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a Department of Pediatrics, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA
b Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was evaluated by RT-PCR and tissue levels (TNF-) were determined by ELISA. Apoptosis was evaluated by TdT-mediated dUTP nick end labeling immunohistochemistry, and nitric oxide (NO) levels were quantified by microelectrode electrochemical detection in explants in culture media. LPS exposure resulted in 43% fetal demise and reduced the size of the surviving fetuses. Placental weight was not altered by LPS. IL-10 attenuated the LPS-induced fetal death rate (to 22%) and growth restriction (P<0.05). In normal rats, IL-10 did not affect fetus size or the incidence of resorptions, although placental size was marginally smaller. Increased uterine TNF- content and NO release and apoptosis of uterine epithelia and muscularis were hallmarks of the LPS model. All were normalized by IL-10. IL-10 may represent a new therapeutic option for the treatment of a variety of perinatal complications. Benefit may result from the suppression of TNF-- and NO-mediated cell death.—Rivera, D. L., Olister, S. M., Liu, X., Thompson, J. H., Zhang, X.-J., Pennline, K., Azuero, R., Clark, D. A., Miller, M. J. S. Interleukin-10 attenuates experimental fetal growth restriction and demise. FASEB J. 12, 189–197 (1998)
Key Words: TNF- • nitric oxide • IL-10 • TUNEL • placenta • cellular infiltration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnancy is a natural condition in which the receipt and tolerance of a foreign tissue, the fetus, must occur for a successful outcome (2–4). Any disturbance of the delicate immune balance within the maternal–fetal interface may result in pregnancy loss or other perinatal complications (5, 6). We have recently described a new model of IUGR that is characterized by the increased release of the vasodilator nitric oxide (NO) after administration of endotoxin (7). Thus, we have proposed that IUGR may result from mechanisms other than vascular insufficiency. A possible mechanism for IUGR in the lipopolysaccharide (LPS) -induced model is the release of cytoxic mediators after activation of resident immune or inflammatory cells. Several cytokines have been implicated in this immune system balance and may influence placental and fetal growth. For instance, second trimester amniotic fluid levels of colony-stimulating factor 1 and tumor necrosis factor-alpha (TNF-) are linked to positive fetal growth and impaired fetal growth, respectively (8, 9). TNF- is also found in uncomplicated uteroplacental units (10). In addition, interleukin-10 (IL-10), an anti-inflammatory cytokine, has been found to be increased in midtrimester amniotic fluid in small-for-gestational age pregnancies (11), but is also expressed in high levels in the placentas of normal pregnancies, which suggests a role in preventing rejection of the fetal allograft by the mother (12).
IL-10 is a newly described pleiotropic cytokine with a range of antiinflammatory properties. It is known to reduce transcription and production of IL-1ß, IL-6, IL-8, and TNF- (13). Conversely, suppression of IL-10 results in increased expression of proinflammatory cytokines (14). In pregnancy, increased IL-10 levels have been associated with preterm labor and infection, suggesting it may act as a inhibitor of intrauterine inflammation (15). More significantly, supplemental IL-10 prevents fetal resorptions in a murine mating combination characterized by increased fetal resorptions, increased inflammatory cytokines, and deficient IL-10 production (16).
Our current model of IUGR is suggestive of a loss of immunological protection of the fetus from the mother. The purpose of this study was to test the efficacy of the anti-inflammatory cytokine, IL-10, as an agent of protection against LPS-induced fetal growth restriction and fetal demise.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-10 by gene expression, TNF- content, and immunohistochemistry. NO production and cellular infiltration were also assessed on tissue samples. In a seperate group of rats, the effects of IL-10 on normal pregnancy were evaluated. Two groups—control (untreated) and an IL-10 alone—were evaluted for placental and fetal size, incidence of resorptions, and uterine myeloperoxidase activity in a 7-day protocol comparable to that described above. All chemicals, unless otherwise specified, were obtained from Sigma Chemical Co. (St. Louis, Mo.). All treatment and surgery protocols were approved by the Institution Animal Care and Use Committee of Louisiana State University Medical Center in New Orleans according to the Declaration of Helsinki and National Institutes of Health guidelines. Rats were housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility.
Detection of IL-10 gene expression (RT-PCR)
Total RNA was isolated from uterine and placental samples by the acid guanidine thiocyanate-phenol-chloroform extraction method. Integrity of RNA extracts was assessed on a 1.2% agarose gel and RNA was visualized by ethidium bromide. First-strand complementary DNAs were synthesized from 1 µg of total RNA using oligo dT (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and Superscript II Reverse Transcriptase (Gibco Laboratories, Grand Island, N.Y.). The first-strand complementary DNA templates were amplified for glyceraldehyde-3-phosphate dehydrogenase and IL-10 by polymerase chain reaction (PCR) using a hot start (Ampliwax, Perkin Elmer, Foster City, Calif.). The primers for IL-10 were as follows: forward 5'-CAT CCG GGG TGA CAA TAA CTG C-3' (a 22-mer oligonucleotide at position 70) and reverse 5'-ACC TGC TCC ACT GCC TTG CTT T-3' (a 22-mer oligonucleotide at position 426), giving rise to a 357 base pair PCR product. These sequence data are available from GenBank under accession number L02926. The primers for glyceraldehyde-3-phosphate dehydrogenase used as an internal standard for rats were as follow: forward 5'-ATT CTA CCC ACG GCA AGT TCA ATG G-3' and reverse 5'-AGG GGC GGA GAT GAT GAC CC-3' (Gen Bank Accession #M17701). The PCR cycle was an initial step of 95° Celsius for 3 min, followed by 94° Celsius for 30 s, 60° Celsius for 45 s, 72° Celsius for 1 min, with 31 cycles and a final cycle of 72° Celsius for 4 min. The negative control was a cDNA reaction that used water instead of RNA.
Detection of TNF- gene expression
With the same RNA extraction conditions, reverse transcriptase (RT) reaction, and PCR program as outlined above, TNF- gene expression was evaluated. The primers were as follows: forward 5'-TAC TGA ACT TCG GGG TGA TTG GTC C-3' and reverse 5'-CAG CCT TGT CCC TTG AAG AGA ACC-3', producing a 295 base pair product. These sequence data are available from GenBank under accession number X66539. Results were visualized on a 2% agarose gel stained with ethidium bromide.
Immunohistochemistry: in situ apoptosis, TdT-mediated dUTP nick end labeling (TUNEL)
The immunohistochemical approach to apoptosis involves detection of digoxigenin-labeled genomic DNA in fixed tissues by an immunoperoxidase. The labeled target is the multitude of 3'OH DNA ends produced by DNA fragments, a hallmark of apoptosis. These fragments typically are localized in morphologically identifiable nuclei and apoptotic bodies. The methods, as supplied by the manufacturer (Oncor, Gaithersburg, Md.), vary slightly for paraffin-embedded fixed tissue, cryostat sections of unfixed tissue, and cultured cells. This technique refers to fixed tissue. In deparaffinized tissue sections, proteinase K (20 µg/ml) was applied for 15 min for protein digestion. Endogenous peroxidase was quenched with 2.0% H2O2 in phosphate-buffered saline (PBS) for 5 min, followed by rinsing and blotting. Equilibration buffer was then added and a coverslip was applied for 10–15 s. The coverslip was then removed and excess fluid was tapped off and blotted around the section. Fifty-four microliters of working strength terminal deoxtidyltransferase were pipetted onto the section and a coverslip was applied. The slide was then incubated at 37° Celsius for 1 h, followed by removal of the coverslip, addition of the stop/wash buffer, and incubation at 37° Celsius for 30 min. During this last step, the section was agitated every 10 min. The slide was then washed three times in PBS and two drops of antidigoxigenin-peroxidase were added; a coverslip was applied and incubated at room temperature for 30 min. The coverslip was then removed and the slide was washed in PBS three times. Finally, DAB (freshly filtered) was added for color development (3–6 min). The slide was then washed and counterstained (hematoxylin), dehydrated in xylene, and mounted (17).
Electrochemical detection of NO
NO production was measured using a microelectrode, electrochemical technique developed in-house as previously described (18). A nafion-coated custom microelectrode sensitive to NO was used as the working electrode and placed in contact with the mucosal surface of the uterine and placental tissue. A platinum wire auxillary electrode and an Ag/AgCl reference electrode were positioned in the media adjacent to the tissue. A current through the working electrode was then recorded with a BAS100B electrochemical analyzer (Bioanalytical Systems, West Lafayette, Ind.), and the concentration of NO released was calculated by comparison to current readings from authentic NO solutions. Tissue explants were placed in tissue culture media (RPMI) and recordings were made within 5 min of dissection in a Faraday cage. Readings were taken at three distinct sites per tissue, and an average reading is reported.
Detection of TNF- content
Uterine and placental tissue collected at death was immediately frozen in liquid nitrogen and then stored at -80° Celsius before analysis. Tissue wet weights were determined on frozen samples that were then transferred to a metal grinder, prechilled in liquid nitrogen, and pulverized to a fine powder. Normal saline was then added to ground frozen samples (1 µl/mg), and suspensions were thoroughly mixed and then centrifuged at 10,000 g for 20 min at 4° Celsius. Aliquots of the resulting supernatant were assayed for TNF- content by using a cytoimmune rat TNF- enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Biosource, Camarillo, Calif.).
Cellular infiltration
Uterine and placental myeloperoxidase activity was measured as an index of tissue granulocyte content, as previously described (19). Briefly, minced uterine and placental tissue (150–200 mg) collected at death was immediately frozen in liquid nitrogen and stored at -80° Celsius. Tissue was thawed and homogenized at 4° Celsius with a Brinkmann polytron (Brinkmann Instruments, Westbury, N.Y.) for 30 s in 50 mmol/l potassium phosphate buffer (pH 6). The homogenate was centrifuged at 20,000 g for 15 min at 4° Celsius, and the resulting pellet was rehomogenized in 50 mmol/l potassium phosphate buffer (pH 6) containing 14 mM hexadecyltrimethylammonium bromide. The homogenate was then sonicated for 20 s at 4° Celsius using a Vibra cell sonicator (Sonics and Materials, Danbury, Conn.), frozen, thawed, and resonicated. This cycle of freezing-thawing-sonicating was repeated twice. Homogenates were then centrifuged at 20,000 g for 15 min at 4° Celsius, and 100 µl of the supernatant was added to 2.9 ml of 5 mmol/l potassium phophate buffer (pH 6) containg O-dianisidine dihydrochloride (0.167 mg/ml) and hydrogen peroxide (5x10-4% v/v). The change in absorbance at 460 nm over 1 min was measured with a Beckman DU-64 spectrophotometer (Beckman Instruments, Fullerton, Calif.). One unit of myeloperoxidase activity was determined as that which degraded 1 mmol/l of hydrogen peroxide in 1 min at 25° Celsius.
Statistical analysis
To compare biochemical values and weights for different treatments, one-way analysis of variance was performed, followed by Duncan's multiple comparisons test or Tukey-Kramer multiple comparisons test using the Instat software package (GraphPad Software, San Diego, Calif.). For all statistical data, a P < 0.05 value was considered to represent a significant difference between the values compared.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1).
Administration of IL-10 (100 µg·kg-1·day-1; s.c.) to dams receiving endotoxin resulted in significant reversal of fetal growth restriction when compared to dams receiving endotoxin with normal saline (s.c.), although IL-10 did not return fetal weights to control levels (P<0.05; Fig. 1). Placental size in all groups was statistically indistinguishable, although there was a trend for IL-10-treated animals to have larger placentas ( Fig. 2).
An additional crucial finding was the attenuation of fetal demise with IL-10. The incidence of fetal demise in the control group was 1% vs. 43% in the endotoxin-treated group vs. 22% in the IL-10-treated group (P<0.05; Fig. 3).
In normal pregnancy, IL-10 treatment had no effect on fetal size ( Table 1),
although there was a modest reduction in placental size (P<0.05). The incidence of resorptions did not differ between the control and IL-10 groups.
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IL-10 and TNF- gene expression
Using RT-PCR, IL-10 and TNF- gene expression were both noted in control pregnant rat uterus and placenta. Though the technique is nonquantitative, placental tissue did show a stronger signal for TNF- and IL-10 after the administration of LPS and a decreased intensity for both genes after the addition of IL-10. Uterine tissue demonstrated a similar stronger signal in IL-10 gene expression with LPS administration, which was restored to control levels after IL-10. Uterine TNF- gene expression did not change during treatment regimens ( Fig. 4
and Fig. 5).
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TUNEL immunohistochemistry
Apoptosis-like DNA fragmentation was determined in the uterus by the TUNEL method. As previously described, there was minimal immunoreactivity in control rats and markedly enhanced positive staining in LPS-treated dams primarily in the uterus, as only minimal apoptosis was evident in the placenta (7). This positive staining was reduced with the administration of IL-10 ( Fig. 6).
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NO production
The effect of LPS and IL-10 on NO release was assessed by directly measuring NO fluxes electrochemically in uterine and placental explants. LPS administration significantly enhanced NO release in the uterus (P<0.05); although IL-10 decreased the LPS response, statistical significance was not achieved. In placental tissue, LPS markedly increased the production of NO when compared to controls. This release was significantly attenuated with the addition of IL-10 (P<0.05; Table 2).
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TNF- content
Uterine and placental TNF- tissue content was measured by ELISA specific for rat TNF-. Uterine tissue obtained from LPS-treated dams contained twice the amount of TNF- when compared to control dams. The administration of IL-10 significantly attenuated the enhanced TNF- content, restoring TNF- to control levels (P<0.05; Fig. 7).
Placental results showed the same trends as uterine TNF- content, but did not reach significance (data not shown).
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Cellular infiltration
Myeloperoxidase activity in uterine tissue was noted to be significantly elevated after administration of LPS (P<0.05; 
Table 2). IL-10 attenuated this response to a small extent, but did not reach significance. In normal pregnancy, IL-10 failed to alter basal myeloperoxidase activity in the uterus (data not shown). There were no significant changes in placental myeloperoxidase activity after the treatments described.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, it underscores the importance of theses mediators as determinants of a successful pregnancy. The attenuation of fetal growth restriction with the anti-inflammatory cytokine IL-10 provides mechanistic insight and may reflect a restoration of balance in the pro- and anti-inflammatory mechanisms. More important, perhaps, fetal demise was significantly reduced with IL-10 administration. The manner in which IL-10 exerts its effects under these circumstances may be multifactorial.
We have evidence to support the role of apoptosis in this model of IUGR (7), a mechanism similar to the handling of a tumor. One effector arm of this process may be NO. NO has been found to be increased in pregnancies complicated by IUGR in both animals and humans (7, 20), and has been implicated as an inducer of apoptosis in cultured macrophages, tumor cells, and epithelial cells (17, 21, 22). In addition, IL-10 is a known inhibitor of inducible NO synthase (iNOS) -dependent NO release (23–25). Our results show a rise in NO release after endotoxin in both uterus and placenta associated with IUGR and a significant decrease (placenta) in NO release with IL-10 associated with significant reversal of IUGR. In this scenario, it seems the role of NO in pregnancy, and more specifically, the placenta, is more complicated than a simple vasodilatory effect. Vasodilation secondary to increased NO (with LPS) would improve placental blood flow and, presumably, fetal growth, whereas decreased NO production (with IL-10) would have the opposite effect. Our results suggest that the role for NO in the uteroplacental unit is complex and perhaps related to the induction of apoptosis. This role is further substantiated with the effect of IL-10 (decreased NO, decreased apoptosis, decreased IUGR) in this model. However, apoptosis was seen predominantly in the uterus, and uterine production of NO was less than that observed in the placenta, as we have previously reported (7). Since high concentrations of NO are usually required to cause apoptosis, if apoptosis is linked to NO it may involve the conversion of NO to the potent oxidant peroxynitrite. We have previously reported the increased formation of nitrotyrosine, a hallmark of peroxynitrite, in this model of IUGR, suggesting that peroxynitrite, and not NO per se, is the species that causes apoptosis (7).
A second factor implicated in this study is TNF-. TNF- is a commonly recognized cytokine associated not only with uncomplicated pregnancy (26, 27), but also in pregnancies marked by infection and IUGR (28). Its role in uncomplicated pregnancy is speculative, but it has been implicated in promoting insulin growth factor-1 release and in early murine growth and lymphoid tissue development (29). Other biological effects in pregnancy include regulation of trophoblast growth and invasion of maternal spiral arteries, as well as cell growth and differentiation (30, 31).
The effects of TNF- in complicated pregnancy are better known. Previous studies have shown its association with LPS-induced fetal resorption in mice (32), bacteria-induced pregnancy loss in rabbits (33), and impaired intrauterine fetal growth in human studies (34). Its mode of action is less clear, but other investigators have shown an adverse effect on blastocyst growth (35), inhibition of decidualization (36), and a role in placental injury (37). Here we consider an additional mechanism in which TNF- may exert its growth inhibiting effect.
As with NO, TNF- is also a known inducer of apoptosis (38). TNF- has been shown to regulate apoptotic death of villous cytotrophoblasts (39). This regulation has been considered to be an additional physiologic effect, but the detrimental implications of this preexisting mechanism at the maternal–fetal interface are readily apparent. We observed a significant increase in TNF- associated with experimental IUGR. Furthermore, the addition of IL-10 attenuated uterine TNF- and apoptosis responses in association with a reversal of IUGR and fetal demise. In essence, this model may have up-regulated normal maternal defenses mediating physiologic apoptotic cell death mechanisms designed to limit the exent to which the fetus invades the maternal tissue. In so doing, growth and survival of the fetus have been compromised.
A secondary effect of TNF- in this scenario may be a stimulation of iNOS transcription and, therefore, increased NO release. Together, TNF- and NO (or peroxynitrite) might further enhance apoptotic cell death. This association has been demonstrated in leukemic cells where proinflammatory cytokines (TNF-) induced apoptosis in tumor targets by a NO-mediated mechanism (40).
The role of TNF--induced apoptosis and the ability of IL-10 to attenuate the response is a viable mechanism underlying poor fetal growth and fetal death. Other investigators have outlined similar effects of IL-10 on TNF- production (19, 41, 42). Moreover, additional studies have shown that IL-10 prevents apoptosis and have further suggested the prevention of TNF--induced apoptosis (43, 44). Nevertheless, the complicated nature of the maternal–fetal interface warrants consideration of other modes of action of IL-10.
In vitro and in vivo studies have shown that IL-10 mediates the phenomenon of endotoxin desensitization via TNF- down-regulation (45), a response not unlike our own. IL-10 has also been shown to have functions other than purely inhibitory, including the role of a growth factor in immature and mature T cells as well as lymph node and spleen cells (46). It is conceivable that IL-10 may function in a similar way in our model; it may actually possess both growth-promoting and cytokine inhibitory properties, and thus accelerate growth in a fetus faced with an adverse environment. However, IL-10 did not affect fetal size in normal pregnancy, suggesting that IL-10's actions were confined to dampening signals for growth restriction and not simply a generalized promoter of fetal growth. Finally, the positive effect of IL-10 on other growth-promoting agents (TGF-ß) and the negative effect on other inflammatory cytokines (IL-6, IFN-
) may be playing an unknown role in this model of endotoxin-induced IUGR.
In conclusion, we propose that our model and underlying mechanisms may be likened not only to clinical IUGR but to other perinatal complications, including premature labor secondary to infection and stillbirths. Several clinical studies of these perinatal complications are similar to our own with regard to NO and TNF- production. One recent study has also associated increased amniotic fluid levels of IL-10 with subsequent poor fetal growth (11). A detrimental effect of IL-10 has been postulated, but here we suggest a therapeutic role for IL-10. In pregnancies marked by poor fetal growth, perhaps IL-10 is produced in an effort to counteract the ‘maternal attack’ on the fetus. Its objective may be to provide an environment that is permissive for ‘tumor growth’ (47): the fetus. However, endogenous IL-10 activation may not be sufficient (as evidenced by minimum gene expression changes) in the face of an overwhelming flow of inflammatory cytokines. The use of supplemental IL-10 in the clinical settings of IUGR, threatened labor, or fetal distress may be a novel therapeutic approach to these perinatal complications.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: TNF-, tumor necrosis factor-alpha; IL, interleukin; IUGR, intrauterine growth restriction; LPS, lipopolysaccharide; NO, nitric oxide; RT-PCR, reverse transcriptase-polymerase chain reaction; iNOS, inducible NO synthase; TUNEL, TdT-mediated dUTP nick end labeling; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.
Received for publication July 18, 1997. Accepted for publication October 27, 1997.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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