Oestradiol regulation of antigen presentation by uterine stromal cells: role of transforming growth factor-beta production by epithelial cells in mediating antigen-presenting cell function

Abstract

We have previously shown that oestradiol treatment of ovariectomized rats for 3 days inhibits antigen presentation by uterine stromal cells at a time when oestradiol increases the numbers of antigen-presenting cells (APC) in the uterine stroma. In the present study, we found that oestradiol treatment for 1 day is sufficient to inhibit antigen presentation by stromal cells. To define the mechanism(s) of this inhibition, we examined the effect of cytokines and found that exogenous transforming growth factor-beta (TGF-beta) inhibits antigen presentation when stromal cells from saline- but not oestradiol-treated animals are incubated with ovalbumin (OVA)-specific T cells and OVA. In contrast, antigen presentation by uterine epithelial cells was not affected by TGF-beta. In other studies, the acute inhibitory effect of oestradiol (1 day) on stromal antigen presentation is fully reversed when anti-TGF-beta antibody is added to the culture media. When given for 3 days, oestradiol inhibition of antigen presentation is partially reversed by anti-TGF-beta antibody at a time when antibodies to tumour necrosis factor-alpha and interleukin-10 have no effect. To determine whether uterine epithelial cells produce TGF-beta, epithelial cells were grown to confluence on transwell inserts. Our findings indicate that uterine epithelial cells produce biologically active TGF-beta which is preferentially released basolaterally in the direction of underlying stromal cells. When oestradiol is given to ovariectomized rats 1 day before sacrifice, TGF-beta production by epithelial cells increases within 24 hr in culture, relative to saline controls. Taken together, these results suggest that oestradiol inhibition of stromal cell antigen presentation is mediated through the stimulatory effect of oestradiol on TGF-beta production by epithelial cells.

Figure 1

Influence of oestradiol on antigen presentation by uterine stromal cells from ovariectomized rats treated with saline (100 µl) or oestradiol (2 µg) for 3 days (a) or 1 day (b) prior to death. Uteri (seven to eight animals/group) were pooled and stromal APC were prepared as described in Materials and Methods. Stromal APC (1 × 10⁵ cells/100 µl) were incubated with OVA-sensitized T cells (1 × 10⁵ cells/100 µl) and OVA (300 µg/ml) for 3 days. [³H]Thymidine was added for the last 24 h of incubation. Values shown are mean ± SE of three wells per group. (Number of experiments: n = 3–5). **Significantly different from saline control (P < 0.01).

Figure 2

Effect of TGF-β on antigen presentation by uterine stromal cells from saline- and oestradiol-treated rats. Isolated stromal cells (APC) from ovariectomized rats (seven to eight animals/group) treated with saline (100 µl) or oestradiol (2 µg) for 1 day were prepared and incubated with OVA-sensitized T cells and OVA for 3 days. TGF-β (0·01, 0·1, 1·0 and 10 ng/ml, final concentration) was added to each well at the start of culture. [³H]Thymidine was added for last 24 hr of incubation. Values shown are mean ± SE of three wells per group. (n = 3) *Significantly different from control (P < 0·05). **Significantly different from control (P < 0·01).

Figure 3

Effect of TGF-β on antigen presentation by stromal cells from rats treated with oestradiol for 3 days. Ovariectomized rats (seven to nine animals/group) received three injections daily (100 µl) of oestradiol (2 µg/rat) or saline prior to death 24 hr after the third injection. Uterine stromal cells from saline- and oestradiol-treated rats were incubated with OVA-specific T cells and OVA, along with TGF-β (10 ng/ml) for 3 days with the addition of [³H]thymidine for the last 24 hr. Values are mean ± SE of three wells per group. (n = 2) *Significantly lower than control group (P < 0·05). **Significantly lower than control (P < 0·01).

Figure 4

Effect of anti-TGF-β1, -2, -3 antibody on antigen presentation by uterine stromal cells from rats treated with oestradiol for either 1 day (a) or 3 days (b) prior to death. Ovariectomized rats received one to three injections daily (100 µl) of oestradiol (2 µg/rat) or saline prior to death 24 hr after the last injection. Stromal cells from saline- and oestradiol-treated rats were incubated with OVA-specific T cells and OVA, along with anti-TGF-β antibody (1 µg/ml) or an isotype (I) control (IgG1; 1 µg/ml), for 3 days with the addition of [³H]thymidine for the last 24 hr. Values are mean ± SE of three wells per group. (n = 2). *Significantly higher than isotype-control group (P < 0·05). **Significantly higher than isotype control (P < 0·01).

Figure 5

Influence of anti-TNF-α antibody and anti-IL-10 antibody on antigen presentation by uterine stromal cells from saline- and oestradiol-treated rats. Ovariectomized rats received three injections at 24-hr intervals of oestradiol (2 µg/rat) or saline (100 µl) prior to death 24 hr after the last injection. APC and T cells were incubated with OVA for 3 days in the presence of (a) anti-TNF-α antibody (80 Units/ml); or (b) anti-IL-10 antibody (2·5 µg/ml). Anti-TNF-α antibody purchased from Genzyme was neat hyperimmune rabbit antiserum which was diluted 1 : 1250 to correspond to an antibody binding capacity of 80 Units/ml. Control normal rabbit serum was similarly diluted. Anti-IL-10 control wells received matched isotype at the same concentration. ³H-thymidine was added for last 24 hr of incubation. Values shown are mean ± SE of three wells per group. (n = 2).

Figure 6

Secretion of TGF-β by polarized uterine epithelial cells in culture. Epithelial cells from intact adult rats were isolated and cultured for 4 days in cell inserts (eight inserts) as described in Materials and Methods. Culture media in apical and basolateral compartments was replaced at 48 h intervals prior to collection on day 4. Following centrifugation at 10 000 g for 5 min and storage at −20°, samples were assayed for TGF-β by bioassay as described in Materials and Methods. Uterine epithelial cells formation of tight junctions was determined by TER measurement in which baseline resistance was 55–60 ohms cm². (n = 3) **Significantly higher than TGF-β present in apical media (P < 0·001).

Figure 7

Effect of oestradiol on TGF-β production by uterine epithelial cells in culture. Ovariectomized rats received one injection (100 µl) of oestradiol (2 µg/rat) or saline prior to death 24 hr later. Isolated epithelial cells (3·5 × 10⁵ cells/900 µl) from saline- and oestradiol-treated rats were cultured for 1, 2 and 3 days in RPMI-1640 media with 10% FBS. Media was collected and replaced from four wells per group at 24 hr intervals, centrifuged at 10 000 g for 5 min and stored at −20° until assayed for TGF-β. (n = 2) *Significantly greater than saline controls (P < 0·05). **Significantly greater than control wells (P < 0·005).