IL-6 trans-signaling system in intra-amniotic inflammation, preterm birth, and preterm premature rupture of the membranes

Abstract

Classic IL-6 signaling is conditioned by the transmembrane receptor (IL-6R) and homodimerization of gp130. During trans-signaling, IL-6 binds to soluble IL-6R (sIL-6R), enabling activation of cells expressing solely gp130. Soluble gp130 (sgp130) selectively inhibits IL-6 trans-signaling. To characterize amniotic fluid (AF) IL-6 trans-signaling molecules (IL-6, sIL-6R, sgp130) in normal gestations and pregnancies complicated by intra-amniotic inflammation (IAI), we studied 301 women during second trimester (n = 39), third trimester (n = 40), and preterm labor with intact (n = 131, 85 negative IAI and 46 positive IAI) or preterm premature rupture of membranes (PPROM; n = 91, 61 negative IAI and 30 positive IAI). ELISA, Western blotting, and real-time RT-PCR were used to investigate AF, placenta, and amniochorion for protein and mRNA expression of sIL-6R, sgp130, IL-6R, and gp130. Tissues were immunostained for IL-6R, gp130, CD15(+) (polymorphonuclear), and CD3(+) (T cell) inflammatory cells. The ability of sIL-6R and sgp130 to modulate basal and LPS-stimulated release of amniochorion matrix metalloprotease-9 was tested ex vivo. We showed that in physiologic gestations, AF sgp130 decreases toward term. AF IL-6 and sIL-6R were increased in IAI, whereas sgp130 was decreased in PPROM. Our results suggested that fetal membranes are the probable source of AF sIL-6R and sgp130. Immunohistochemistry and RT-PCR revealed increased IL-6R and decreased gp130 expression in amniochorion of women with IAI. Ex vivo, sIL-6R and LPS augmented amniochorion matrix metalloprotease-9 release, whereas sgp130 opposed this effect. We conclude that IL-6 trans-signaling molecules are physiologic constituents of the AF regulated by gestational age and inflammation. PPROM likely involves functional loss of sgp130.

Figure 1. Diagram illustrating the downstream pathways of IL-6 signaling

In the classic pathway, binding of the IL-6 to its transmembrane receptor IL-6R induces dimerization of the signal transducing receptor subunit gp130 (A). In the trans-signaling pathway, IL-6 binds a soluble form of IL-6R (sIL-6R). The complex IL-6/sIL-6R complex enables activation of cells expressing solely gp130 (B). A soluble form of gp130 (sgp130) selectively blocks trans-signaling (C).

Figure 2. Relationships between amniotic fluid (AF) levels of IL-6, sIL-6R, sgp130, sIL-R/ sgp130 molar ratio and gestational age (GA) in pregnancies with normal outcomes (n=110)

We restricted this analysis to amniocenteses samples from women with normal outcomes: second trimester genetic (n=39), third trimester lung maturity (n=40) and preterm women with amniocentesis to rule-out infection who ultimately delivered at term (n=31). Data for IL-6 (x-axis of A) and sIL-6R (x-axis of B) demonstrated no significant correlations between the levels of these two analytes and GA (y-axes of all). The AF levels of sgp130 were inversely correlated with GA with significantly lower levels at term (x-axis of C). Using the two-step clustering method, we identified unbiased GA separation points that partitioned AF sgp130 concentration into 3 clusters: “high”: 104 [100–120] ng/mL, GA: 22 [18–26] weeks “mid”: 67 [60–74] ng/mL, GA: 27 [19–35] weeks and “low”: 39 [34–45] ng/mL, GA: 35 [31–37] weeks. There was a significant GA regulation of the sIL-6R/sgp130 molar ratio (x-axis of D) with higher values at term. This trend was primarily due to the decrease in sgp130 as gestation progressed. For each graph the Spearman correlation coefficient R and the level of statistical significance is shown. Data presented in logarithmic format. Panels A-D: Thick lines: 1st order regression line; Thin continuous lines: 95% confidence intervals; dotted lines: 95% prediction intervals.

Figure 3. Levels of amniotic fluid (AF) IL-6, sIL-6R, sgp130 and sIL-6R/sgp130 molar ratio in pregnancies complicated by intra-amniotic inflammation (IAI) and preterm premature rupture of the membranes (PPROM)

The following groups were studied: no IAI & intact membrane (n=85); no IAI & PPROM (n=61); IAI & intact membrane (n=46); IAI & PPROM (n=30). The AF IL-6 levels were significantly elevated in women with IAI. IL-6 concentrations were lower in women with IAI & PPROM compared to those with IAI & intact membranes (A). The levels of AF sIL-6R were elevated in women with IAI independent of PPROM (B). Pregnancies complicated by PPROM were characterized by lower AF levels of sgp130 and this phenomenon was observed in both pregnancies complicated or not by IAI (C). The sIL-6R/sgp130 molar ratio was significantly elevated by PPROM and further elevated in the setting of PPROM & IAI (D). All results maintained following correction for GA. Different letters reflects statistically significant differences among groups. Thick line represents the median value for the group. Data presented in logarithmic format. Data were analyzed by two-way ANOVA.

Figure 4. mRNA (quantitative RT-PCR) and protein (Western blot) expression of IL-6R and gp130

Real time quantitative RT-PCR was used to study tissues retrieved from 9 term (T) third trimester healthy women and a subset of 20 preterm birth (PTB) cases [no intra-amniotic inflammation (IAI) & no histological chorioamnionitis n=5; yes IAI, & yes histological chorioamnionitis n=15]. IL-6R and gp130 mRNA was identified in both fetal membranes and placental villous trophoblast (A). gp130 mRNA was more abundant than the IL-6R mRNA in both fetal membranes and placental villous tissues. In the fetal membranes the gp130 mRNA levels were significantly decreased at term (T). There was a significant increase in IL-6R mRNA (B) and a significant decrease in the expression of gp130 (C) in the fetal membranes of women with IAI. Relative quantitation (RQ) ΔCT values are reported relative to expression of the housekeeping genes for each tissue (A). ΔΔCT RQ values were reported relative to a reference RNA pool of the same tissue (B-C). Data presented as mean + SEM and analyzed by one-way ANOVA followed by post-hoc Student-Newman-Keuls tests. Means marked with different superscripts are statistically significant (P<0.05). Panels D and E show representative Western blots of AF (AF), fetal membrane (FM) and placental (Plac) proteins. Membranes were probed with compatible polyclonal anti-IL-6R (D) or monoclonal anti-gp130 antibody (E). Specificity was confirmed in identical blots with omitted primary antibodies.

Figure 5. Representative photomicrographs of IL-6R and gp130 immunoreactivity in fetal membranes and placental sections of preterm and term pregnancies

Preterm amnion (am) stained higher for IL-6R in the presence of IAI compared to no IAI (A-B) and term fetal membranes (C). Intense IL6-R immunostaining was identified in extravillous trophoblasts (evt, arrows) and decidual cells (open arrowheads) of the choriodecidua (cd) as identified by cytokeratin or vimentin positive staining (data not shown). In the placenta (D), IL-6R was identified in evt and villous synctiotrophoblasts (st) without notable changes with either IAI or GA (data not shown). Localization of IL-6R staining in extravillous trophoblasts (evts) was both peri-membranar and intra-cytoplasmic consistent with the ability of the antibody to recognize both membrane bound IL6-R and sIL-6R. The amnion (am) stained less positive for gp130 than choriodecidua (cd) with no discernable differences with histological inflammation or GA (E-G). Both extravillous trophoblasts (arrows) and decidual cells (open arrowheads) stained intensely for gp130 in the absence of histological inflammation (E). In the placenta (H), the strongest gp130 signal was noted in villous cytotrophoblasts (ct) and extravillous trophoblasts (evt, arrows) of chorionic plate. The outlined area in B is shown in panels I&J at higher magnification aimed to illustrate concurrent homing of neutrophils (CD15⁺) (I) and T lymphocytes (CD3⁺, closed arrowheads) (J) cells in the inflammatory infiltrate of the choriodecidua (cd) of women effected by IAI. CD15⁺ and CD3⁺ cells were also identified in the vicinity of choriodecidual (cd) blood vessels (bv) suggestive of trans-endothelial migration of inflammatory cells (L-M). Specificity of staining was confirmed by incubation of slides with rabbit IgG as control for IL- 6R & CD3 staining (K). Mouse non-immune IgG served as negative control for gp130 and CD15 mono-clonal antibodies (N). IL-6R and gp130 histological scores in the amnion and choriodecidua of preterm (NO & YES IAI) and term patients (T) is shown in panel O and P, respectively. Data presented as mean + SEM and analyzed by one-way ANOVA followed by post-hoc Student-Newman-Keuls tests. Means with at least one common superscript are not statistically significant (P > 0.05). Scale bars: 30 µm for all panels (A-N).

Figure 6. Ex-vivo production of MMP-9 and IL-6 following incubation of the fetal membranes with sgp130, sIL-6R and LPS

Recombinant sIL-6R and LPS stimulated the release of MMP-9 (A). Addition of recombinant sgp130 over sIL-6R or LPS reversed the release of MMP-9. Incubation of the tissues with sgp130, sIL-6R and their combination had no effect of IL-6 release in the conditional media of the amniochorion explant (B). The IL-6 levels were significantly upregulated following incubation with LPS. sgp130 did not impact the stimulatory effect of LPS. These results indicate that sIL-6R and sgp130 modulate MMP-9 activity through mechanisms independent of IL-6 levels in the system. Data presented as mean + SEM of 7 independent experiments and analyzed by one-way ANOVA followed by post-hoc Student-Newman-Keuls tests. Means with at least one common superscript are not statistically significant (P > 0.05).