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).

BMS2, a bone marrow stromal cell line that supports the growth and differentiation of granulocytes, pro/pre-B, and pre-B cells 834 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 49. 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.

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 C_T 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 50. 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'-GATCCAAGTC

CGGGTTTTCCCC

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.

Bone marrow stromal cells support the growth and differentiation of early B cells 8. 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 28293032333452. 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.

RPAs with RNA from BMS2 cells consistently demonstrated significant expression of IL-11, monocyte colony stimulating factor (M-CSF), leukemia inhibitory factor (LIF), IL-6, transforming growth factor-β1 (TGF-β1), TGF-β3, and macrophage migration inhibitory factor (MIF) mRNAs (Figure 1). Low but detectable levels of IL-7 mRNA also were noted. Addition of LPS consistently induced LIF and IL-6 mRNAs within 7 hours (Figure 1 and Table 1). GM-CSF and G-CSF mRNAs were clearly induced in two of three and three of four experiments respectively. As suggested by observations made with other stromal cell lines 4445, these studies demonstrate a potent innate inflammatory cytokine response against bacterial LPS. It should be noted that these studies do not exclude possible LIF or other cytokines at other time points.

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.

Neither DMBA nor TCDD had a consistent effect on baseline levels of any cytokine mRNA assayed including IL-6 (Figure 2A and 2B). This result contrasts with that obtained with vascular endothelial cells in which AhR agonists (coplanar PCBs) alone induced IL-6 53. As in previous experiments, LPS significantly induced LIF and IL-6 mRNAs (Figure 2A). (RNA encoding GM-CSF and G-CSF also were increased, although bands representing these RNAs were sometimes difficult to see when under-exposing blots to emphasize IL-6 levels (e.g. Figure 2)). Importantly, in five experiments, both DMBA and TCDD significantly reduced the LPS-mediated IL-6 mRNA increase in BMS2 cells (Figure 2A and 2B; p < 0.05). Other genes activated by LPS, i.e. LIF, GM-CSF, and G-CSF, were not affected by either of these AhR agonists (Figure 2A and data not shown) demonstrating the selectivity of AhR agonist-mediated effects on stromal cells.

Kinetics studies were performed using real-time PCR as a readout to quantify this change in IL-6 mRNA levels. As seen in Figure 3, IL-6 mRNA levels increased within 1 hr of LPS stimulation and reached a plateau at the four hour time point. Inclusion of DMBA in the cultures reduced IL-6 mRNA induction significantly at every time point, including at 1 hour (p < 0.02). These results are consistent with those obtained by RPA and demonstrate that AhR ligands suppress IL-6 mRNA levels by approximately 40–60%.

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.

Supernatants from BMS2 cultures established 18–24 hrs previously contained approximately 700 pg/ml IL-6 (Figure 4A). Addition of DMBA had no effect on IL-6 levels at any time point assayed. However, TCDD significantly (p < 0.05) reduced the background level of IL-6 as early as 24 hrs after its addition to the cultures. Control experiments in which recombinant IL-6 was assayed in the presence of TCDD indicated that TCDD did not interfere with the IL-6-specific ELISA (data not shown). Therefore, TCDD is capable of reducing baseline IL-6 protein levels early in the response to LPS.

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.

LPS-induced IL-6 gene transcription in murine monocytes is controlled primarily by NF-κB, although other transcription factors may play minor roles 48. Since we and others have demonstrated interactions between the AhR and the NF-κB signaling pathways 46475354, 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 29.

Data presented in Figure 5A extend previous studies performed with monocytes by demonstrating that LPS induces NF-κB-DNA binding in bone marrow stromal cells. The ability to supershift NF-κB-DNA complexes with antibodies specific for the p50 or RelA/p65 subunits of NF-κB (Figure 5B) indicates that LPS primarily activates a conventional NF-κB complex probably consisting of p50/RelA. While DMBA alone had no effect on the low baseline levels of NF-κB-DNA binding, it modestly suppressed the NF-κB activity induced with LPS (Figure 5A). Quantification of normalized band densities indicated that DMBA significantly decreased LPS-induced NF-κB-DNA binding by 33% (p < 0.05) (Figure 5C). These data demonstrate a correlation between NF-κB activity and IL-6 mRNA levels.