Inflammatory cytokines differentially up-regulate human endometrial haptoglobin production in women with endometriosis

Abstract

Background: Evidence suggests that eutopic endometrium from women with endometriosis (US-E) has intrinsic functional anomalies compared with women without endometriosis (US-C). We hypothesized that differences in endometrial haptoglobin (eHp) mRNA and protein levels exist between eutopic endometrium from US-E and US-C and that inflammatory mediators may be involved.

Methods: Endometrial stromal cells and tissue explants from US-E (n = 18) and US-C (n = 18) were cultured (24 h/48 h for cells/explants) with interleukin (IL)-1alpha, -1beta, -6, -8 or tumor necrosis factor-alpha (TNF-alpha) at 0-100 ng/ml. eHp protein in media and mRNA levels were quantified by enzyme-linked immunosorbent assay and quantitative PCR.

Results: In eutopic endometrial stromal cells from US-E, IL-1beta, IL-6 and TNF-alpha (10 ng/ml) increased eHp mRNA levels (P = 0.002, P < 0.001 and P < 0.001, respectively) and eHp protein (P = 0.023, 0.031 and 0.006, respectively) versus control. In endometrial tissues from US-E, IL-1beta, IL-6 and TNF-alpha increased eHp mRNA (P < 0.001, P = 0.017 and P < 0.001, respectively) and eHp protein (P < 0.001, P = 0.007 and 0.039, respectively) versus control. IL-1alpha and IL-8 had small or no effects on isolated endometrial cells or tissues. In US-C, IL-1beta, IL-8 and TNF-alpha each reduced eHp mRNA in endometrial stromal cells (all P < 0.001) versus control; IL-1alpha and IL-6 had no effect. eHp mRNA increased in endometrial tissues from US-C in response to IL-1beta (P = 0.008), IL-6 (P = 0.015) and TNF-alpha (P = 0.031) versus control; IL-1alpha or IL-8 had no effect.

Conclusions: Endometrium from US-E differentially responds to specific inflammatory cytokines by production of eHp. We propose that up-regulation of endometrial eHp by inflammatory mediators disrupts normal endometrial function and may facilitate the pathogenesis of endometriosis.

Figure 1

Fold change in eHp protein production by endometrial stromal cells from women with endometriosis (US-E) in response to IL-1α, IL-1β, IL-6, IL-8 or TNF-α. Each horizontal interval bar depicts the estimated 95% confidence interval (CI) for the mean fold change for a given dose of cytokine (0.1, 1, 10 or 100) versus no dose. The black circle in each interval is the estimated mean fold change. A broken vertical line is shown at fold change = 1, as this aids in the interpretation of the CIs. Intervals which overlap the broken line imply that the associated dose for that cytokine is not significantly different from the control (α = 0.05). The fold change was tested using the least-square means of pairwise differences.

Figure 2

Relative fold change in eHp protein production (A, B) and mRNA levels (C, D) from endometrial stromal cells (A, C) and endometrial tissue explants (B, D) from women with (gray bars) and without (white bars) endometriosis in response to 0 or 10 ng/ml cytokine treatment. The mRNA levels were quantified by real-time PCR and calculated using the 2^−ΔΔCt method, relative to no treatment and therefore by nature are log base two. Hence for comparison purposes, the eHp protein data [eHp (pg/ml)/total protein (mg/ml)] were transformed to log base two. (Fold = eHp at 10 ng/ml cytokine/eHp at 0 ng/ml cytokine). Both isolated US-E (A) and endometrial tissue explants (B) from women with endometriosis (gray bars) produced more significant eHp protein (A, B) and had higher levels of eHp mRNA (B, D) in response to 10 ng/ml IL-1β, IL-6 and TNF-α, but not IL-1α or IL-8 compared with culture media without cytokines and compared with women without endometriosis (A, B, C, D; white bars). Large (>4-fold; P < 0.001) decreases in eHp mRNA levels were noted when US-C (C, white bars) were treated IL-1β (P < 0.001), IL-8 (P < 0.001) or TNF-α (P < 0.001). Each bar represents the mean and SE analyses from samples of 4 to 6 women per cytokine (Raw data shown in Table 1).