Induction of the aryl hydrocarbon receptor-responsive genes and modulation of the immunoglobulin M response by 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary human B cells

Abstract

Past studies in rodent models identified the suppression of primary humoral immune responses as one of the most sensitive sequela associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Yet, the sensitivity of humoral immunity to TCDD in humans represents an important toxicological data gap. Therefore, the objectives of this investigation were two-fold. The first was to assess the induction of known aryl hydrocarbon receptor (AHR)-responsive genes in primary human B cells as a measure of early biological responses to TCDD. The second was to evaluate the direct effect of TCDD on CD40 ligand-induced immunoglobulin M (IgM) secretion by human primary B cells. The effects of TCDD on induction of AHR-responsive genes and suppression of the IgM response were also compared with B cells from a TCDD-responsive mouse strain, C57BL/6. AHR-responsive genes in human B cells exhibited slower kinetics and reduced magnitude of induction by TCDD when compared with mouse B cells. Evaluation of B-cell function from 12 donors identified two general phenotypes; the majority of donors exhibited similar sensitivity to suppression by TCDD of the IgM response as mouse B cells, which was not attributable to decreased B-cell proliferation. In a minority of donors, no suppression of the IgM response by TCDD was observed. Although donor-to-donor variation in sensitivity to TCDD was observed, human B cells from the majority of donors evaluated showed impairment of effector function by TCDD. Collectively, data presented in this series of studies demonstrate that TCDD impairs the humoral immunity of humans by directly targeting B cells.

FIG. 1.

Time-dependent induction of CYP1A1 gene expression by TCDD in naive human B cells. Naive human B cells (1 × 10⁶/ml) were treated with 30nM of TCDD or VH (0.05% DMSO) for 2, 4, 8, and 12 h. Total RNA was isolated, and steady-state mRNA levels of CYP1A1 were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group at the same time point. The results are the mean ± SE as determined for each group. Data from three individual donors are presented.

FIG. 2.

Time-dependent induction of AHR repressor gene expression by TCDD in naive human B cells. Naive human B cells (1 × 10⁶/ml) were treated with 30nM of TCDD or VH for 2, 4, 8, and 12 h. Total RNA was isolated, and steady-state mRNA levels of AHR repressor were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group at the same time point. The results are the mean ± SE as determined for each group. Data from three individual donors are presented.

FIG. 3.

Time-dependent induction of CYP1B1 gene expression by TCDD in naive human B cells. Naive human B cells (1 × 10⁶/ml) were treated with 30nM of TCDD or VH for 2, 4, 8, and 12 h. Total RNA was isolated, and steady-state mRNA levels of CYP1B1 were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group at the same time point. The results are the mean ± SE as determined for each group. Data from three individual donors are presented.

FIG. 4.

Time-dependent induction of TIPARP gene expression by TCDD in naive human B cells. Naive human B cells (1 × 10⁶/ml) were treated with 30nM of TCDD or VH (0.05% DMSO) for 2, 4, 8, and 12 h. Total RNA was isolated, and steady-state mRNA levels of TIPARP were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group at the same time point. The results are the mean ± SE as determined for each group. Data from three individual donors are presented.

FIG. 5.

Time-dependent induction of (A) CYP1A1, (B) AHR repressor, (C) CYP1B1, and (D) TIPARP gene expression by TCDD in mouse B cells. Mouse B cells (1 × 10⁶/ml) were treated with 30nM of TCDD or VH (0.05% DMSO) for the indicated time periods. Total RNA was isolated, and steady-state mRNA levels of (A) CYP1A1, (B) AHR repressor, (C) CYP1B1, and (D) TIPARP were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group at the same time point. Data are representative of two separate experiments with three experimental replicates per group.

FIG. 6.

Concentration-dependent induction of CYP1A1 and AHR repressor expression by TCDD in naive human B cells. Naive human B cells (1 × 10⁶/ml) were treated with TCDD at indicated concentrations or VH (0.05% DMSO) for 12 h. Total RNA was isolated, and steady-state mRNA levels of (A) CYP1A1 and (B) AHR repressor were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group. **p < 0.01, compared with VH control group. These data are representative of three separate experiments using B cells isolated from three individual donors.

FIG. 7.

Concentration-dependent induction of CYP1A1 and AHR repressor expression by TCDD in mouse B cells. Mouse B cells (1 × 10⁶/ml) were treated with TCDD at indicated concentrations or VH (0.05% DMSO) for 2 h. Total RNA was isolated, and steady-state mRNA levels of (A) CYP1A1 and (B) AHR repressor were measured by TaqMan real-time PCR and normalized to endogenous 18S ribosomal RNA. Data are presented as fold change compared with the VH control group. **p < 0.01, compared with VH control group. Data are representative of two separate experiments with three experimental replicates per group.

FIG. 8.

Effect of TCDD on the CD40L-induced IgM response in human B cells: (A) “nonresponsive” donors versus (B) “responsive” donors. Naive human B cells (1 × 10⁶/ml) were treated with TCDD at indicated concentrations or VH and then cultured with irradiated CD40L-L cells (1.5–3 × 10³ cells per well) in the presence of recombinant human IL-2 (10 U/ml), IL-6 (100 U/ml), and IL-10 (20 ng/ml) for 4 days, and CD40L stimulation was removed on day 4. Cells were cultured for another 3 days prior to being harvested on day 7 to enumerate IgM-secreting cells by ELISPOT. The cell number was determined by a particle counter, and the viability was assessed by the pronase activity assay. Data were normalized to no TCDD group (100%) and presented as percentage of control. B cells from multiple human donors were assessed.

FIG. 9.

Analysis of TCDD-mediated effect on the CD40L-induced IgM response in human B cells from multiple “responsive” donors. **p < 0.01, compared with the VH control group.

FIG. 10.

Effect of TCDD on the CD40L-induced IgM response in mouse B cells. Mouse B cells (1 × 10⁶/ml) were treated with TCDD at concentrations indicated or VH and then cocultured with irradiated CD40L-L cells (5 × 10⁴ cells per well) in the presence of recombinant mouse IL-2 (10 U/ml), IL-6 (100 U/ml), and IL-10 (20 ng/ml) for 3 days, and CD40L stimulation was removed on day 3. Cells were cultured for another 3 days prior to being harvested on day 6 to enumerate IgM-secreting cells by ELISPOT. The cell number was determined by a particle counter, and the viability of cells was determined by pronase activity assay. **p < 0.01, compared with the VH control group. Data are representative of three separate experiments with at least three experimental replicates per group.

FIG. 11.

CD40L-induced proliferation of human and mouse B cells. Human or mouse B cells (1 × 10⁶/ml) were labeled with the proliferation dye and then cocultured with irradiated CD40L-L cells (1.5 × 10³ cells per well for human and 5 × 10⁴ cells per well for mouse) in the presence of recombinant human or mouse IL-2 (10 U/ml), IL-6 (100 U/ml), and IL-10 (20 ng/ml) for 4 or 3 days, and CD40L stimulation was removed on day 4 or 3. Cells were cultured for one additional day prior to being harvested on day 5 or 4 to flow cytometric analysis. Dead cells were excluded from the analysis using the Live/Dead Near-Infrared Dead Cell Staining Kit. Data are representative of two separate experiments (for human, two experiments using B cells derived from two separate donors) and are concatenated from three experimental replicates per group.