In the present study we used a previously published ex-vivo
model that resembles the presence of two independent compartments with a fully functional chorioamnion; its upper chamber is delimited by the choriodecidua and the lower chamber by the amnion, allowing us to test both compartments simultaneously [13
]. We included three different conditions of membrane stimulation with Escherichia coli
, resembling clinical scenarios. 1) Microorganisms arrive to the choriodecidua through ascendant colonization (choriodecidua stimulation), 2) bacteria reach the amnion side by iatrogenic introduction during amniocentesis (amnion stimulation), and 3) bacteria are in contact with both sides of the membranes at late stages of colonization, which enables them to cross the membranes.
With this system, we were able to show that the basal secretions of IL-1β, IL-6, IL-10, and IL-8 by the choriodecidual tissue increased significantly (P < 0.05) in response to Escherichia coli stimulation, regardless of whether the stimulus was applied directly to this region, the amnion, or to both regions simultaneously. In addition, TNFα increased markedly in both the choriodecidua and the amnion sides when the bacterium was applied to both membranes at the same time. IL-1β rose mildly when the amnion was directly stimulated by Escherichia coli whereas IL-8 secretion rose markedly in the amnion regardless of the primary side stimulated. A marked rise in TNFα secretion in the amnion side was also observed but only when the bacterium was simultaneously applied to the amnion and choriodecidua tissues.
Previous works have evidenced that inoculation of the amniotic cavity with Escherichia coli
induces a toxic response, characterized by the overproduction of pro-inflammatory cytokines [20
], such as IL-1β, a potent immunomodulator able to induce preterm labor after its experimental infusion in pregnant rhesus monkeys [22
]. However, the used animal models do not allow characterizing the particular and specific contribution of each of the chorioamniotic membranes in this response and cannot be compared with our results. In addition, we demonstrated that in vitro
stimulation of chorioamniotic membranes with lipopolysaccharide (LPS) and Streptococcus agalactiae
induces IL-1β increase in the choriodecidua region [13
On the other hand, the IL-6 concentration in the amniotic fluid is considered a marker of intra-amniotic inflammation and is frequently associated with an infectious process in either the amniotic fluid or the chorioamniotic space [23
]. Previous evidence has demonstrated that the concentration of this cytokine is increased in human decidua [25
] and chorion cells [26
] after treatment with IL-1β and TNFα.
Similar findings have been obtained with IL-10, an anti-inflammatory cytokine, to which a role as therapeutic factor in preterm labor has been ascribed [27
] and whose concentration increases in patients with preterm labor associated with intrauterine infection [28
The differential secretion capacities of pro-inflammatory cytokines by the amnion and the choriodecidua after Escherichia coli
stimulation here reported suggest the existence of a complex interactive regulation network. There is evidence indicating that IL-1β [29
], TNFα [30
], IL-6 [31
], and IL-10 [32
], in different gestational tissues, are able to induce biosynthesis and secretion of prostaglandin E2
) and PGF2α
; these uterotonic factors play key roles in the onset and progression of labor [33
] in normal and pathological conditions. In a previous work, we demonstrated that the amnion's epithelium is the main source of PGE2
secretion after stimulation with Candida albicans
, yeast that has been associated with cervico-vaginal infections [14
Under the experimental conditions used in our study, it was clear that IL-1β, IL-6, and IL-10 were mainly secreted to the choriodecidual compartment regardless of the primary zone stimulated with Escherichia coli. Interestingly, when the amnion was primarily stimulated, its contribution to increase the secretion of theses cytokines was small if any; whereas the secretion of theses cytokines by the choriodecidual region was significantly increased. This observation suggests the existence of a communication or "cross-talk" between both regions and, thus, it is possible that the amnion's epithelium might be an important indirect factor in the whole cytokine response.
On the other hand, TNFα, whose negative effects on the course of pregnancy have been characterized [34
], increases in the amniotic fluid of women with preterm labor and intra-amniotic infection [30
]. There is also experimental evidence demonstrating that the administration of a TNFα bolus to pregnant animals causes profuse hemorrhage and pregnancy termination [36
]. In our model, the increase in TNFα secretion in both the amnion and choriodecidua compartments was significant (P < 0.05) only after simultaneous stimulation of both membrane sides. A possible interpretation of the biological significance of these findings is that, since TNFα is a pro-inflammatory cytokine with major immuno-toxic properties, the chorioamniotic membrane coordinates its secretion in response to a very complicated infectious scenario, as represented by chorioamnionitis, in which both the fetal and maternal sides are insulted by an infectious agent.
It is possible that the secretion of IL-1β, IL-6, IL-10, and TNFα in the choriodecidua region after infection with Escherichia coli
would favor their trans-membranal translocation to the amnion [38
] and thereby exert their effect on the whole membrane.
The IL-8 secretion pattern was also interesting, because both the amnion and the choriodecidua were active in the secretion of this chemokine. This ubiquitous production might play a key role in the recruitment and activation of professional cells of the immune system, such as neutrophils whose migration toward the cervix, the placenta, and chorioamniotic membranes is a clinical/histological characteristic of infection/inflammation [39
]. The present results show that the IL-8 response in the amnion was mild as compared to the choriodecidua region, which is the first tissue to be colonized by the microbial pathogen during an ascending intrauterine infection and is the main barrier to progression of infection into the amniotic cavity. Therefore, the tissue-specific capacities could be important factors in determining the severity of the inflammation in fetal membranes infected with Escherichia coli
It is tempting to hypothesize that Escherichia coli infection of the chorioamniotic membrane may induce a precocious onset of the overproduction of pro-inflammatory cytokines (an "anticipation" of the normal parturition cascade?), leading to PROM and preterm labor.