Corresponding results were obtained when: 1) the incubation period with TNF and IL1B was extended to 48 h; 2) the cytokine concentration was increased to 10 ng/ml; and 3) cultures were not treated with steroids.
To determine whether this differential response to inflammatory cytokines was present throughout gestation, TNF and IL1B were tested on cultured first trimester decidual cells.
Consistent with our observations for term decidual cells, in first trimester decidual cell cultures maintained in E2 or E2 + MPA, 1 ng/ml of TNF or IL1B increased the CSF1 output (Fig. 4), whereas these cytokines did not significantly alter the MIF protein levels (Fig. 5).
Given the absence of a steroid effect, further evaluations of the effects of TNF and IL1B on CSF1 expression were carried out with term decidual cells that were primed with E2 + MPA. The dose-response relationships of CSF1 to each cytokine added at concentrations between 0.1 and 10 ng/ml are shown in Figure 6. Both TNF (A) and IL1B (B) elevated CSF1 output over the entire range of concentrations tested, with maximum effects for both cytokines obtained at 10 ng/ml.
Following experimental incubations of the leukocyte-free term decidual cells, the levels of CSF1 and MIF mRNA were determined in extracted RNA samples by quantitative RT-PCR. As indicated in Figures 7 and 8, changes in the patterns of CSF1 and MIF mRNAs corresponded to those established for their respective proteins. Thus, TNF and IL1B each induced a statistically significant increase in CSF1 mRNA levels, irrespective of whether they were added with E2 or with E2 + MPA, whereas in the absence of cytokines, similar CSF1 mRNA levels were observed in parallel incubations with E2 or with E2 + MPA (Fig. 7). In contrast, the MIF mRNA levels were not significantly altered by TNF, IL1B or MPA (Fig. 8).
FIG. 6. Dose-response effects of TNF (A) and IL1B (B) on CSF1 production by term decidual cell monolayers maintained in the presence of E2 + MPA. Confluent decidual cells were incubated for 7 days in 10~8 M E2 + 10~7 M MPA, then switched to DM with the steroids with or without the indicated amount of TNF or IL1B. The CSF1 levels in conditioned DM were measured by ELISA and normalized to the level of cell protein (n = 3, mean ± SEM). **, versus E2 + MPA (P < 0.05).
FIG. 7. Quantitative RT-PCR of effects of E2 MPA, TNF, and IL1B on CSF1 mRNA levels in term decidual cell monolayers. Confluent decidual cells were incubated for 7 days with 10~8 M E2 or with E2 + 10~7 M MPA, and then switched to DM with the corresponding steroid(s) with or without 1 ng/ml of TNF or IL1B for 5 h. The CSF1 mRNA levels were measured by quantitative RT-PCR and normalized to the ACTB mRNA levels. Ordinate: CSF1 mRNA/ACTB mRNA. *, versus E2; **, versus E2 + MPA (P < 0.05, mean ± SEM;n = 3).
FIG. 8. Quantitative RT-PCR analysis of the effects of E2, MPA, TNF, and IL1B on MIF mRNA levels in term decidual cell monolayers. Confluent decidua cells were incubated for 7 days with 10~8 M E2 or with E2 + 10~7 M MPA, and then switched to DM with the corresponding steroid(s) with or without 1 ng/ml of TNF or IL1B for 5 h. The MIF mRNA levels were measured by quantitative RT-PCR and normalized to the ACTB mRNA levels. Ordinate: MIF mRNA/ACTB mRNA (mean ± SEM;n = 3).