PMC

BM cell isolated from OVX mice were cultured in sex hormone-deficient medium with murine GM-CSF alone, or in the presence of varying concentrations of E2 (A, B) or P4 (C, D). At end of culture, cells were collected and stained with Abs for CD11b, CD11c, MHC II and CD40 antibodies. Cell culture were treated with (dashed) or without (solid) LPS for last 24 hrs. Y axis represents the percentage of CD40+ (A, C) or MHCII+ (B, D) CD11b⁺CD11c⁺ DCs. Data shown are Mean± SD of 6 independent experiments. Statistical significance is indicated by * p<0.05; **p<0.01 compared with untreated control.

BM cells isolated from OVX mice were cultured with GM-CSF in the presence of E2 at a low (10^-11M) (A) or high (10^-9M) (B) concentration with varying doses of P4 (10^-9M to 10^-5M), with (dashed) or without (solid) LPS. At the end of culture, cells were harvested and stained with Abs to detect CD11b, CD11c, CD40 and MHCII. Data are percentages of CD11b⁺CD11c⁺ DCs (top panels); percentages of CD40⁺/CD11b⁺CD11c⁺DCs (middle panels); and percentages of MHCII⁺/CD11b⁺CD11c⁺ DCs (bottom panels). Data shown are Mean ± SD of 6 independent experiments. Statistical significance is indicated by *p<0.05; **p<0.01;*** p<0.001 compared with E2 only treated controls.

BM cell isolated from OVX mice were cultured in sex hormone-deficient medium with murine GM-CSF alone, or in the presence of varying concentrations of P4. Data in (A, B, C) are from cells collected at end of culture, and stained with Abs for CD11b and CD11c. Initial gating for monocytes as shown in . Representative dot plot data are shown in (A). Percentages of CD11b⁺CD11c⁺ APCs from cultures grown in presence of different concentrations of P4, treated with (dashed) or without (solid) LPS are shown in (B). Absolute numbers of CD11b⁺ CD11c⁺ APCs cultured with P4 are shown in (C). Data show Mean ± SD of 6 independent experiments. Statistical significance is indicated by * p<0.05; **p<0.01; *** p<0.001 compared with untreated control.

BM cell isolated from OVX mice were cultured in sex hormone-deficient medium with murine GM-CSF alone, or in the presence of varying concentrations of E2 (A, B, C, D). Initial gating for mononuclear cells is shown in (A). Data in (B, C, D) are from cells collected at end of culture, and stained with Abs for CD11b and CD11c. Representative dot plot data are shown in (B). Data for percentages of CD11b⁺CD11c⁺ DCs from cultures with different concentrations of E2 and treated with (dashed) or without (solid) LPS are shown in (C). Absolute numbers of CD11b⁺ CD11c⁺ DCs cultured with different concentrations of E2 are shown in (D). Data shown is Mean ± SD of 6 independent experiments. Statistical significance is indicated by * p<0.05; **p<0.01; *** p<0.001 compared with untreated control.

BM cells isolated from OVX mice were cultured with GM-CSF in the presence of varying doses of E2 or P4. On day 6, 5ng/ml LPS was added to the culture for further 24h culture and supernatants were collected and cytokine concentrations were determined (IL-12, IL-10, IL-8, IL-10, TNF-α, IFN-γ and IL-1β) by multi-analyte cytokine and chemokine assays. TGF-β in the supernatants was determined by ELISA. The values (pg/ml, mean ±SD) were normalized to the percentage of CD11b⁺ CD11c⁺ DCs in each treatment. Data represent triplicate samples from 2 independent experiments. Statistical significance is indicated by *p<0.05; ** p<0.01; ***p<0.001 compared with untreated control.

BM cells isolated from OVX mice were cultured with GM-CSF in the presence of E2 (10^-11M and 10^-9M) or P4 (10^-8M and 10^-6M) or 10^-9M E2 + 10^-6M P4 and FITC-Dextran (200μg/ml) was added to the culture on day 5 for further 2 hrs at 37°C. Negative control was FITC-Dextran added to BMDCs and incubated at 4°C for 2 hrs. Cells were collected and stained with antibodies for CD11b and CD11c. Percentage of FITC-Dextran-positive CD11b⁺CD11c⁺ DCs were obtained by gating on CD11b⁺CD11c⁺ DCs. Representative flow cytometry data are shown in (A) and corresponding data graph are shown in (B). Data shown are Mean ± SD from 4 independent experiments. Statistical significance is indicated by *p<0.05; ** p<0.01; ***p<0.001 compared with untreated control or the comparison of combination treatment with single hormone treatment.

BM cells isolated from OVX mice (B, C, D) or ER KO mice (A) were cultured with GM-CSF in the presence of 10^-11M (A) or 10^-9M E2 (A, B) or (10^-6M) P4 (C, D). Varying concentrations of E2 inhibitor ICI 182 780 (B), P4 inhibitor RU-486 (C) or Glucocorticoid receptor AL082D06 (D) were used in E2- or P4-treated BM cultures, beginning at the first day of culture. At the end of culture, cells were collected and stained with antibodies against CD11b and CD11c (A, B) and plus MHC II (C, D). Percentage of CD11c+ cells are shown in (A, B) for E2 cultured cells and P4 cultured cells (C, D). Data show Mean ± SD of triplicate determination each time of 3 independent experiments. Statistical significance is indicated by *p<0.05,** p<0.01, ***p<0.001 compared with untreated control.

BM cells isolated from OVX mice were cultured with GM-CSF in the presence of varying doses of E2 or P4 or combination of both. On day 6, 5ng/ml LPS was added to the culture for 24hrs. Culture treated with LPS alone (+LPS-E2) was negative control and untreated culture (-LPS -E2) was background. The protein transport inhibitor (BD Golgi Plug) was added for the last 6 hrs of culture. Cells were stained for intracellular IL-12, TNF- α and IL-6 with respective APC-conjugated antibodies. The top panel in A is gating scheme and isotype control. The rest of the panels in A are the percentage of IL-12-secreting CD11b⁺CD11c⁺ DCs by intracellular staining under experimental conditions. Percentage of CD11b+CD11c+ DCs positive for IL-12, TNF-α and IL-6 are showed in B, C and D, respectively. Data represent samples in triplicate from 2 independent determinations. *p<0.05; ** p<0.01; ***p<0.001 compared with non-hormone treated control or the comparison of combination treatment with single hormone treatment.