Fig. 1. Effect of EPF on cell surface MHC-II expression level by human monocytes and THP-1 cells. THP-1 cells (A) and peripheral blood monocytes from healthy donors (B) (1 × 10⁶) were incubated with or without 10% PF from 10 different endometriosis patients (P1– P10) for 48 h. Ten different control samples were divided into two groups of mixture of 5 different CPFs (C1 and C2) and added to the culture medium. Cells were stained with FITC-conjugated anti-HLA-DR, -DP, -DQ Abs and expression levels were analyzed by flow cytometry. Data are expressed as percentages of stained cell on total cells (10⁴ cells). Results are the means ± SD of three separate experiments.

Fig. 2. Dose-dependent down-regulation of MHC-II expression level by EPF. THP-1 cells were cultured for 48 h in RPMI-1640 medium containing various concentrations of mixtures of 5 different EPFs (P1–P5). The cells were then harvested and the surface expression levels of MHC-II were measured, as shown in Fig. 2. Results are means ± SD of three separate experiments.

Fig. 3. Effect of EPF on the mRNA and protein levels of HLA-DR in THP-1 cells. (A) THP-1 cells (1 × 10⁶) were treated with media containing 10% of PF mixtures or 10 ng/ml of IFN-γ for 12 h. In the right panel, densitometric analysis of each band is shown. (B) THP-1 cells (1 × 10⁷) were treated with media containing 10% of mixtures of 5 different CPFs or 5 different EPFs for 24 h. The cell lysates were prepared and total proteins (75 μg) were subjected to SDS–PAGE and immunoblotted using anti-HLA-DR Ab. The results are representative of three independent experiments. (C) THP-1 cells were incubated as shown in panel B, and the cytospin preparations of the cells were stained using anti-HLA-DR Ab. A representative of three separate experiments is shown.

Fig. 4. Effect of EPF on the expression levels of adhesion molecules and costimulatory molecules on monocytes. Monocytes were treated with 10% CPF or 10% EPF for 48 h. The expression levels of MHC-II, CD14, CD54, CD80, and CD86 were then analyzed by flow cytometry using FITC-conjugated specific antibodies. Data are expressed as the percentages of stained cell in total cells (10⁴ cells) compared to stained cells with isotype control Abs (gray filled peaks). The result of a representative experiment from three independent experiments is shown.

Fig. 5. Inverse correlation between MHC-II expression levels on THP-1 cells and the IL-10 concentrations in EPF or cultured supernatant with EPF. (A) Peritoneal concentrations of IL-10, TGF-β, and VEGF in EPF were measured as described in “Materials and Methods”. (B) Cytokine levels in the supernatant of THP-1 cells cultured in 10% PF were measured, as shown in panel A. Each value was obtained from a mean of triplicate assays. The Pearson correlation coefficient was used to determine the nature of the correlation between MHC-II expression levels on THP-1 cells and cytokine levels of EPF (A) or supernatants (B). C: correlation efficient, P: probability.

Fig. 6. Effects of heat-treatment and IL-10 neutralizing antibody on the down-regulation of MHC-II expression level by EPF. (A) EPF mixture was pre-heated for 5 min at 90 °C and added to the media containing THP-1 cells (heat-treated). The cells were pre-treated with (PTX + EPF) or without (EPF) pertussis toxin (1 μg/ml) for 4 h at 37 °C and treated with 10% of EPF mixture (n = 5). As a negative control, the cells were treated with 10% of CPF mixture (n = 2). (B) The THP-1 cells were pre-treated with 1 μg/ml of control IgG, anti-IL-10, and anti-VEGF Abs for 1 h and treated with 10% of EPF mixture. After 48 h, cells were harvested and the MHC-II expression level was measured, as described in Fig. 1. Results are means ± SD of three separate experiments. *p < 0.05 vs. EPF alone.

Fig. 7. Proliferation of T cells in the presence of SEB and monocytes pre-treated with EPF. The monocytes were pre-treated with 10% of EPF mixture from 5 patients for 48 h, treated with mitomycin c, and then mixed with T cells (1 × 10⁵) at the indicated ratios. T cells alone, monocytes alone, and cell mixtures were cultured in the presence or absence of SEB (1 μg/ml) for 24 h and then further cultured for 18 h after adding 1 μCi [³H]thymidine. The incorporation of [³H]thymidine was determined using a scintillation counter. Results are presented as the means ± SD of triplicate cultures. *p < 0.05 vs. without EPF.

Fig. 8. Effect of EPF on cytokine secretion in culture of monocytes and T cells and the expression levels of negative costimulatory molecules on monocytes. (A) EPF-treated or CPF-treated monocytes were mixed with T cells (1 × 10⁵) at the indicated ratios and cultured for 48 h. The supernatants were withdrawn and the IFN-γ (left panel) and IL-12 (right panel) concentrations were measured by ELISA. The results are presented as a mean ± SD of triplicate assays from two separate cultures. (B) The monocytes were treated with or without 10% of the EPF or CPF mixture for 48 h. The expression levels of B7-H1 and -H2 were then analyzed by flow cytometry using FITC-conjugated specific Abs. The staining of the cells with isotype control Abs was shown as gray filled peak. The result of a representative experiment from three independent experiments is shown. *p < 0.05 compared with CPF.