AhR agonists, including an environmentally ubiquitous dioxin, specifically suppress the LPS-induced production of IL-6 by bone marrow stromal cells. The more potent AhR agonist, TCDD, also reduced baseline levels of IL-6 protein. Inhibition of IL-6 production in response to LPS occurs at least at the level of mRNA expression and may involve down-regulation of NF-κB, a transcription factor known to play a major role in regulating IL-6 gene transcription. Collectively, the results demonstrate the potential for common environmental AhR ligands to compromise the ability of bone marrow stromal cells to generate important inflammatory responses and to support production of blood cell lineages.
aryl hydrocarbon receptor/transcription factor
- B [a]P:
granulocyte colony stimulating factor
granulocyte/monocyte colony stimulating factor
halogenated aromatic hydrocarbons
leukemia inhibitory factor
macrophage migration inhibitory factor
polycyclic aromatic hydrocarbon
tumor necrosis factor
transforming growth factor
The authors thank Ilga Wohlrab for performing all the administrative duties related to this project and to Maryam Shansab for assisting in manuscript preparation. Supported by NIH RO1-ES06086, Superfund Basic Research Grant #1P42ES-07381, and PO1 HL 68705.
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