Timothy A. Sato

Cytokine abundance in placental tissues: Evidence of inflammatory activation in gestational membranes with term and preterm parturition ☆ ☆☆ ★

OBJECTIVES This study of the changes in cytokine concentrations in gestational tissues from women with term and preterm labor was undertaken to assess the extent of inflammatory activation associated with spontaneous labor and delivery. STUDY DESIGN Extracts of amniotic, chorionic-decidual, and placental tissues from women delivered at term before labor (n = 15), at term after labor (n = 15), and preterm (n = 31) were assayed for interleukin 1beta, interleukin 6, and interleukin 8. RESULTS In amniotic tissues of women delivered by spontaneous labor at term the median interleukin-6, interleukin-8, and interleukin-1beta concentrations were 3.8 to 5.4 times those of tissues from women delivered at term without labor (P <.05, Mann-Whitney U test). Interleukin-6 and interleukin-8 concentrations were also significantly increased (3. 3-4 times) in chorionic-decidual tissues. Marked increases (approximately 3-6 times) in the concentrations of all 3 cytokines were observed in both amniotic and chorionic-decidual tissues from women with preterm deliveries with respect to those from women with term deliveries after labor. Cytokine concentrations were significantly correlated within amniotic tissues from both women with term delivery after labor and women with preterm delivery and also in preterm chorionic-decidual tissues but not preterm placental tissues. Concentrations of cytokines in the tissues of women delivered preterm were not significantly affected by mode of delivery, treatment with antibiotics, or twin birth. In preterm tissues with evidence of intrauterine infection only amniotic interleukin-1beta concentrations were significantly elevated (P <. 05). Little or no labor-related change in cytokine concentrations was seen within placental tissues. CONCLUSIONS Increased cytokine abundance in gestational membranes associated with labor supports the view that an inflammatory process is involved in both term and preterm labor. This process does not, however, appear to be evident in the villous placenta.

Critical Paracrine Interactions Between TNF-α and IL-10 Regulate Lipopolysaccharide-Stimulated Human Choriodecidual Cytokine and Prostaglandin E2 Production

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 μg/ml) induced a rapid increase in TNF-α production, peaking at 4 h. In contrast, IL-10, IL-1β, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-α was a primary regulator of IL-1β, IL-10, and PGE2 production, while IL-1β stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-α and PGE2 production. IL-10 treatment markedly decreased TNF-α and IL-1β production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-α may be a potential target for treatment intervention; IL-10 could be one such therapeutic.

Epithelial Cell-Derived Neutrophil-Activating Peptide-78 Is Present in Fetal Membranes and Amniotic Fluid at Increased Concentrations with Intra-amniotic Infection and Preterm Delivery

Abstract Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased (∼4-fold) with term labor in amnion (n = 15), and with preterm labor (∼30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r2 = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1β and tumor necrosis factor α. Production of ENA-78 by choriodecidual explants was increased modestly after 2–4 h of exposure to lipopolysaccharide (5 μg/ml). An immunoreactive doublet (∼8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.

Hypothermia suppresses inducible nitric oxide synthase and stimulates cyclooxygenase-2 in lipopolysaccharide stimulated BV-2 cells

Hypothermia is neuroprotective, possibly through suppression of microglial activation. We investigated the effects of hypothermia on lipopolysaccharide (LPS) stimulated BV-2 cells. At 37 degrees C, LPS elicited strong increases in inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6), accompanied by translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus. Hypothermia (33 degrees C) caused complete suppression of iNOS and NO, a partial reduction of IL-6 but did not prevent TNF-alpha production or NF-kappaB translocation. In contrast, LPS induced cyclooxygenase-2 (COX-2) to higher levels under hypothermic conditions. These results show that hypothermia selectively suppresses iNOS in microglia.

Anti‐inflammatory Effects of Interleukin‐4, Interleukin‐10, and Transforming Growth Factor‐β on Human Placental Cells In Vitro

PROBLEM: To determine the effects of anti‐inflammatory cytokines on the production of inflammatory mediators by placental cells.

Delayed allograft rejection in mice transgenic for a soluble form of the IL-4 receptor

Accumulating evidence suggests that in serum and other biological fluids, cytokine binding is a property associated with soluble proteins, including a high-affinity soluble version of the IL-4 receptor (sIL-4R). While it is tempting to speculate that sIL-4R might act as a serum carrier protein or serve to inhibit or modulate IL-4 action, specific biological roles for sIL-4R remain to be established. To further assess the immunoregulatory and therapeutic potential of sIL-4R and other soluble receptors, we have created transgenic mice which constitutively express elevated levels of biologically active sIL-4R. Phenotypic characterization of lymphoid organs in sIL-4R transgenic mice revealed normal numbers of B and T cells and normal surface marker expression. Splenic lymphocytes displayed normal in vitro activities as measured by the PFC response and generation of cytotoxic T cells. In addition, antigen-specific IgE and IgG1 in vivo responses were similar in control and transgenic mice. Despite the apparent developmental normality of the sIL-4R transgenic mice, these animals were markedly deficient in the ability to reject cardiac allografts, suggesting that IL-4 is critical for the generation of alloreactivity. The results further suggest that the ability of sIL-4R to regulate IL-4 activities may be under the control of complex interactions that remain to be elucidated.

15-deoxy-Δ12,14-prostaglandin J2-induced apoptosis in amnion-like WISH cells

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-Delta(12,14)PGJ(2) (15d-PGJ(2)), Delta(12)PGJ2(,) ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ(2) (10 muM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50 of approximately 7 muM 15d-PGJ(2). Delta(12)-PGJ(2) also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ(2) and Delta(12)PGJ(2), which may be independent of PPAR-gamma activation, requires further elucidation

Regulation of interleukin (IL)-6 and IL-8 production in an amnion-derived cell line by cytokines, growth factors, glucocorticoids, and phorbol esters.

PROBLEM: To determine whether amnion cells produce interleukin (IL)−6 and −8 and thus may contribute to the high concentrations of these cytokines in amniotic fluid at term.

In Vivo Biological Effects of Recombinant Soluble lnterleukin-4 Receptor

Abstract We investigated the role of soluble interleukin-4 receptor (slL-4R) as a regulator of IL-4 mediated activities in vivo. Administration of recombinant sIL-4R to mice resulted in (i) prolonged survival of heterotopic cardiac allografts; (ii) decreased popliteal lymph node enlargement in response to allogeneic cells; and (iii) inhibition of IgE secretion in response to anti-lgD treatment. Transgenic mice constitutively expressing elevated levels of biologically active SIL-4R displayed prolonged cardiac allograft survival compared with control animals. However the SIL-4R transgenic mice were capable of mounting normal antigen-specific IgE responses despite the presence in serum of up to 3 μg/ml slL-4R. Surprisingly, coadministration of IL-4/slL-4R or IL-4/anti-IL-4 mAb complexes caused a superinduction of IgE secretion in anti-lgD-treated normal mice and subsequently in other IL-4-dependent biological activities. Thus, recombinant slL-4R can not only antagonize functions mediated by endogenous IL-4, but also potentiate the biological activity of exogenously administered IL-4. These dual roles may have possible clinical implications for the recombinant molecule, as well as for natural slL-4R immunoregulation.

Prostanoid stimulation of cytokine production in an amnion-derived cell line: Evidence of a feed-forward mechanism with implications for term and preterm labor

Objective: To test the hypothesis that amnion cytokine production might be regulated by prostanoids. Methods: Amnion-derived WISH cells were treated with a range of prostanoids and their effects on production of interleukin (IL)-6 and IL-8 were determined by enzyme-linked immunosorbent assay and Northern analysis. The effect of thromboxane inhibitors on cytokine production by term primary amnion explants also were examined. Results: Prostaglandin (PG)A2, PGD2, PGF2α PGE2, PGJ2, and the PGI2 analogue carbaprostacyclin (1-1000 nmol/L) exhibited no significant effects on cytokine production. However, the thromboxane A2 (TXA2) agonists U46619 and carbocyclic (c)TXA2 both stimulated WISH cytokine production with similar potencies under basal or cytokine-stimulated conditions. Significant stimulation of IL-6 production was observed at concentrations ≥8 nmol/L (P < .05 by analysis of variance), whereas IL-8 production was stimulated significantly but to a lesser extent. The effects of U46619 and cTXA2 were rapid; maximal stimulation of cytokine production occurred within 4 to 8 hours of treatment. U46619 augmented IL-1β-stimulated IL-6 and IL-8 mRNA expression within 2 hours of treatment. In amnion explants inhibitors of TX synthesis and action abrogated the stimulatory effect of IL-1β on cytokine production. Conclusion: These results are consistent with the presence of a feed-forward loop in amnion involving TXA2 and cytokines, which could play a significant role in the progression of the inflammatory response involved in the mechanism of infection-driven preterm labor.