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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, Saint Barnabas Medical Center, Livingston, New Jersey; North Texas Perinatal Associates, Dallas, Texas; and Departments of Physiology and Biophysics and Obstetrics and Gynecology, University of Mississippi Medical Center, Jackson, Mississippi.
Address reprint requests to: Dom A. Terrone, MD, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Saint Barnabas Medical Center, Old Short Hills Road, Suite 402, East Wing, Livingston, NJ 07039; E-mail: terrone{at}excite.com.
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ABSTRACT |
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METHODS: Pregnant rats underwent implantation of uterine catheters and were randomly assigned to receive intrauterine infusion of either normal saline, 50 µg lipopolysaccharide endotoxin, or 50 µg lipopolysaccharide with 500 ng interleukin-10 administered either concurrently or 24 hours later. The interval from infusion to delivery for each group was recorded, along with the number of live born pups and their birth weight. 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.
RESULTS: In females receiving lipopolysaccharide (50 µg) alone, the interval to delivery (P < .05), live birth rate (P < .05), and pup weight (P < .001) were reduced compared with the saline-infused controls. In contrast, females receiving interleukin-10 at the time of the endotoxin challenge or 24 hours after delivered at term with no difference in litter size or live birth weight compared with the controls.
CONCLUSION: Animals treated with both lipopolysaccharide and interleukin-10, administered concurrently or 24 hours after the endotoxin challenge, delivered normal weight pups at term with a similar litter size as the saline-infused controls. Interleukin-10 appears to be effective in preventing endotoxin-induced preterm birth and fetal wastage in pregnant rats.
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-1ß,6 tumor necrosis factor-
,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
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MATERIALS AND METHODS |
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).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).
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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-ß, 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.
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RESULTS |
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. 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.
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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.
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DISCUSSION |
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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- 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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Footnotes |
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This study was presented at the Society for Maternal-Fetal Medicine Annual Meeting, Miami Beach, Florida, February, 2000.
Received December 11, 2000. Received in revised form March 23, 2001. Accepted March 30, 2001.
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REFERENCES |
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2. McGregor JA, French JI. Bacterial vaginosis in pregnancy. Obstet Gynecol Surv 2000;55:S1–19.[Medline]
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14. Allbert JR, Naef RW, Perry KG, Magann EF, Whitworth NS, Morrison JC. Amniotic fluid interleukin-6 and interleukin-8 levels predict the success of tocolysis in patients with preterm labor. J Soc Gynecol Invest 1994;1:264–8.[Medline]
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17. Bry K, Hallman M. Transforming growth factor beta-2 prevents preterm delivery by interleukin-1 and TNF-alpha in the rabbit. Am J Obstet Gynecol 1993;168:1318–22.[Medline]
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19. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse helper T-cells. IV. TH2 clones secrete a factor that inhibits cytokine production by TH1 clones. J Exp Med 1989;70:2081–95.
20. Chaouat G, Menu E, Dy M, Minkowshi M, Clark DA, Wegmann TG. Control of fetal survival in CBA x DBA/2 mice by lymphatic therapy. J Reprod Fertil 1990;89: 447–58.
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24. Bennett WA, Lagoo-Deenadayalan S, Whitworth NS, Brackin MN, Hale E, Cowan BD. Expression and production of interleukin-10 by human trophoblast: A model for the cytokine regulation of pregnancy immunotolerance. Early Pregn 1997;3:190–8.
25. Bennett WA, Lagoo-Deenadayalan S, Stopple JA, Barber WH, Hale E, Brackin MN, et al. Cytokine expression by first-trimester human chorionic villi. Am J Reprod Immunol 1998;40:309–18.
26. Bennett WA, Lagoo-Deenadayalan S, Whitworth NS, Stopple JA, Barber WH, Hale E, et al. First-trimester human chorionic villi express both immunoregulatory and inflammatory cytokines: A role of interleukin-10 in regulating the cytokine network of pregnancy. Am J Reprod Immunol 1999;41:70–8.
27. Rivera DL, Olister SM, Liu X, Thompson JH, Zhang XJ, Penmoline K, et al. Interleukin-10 attenuates experimental fetal growth restriction and demise. FASEB J 1998;12: 189–97.
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29. Fortunato SJ, Menon R, Lombardi SJ. The effect of transforming growth factor and interleukin-10 on interleukin-8 release by human amniochorion may regulate histologic chorioamnionitis. Am J Obstet Gynecol 1998;179:794–9.
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31. Fortunato SJ, Menon R, Lombardi SJ. Interleukin-10 and transforming growth factor-beta inhibit amniochorion tumor necrosis factor-alpha production by contrasting mechanisms of action: Therapeutic implications in prematurity. Am J Obstet Gynecol 1997;177:803–9.[Medline]
32. Yoon BH, Jun JK, Romero R, Park KH, Gomez R, Choi JH, et al. Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-1b, and tumor necrosis factor-a), neonatal brain white matter lesions, and cerebral palsy. Am J Obstet Gynecol 1997;177:19–26.[Medline]
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