Preterm birth continues to be a leading cause of perinatal morbidity and mortality in the United States despite intensive research concerning its diagnosis and treatment. In light of the minimal improvement in the rate of preterm birth during the past 40 years, the focus has shifted to understanding the process at the cellular level. As the events leading to preterm delivery have become better understood, it appears that infection plays a prominent role.1 Numerous authors have reported an association between lower genital tract infection and preterm delivery.2,3 Whether these organisms are directly responsible for upper genital tract infections or facilitate colonization by other pathogens has been a topic of debate. Nonetheless, upper genital tract colonization resulting in infection of gestational tissues is clearly linked to preterm birth.4
Inflammatory cytokines are widely viewed as mediators of preterm labor because of infection and are elevated in amniotic fluid of patients with intraamniotic infection.5–7 The cytokines commonly cited as mediators of infection-induced preterm labor include interleukin-1β,6 tumor necrosis factor-α,5 interleukin-6,7 and interleukin-8.8 Both interleukin-6 and interleukin-8 have also been proposed as possible clinical markers for chorioamnionitis.9–11 In addition, inflammatory cytokines increase prostaglandin production by gestational tissues,12,13 viewed as a critical step in the initiation and progression of human labor. Most current tocolytic therapies act either at the level of the uterine smooth muscle cells (ie, magnesium sulfate) or through inhibiting prostaglandin production (ie, indomethacin). Because these agents act late in the chain of events leading to preterm labor, this may explain their inefficacy when infection is present.14
Inflammatory processes lead to infection-induced preterm labor; thus, development of an immune-based approach to tocolysis would seem appropriate. Fortunato et al15 recently examined the effects of anti-inflammatory cytokines in blunting the cytokine cascade that leads to prostaglandin production and preterm labor. They reported that interleukin-10 appears to have promise in limiting amniochorionic membrane production of interleukin-8, a cytokine known to be associated with infection-induced preterm labor.14 On the basis of these findings, we hypothesized that interleukin-10 might block the cytokine cascade that leads to preterm labor when infection is present. To test this hypothesis, we examined the effects of interleukin-10 on endotoxin-induced preterm birth using a novel rat model recently established in our laboratory.16
MATERIALS AND METHODS
Before this study, approval of all experimental protocols was obtained from the Animal Use Committee at the University of Mississippi Medical Center. Timed pregnant Sprague-Dawley rats (n = 32) were received on day 12 or 13 of gestation (term = 22 days) and were allowed to recover from transport for a minimum of 48 hours. On day 15, all rats underwent laparotomy under general anesthesia (isofluorane) with placement of an intrauterine catheter in the distal region of one uterine horn (Figure 1).16 The catheters were then tunneled subcutaneously and externalized between the scapulae. After closure of the abdominal wall, the animals were placed back into their cages and allowed to recover from the general anesthesia. On day 17 of gestation, the rats were randomly assigned, with eight in each group to receive saline, 50 μg lipopolysaccharide, 50 μg lipopolysaccharide with 500 ng interleukin-10, or 50 μg lipopolysaccharide followed 24 hours later by 500 ng interleukin-10. Randomization was performed using a random number generator program. The infusions were performed during a 4-hour period in equal volumes of saline (1 mL) through the intrauterine catheter using a Harvard infusion pump (Harvard Instruments, Holliston, MA).
The lipopolysaccharide used for this study was isolated from Escherichia coli, serotype 026:B6 (Sigma Chemical, St. Louis, MO). In the initial study with this model, the effects of a wide range of concentrations (750–25 μg) of lipopolysaccharide were examined (Terrone DA, Rinehart BK, Martin JN Jr, Miller MT, Granger JP, Bennett WA. A rat model of infection-induced preterm labor (Abstract). Am J Obstet Gynecol 1999;180:598). Intrauterine infusion of lipopolysaccharide at concentrations 100 μg or greater resulted in fetal reabsorption or stillbirth. In contrast, treatment with 50 or 25 μg lipopolysaccharide resulted in preterm birth of low birth weight pups, with a live birth rate approximately 50% that of the saline-infused controls.16 We calculated that to obtain a power of 80%, assuming a 24-hour difference in the treatment to delivery times between the test and control subjects, at least six animals would be needed in each group.
The rat recombinant interleukin-10 used for this study was obtained commercially (Biosource International, Wilmington, NC). The dose of interleukin-10 was calculated from previous studies in which 50 ng per conceptus of transforming growth factor-β, a cytokine with similar anti-inflammatory properties as interleukin-10, blocked cytokine-induced of preterm labor in rabbits.17 Because the Sprague-Dawley rats average approximately ten pups per litter, we chose 500 ng per infusion as our treatment dose for this study. In the preliminary studies, intrauterine infusion of this dose of interleukin-10 did not affect the rat pregnancy in terms of interval to delivery, litter size, or pup birth weight. Interleukin-10 was administered concurrently with the lipopolysaccharide challenge or 24 hours later in a volume of 1 mL normal saline.
After administration of the respective treatments, the animals were observed in their individual cages until delivery. All rats were housed in our animal care facilities and given free access to food and water. At the time of delivery, the number of live pups, birth weight, and interval from infusion to delivery were recorded. After delivery, all the animals were killed by an overdose of pentobarbital.
Statistical analysis was performed using analysis of variance, with the Student-Newman-Keuls test used for multiple comparisons.18 A probability value of ≤ .05 was considered statistically significant.
A total of 32 rats were used for this study, with eight animals randomly assigned to each of the four treatment groups. All animals tolerated the surgery and anesthesia well, with no obvious complications. The results of the interval to delivery, number of live pups, and birth weight of the live born pups are detailed in Table 1. A global analysis of variance revealed significant differences among the four groups with respect to the interval from treatment to delivery, live birth rate per animal, and birth weight. Using the Student-Newman-Keuls test for multiple comparisons, the lipopolysaccharide group displayed a reduced interval from treatment to delivery compared with both the lipopolysaccharide plus interleukin-10 groups and the saline controls (P < .05 each). Animals treated with lipopolysaccharide delivered approximately 24 hours earlier than did the controls.
The addition of interleukin-10, either concurrently with or 24 hours after the lipopolysaccharide infectious stimulus, reversed the trend of preterm birth demonstrated in animals treated with lipopolysaccharide alone. No difference in the number of live pups born to the rats receiving either lipopolysaccharide plus interleukin-10 or those receiving saline occurred. In contrast, the lipopolysaccharide endotoxin-treated rats delivered fewer (P < .05) live pups per litter than any of the other three treatment groups.
The infusion of lipopolysaccharide alone also resulted in a reduction in the birth weight of the pups compared with the saline controls (P < .001). In contrast, no statistical difference in the birth weight between the lipopolysaccharide plus interleukin-10 groups and the saline-infused controls was found.
Despite advances in tocolytic agents, identification of patients at greatest risk of preterm birth, and easier access to prenatal care, we have had essentially no impact on the preterm birth rates during the past 40 years. The key to resolving the dilemma of preterm birth may lie in obtaining a better understanding of the processes involved. As we better understand the role that cytokines play in preterm labor with infection, new approaches to treating these women may be developed. Rather than waiting to treat the cytokine-driven prostaglandin production that causes uterine contractions, an approach focused on preventing the induction of this cascade may prove more effective. The development of such therapies will require establishing which cytokines are effective in blocking the activity of those inflammatory mediators linked to preterm labor.
Interleukin-10 was originally described as a factor produced by class-2 T-helper cells that suppressed inflammatory cytokine production and proliferation by class-1 T-helper cells.19 Several lines of experimental evidence support the hypothesis that the ratio of class-1 T-helper (inflammatory) to class-2 T-helper (anti-inflammatory) cytokines may be critical to pregnancy immunotolerance.20–23 Our laboratory has recently reported that interleukin-10 is both expressed and produced by human trophoblast-derived choriocarcinoma cell lines, purified term cytotrophoblast, and first trimester chorionic villi.24–26 On the basis of these results, we hypothesized that interleukin-10 may suppress the inflammatory cytokine production by macrophages and other cell types that occurs in the setting of maternal infection.
In the current study, interleukin-10 appeared to be effective in preventing preterm birth, fetal loss, and low birth weight pups born to pregnant rats receiving intrauterine infusion of lipopolysaccharide. These results are in agreement with those of Rivera et al27 who reported that the lipopolysaccharide-induced fetal death rate and growth restriction are attenuated by interleukin-10 administration. This action of interleukin-10 is likely mediated through a suppression of cytokine-derived prostaglandin production. This concept is supported by in vitro studies in which cytokine-induced prostaglandin production by human amniotic cells was suppressed by interleukin-10.28,29 In addition, interleukin-10 also decreased tumor necrosis factor-α and interleukin-6 release from lipopolysaccharide-treated human amniochorionic membranes.30,31
The endotoxin-induced model of preterm birth used for this study has been shown to reliably induce delivery of pups approximately 24 hours earlier than controls. Although this would relate to a period of only approximately 2 weeks in human pregnancy, we believe that this degree of prematurity is clinically significant in the rat, as evidenced by a significantly lower birth weight and an increased fetal loss rate. Although the results of this study suggest that interleukin-10 can block endotoxinmediated preterm labor, the efficacy of interleukin-10 in preventing preterm labor caused by live bacteria needs to be established. Additionally, with increasing evidence suggesting an association between amniotic fluid infection and cerebral palsy in humans, the effects of prolonging pregnancy in the setting of infection may be detrimental.32 Future studies are planned to examine the effects of interleukin-10 administered with or without antibiotics on the incidence of neonatal brain lesions in pups born to experimentally infected mothers.
Another important question that must be addressed before the implementation of cytokine tocolytics is the effect that higher than physiologic concentrations of these mediators might have on the fetus. In our study, interleukin-10 administered with lipopolysaccharide had no significant effect on birth weight or the number of live pups per litter compared with controls. Future studies will need to address the effects of interleukin-10 on long-term perinatal development.
Finally, we believe interleukin-10 has shown promise as a cytokine-based tocolytic agent for preventing preterm birth when infection is present. This study may represent an early step in a new approach to addressing the problem of preterm birth.
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