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Interleukin-10 Inhibition of Gelatinases in Fetal Membranes: Therapeutic Implications in Preterm Premature Rupture of Membranes

From The Perinatal Research Center of the Women’s Health Research and Education Foundation, The Maternal-Fetal Group and Aquinas College, Division of Biostatistics, Department of Preventive Medicine, Vanderbilt University Medical Center, Nashville, Tennessee.

Address reprint requests to: Stephen J. Fortunato, MD, The Perinatal Research Center, Women’s Health Research and Education Foundation, 2300 Patterson Street, Nashville, TN 37203; E-mail: fortunat{at}edge.net.

	ABSTRACT

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OBJECTIVE: To examine the effect of interleukin-10 on production and regulation of gelatinases by amniochorion in an in vitro model of infection.

METHODS: We placed amniochorionic membranes collected from eight women who had elective repeat cesareans at term in an organ explant culture system. After 48 hours in culture, the membranes were stimulated with lipopolysaccharide (50 ng/mL), and some were costimulated with interleukin-10 (500 ng/mL). Tissue and media samples were collected after 24-hour stimulation. Quantitative polymerase chain reactions and enzyme-linked immunosorbent assays were used to evaluate matrix metalloproteinase 2 and matrix metalloproteinase 9 messenger RNA and proteins, respectively.

RESULTS: Lipopolysaccharide stimulation induced 55.14 transcripts of matrix metalloproteinase 9, compared with 0.83 in control tissues (P < .001). Costimulation with interleukin-10 and lipopolysaccharide significantly reduced matrix metalloproteinase 9 messenger RNA levels to 10 transcripts (P < .001). Lipopolysaccharide stimulation produced 29.25 ng/mL of immunoreactive matrix metalloproteinase 9, which was reduced to 6.3 ng/mL (P_adj ⁼ .016) after costimulation with interleukin-10. Although not significant, matrix metalloproteinase 2 messenger RNA levels were higher in lipopolysaccharide-stimulated tissues (4.37 x 10⁶ transcripts) compared with control (2.8 x 10⁵ transcripts; P_adj ⁼ .08), with a significant decrease in matrix metalloproteinase 2 messenger RNA levels in interleukin-10- costimulated tissues (2.9 x 10⁶; P_adj ⁼ .007). Interleukin-10 costimulation resulted in a significant decrease in matrix metalloproteinase 2 protein production (203.1 [lipopolysaccharide] and 149.75 [with interleukin-10]; P_adj < .001).

CONCLUSION: Interleukin-10 eliminated lipopolysaccharide induction of matrix metalloproteinase 2 and 9 in amniochorion.

Fetal membranes comprising amnion and chorion cell layers connected by an extracellular matrix region are partly composed of various types of collagens.¹ Abnormal complications such as intra-amniotic infection during pregnancy are believed to contribute to destruction of those collagens by substrate-specific enzymes called matrix metalloproteinases, causing membrane degradation.² During infection and other complications of pregnancy, our laboratory documented an imbalance between matrix metalloproteinases and tissue inhibitors of metalloproteinases, which might indicate matrix metalloproteinase activity causing destruction of extracellular matrix components, rather than remodeling.^3,4 We proposed that increased degradation of extracellular matrix weakens the membrane and causes rupture.

Matrix metalloproteinase 2 and 9 (gelatinases A and B) are two of the best studied proteases involved in preterm premature rupture of membranes (PROM).^5–7 Matrix metalloproteinase 2 (gelatinase A) is a constitutively expressed protein throughout pregnancy and is likely involved in membrane remodeling. Matrix metalloproteinase 9 (gelatinase B) is an inducible gene in human fetal membranes, the expression of which increases during infection, PROM, and active term labor.⁷ Induced expression and its increased active form is seen during PROM, suggesting its possible effect on membrane degradation. We and several other investigators documented that gelatinases and other matrix metalloproteinases crucially affect PROM and are involved in the labor process, causing cervical ripening and membrane rupture.^3,4,8–10

Earlier, we described increased active and inhibitor-free forms of both enzymes in amniotic fluid (AF) of women with PROM.³ We used lipopolysaccharide to reproduce in vitro changes in intra-amniotic infections in vitro. The increase in matrix metalloproteinase levels in tissue-conditioned media after lipopolysaccharide treatment created a stoichiometric imbalance in the matrix metalloproteinase-tissue inhibitor of matrix metalloproteinase molar ratio that favored matrix metalloproteinase activity over inhibition.³ The matrix metalloproteinases can initiate a vicious pathway through activation of other matrix metalloproteinases (collagenases and stromelysins), which can degrade additional extracellular matrix collagens, causing weakening of the membrane and predisposition to rupture.

Down-regulation of matrix metalloproteinase expression and production might reduce significantly extracellular matrix degradation and thereby risk of PROM. Herein, we report the effectiveness of interleukin-10 for controlling gelatinase expression and production.

	MATERIALS AND METHODS

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This study was approved by the institutional review board at The Women’s Hospital at Centennial Medical Center, Nashville, TN. Fetal membranes were collected from women who had elective repeat cesareans at term (n = 8; mean gestational age 37.5 weeks) after uncomplicated gestations. The placentas from six white and two black women were included. We excluded women with pregnancy complications, infections, or on antibiotic treatment. Amniochorionic membranes were cleansed in phosphate-buffered saline (pH 7.4), and 6-mm diameter sections were cut using a biopsy punch. Those membranes were harvested and placed in an organ explant system as described.^4,7 After preincubation for 48 hours (as described),^4,7 membranes were stimulated with 50 ng/mL of lipopolysaccharide (Escherichia coli O55: B5; Sigma, St. Louis, MO). Forty-eight-hour preincubation was chosen because genes of interest were induced by physical manipulation in preparation for culture. A return to baseline value of gene expression was achieved over 48 hours. In each of eight cultures, a selected group of membranes was treated simultaneously with 500 ng/mL of recombinant interleukin-10 (R&D Systems, Minneapolis, MN). Tissue and media samples were collected after 24-hour treatment and frozen at -80C for messenger RNA and protein analysis. Fetal membranes were examined histologically after hematoxylineosin staining to rule out histologic chorioamnionitis (more than four polymorphoneutrophils per high power field).

Total RNA was extracted by Trizol (Life Technologies, Bethesda, MD) method, and 0.5 µg of total RNA was subjected to oligo (dT)-primed reverse transcriptase reaction. Complementary DNA was then subjected to polymerase chain reaction (PCR) using matrix metalloproteinase 2 and 9 specific primers.⁷ Quantitative competitive PCR was used to measure messenger RNA for matrix metalloproteinase 2 and 9 after lipopolysaccharide and interleukin-10 stimulation. In the competitive PCR assay, one set of primers was used to amplify target gene complementary DNA and another neutral DNA fragment. A known concentration of neutral DNA fragment competes with the target complementary DNA fragment (unknown) for the same primers (primer sequences for matrix metalloproteinase 2 and 9 as published⁷) and acts as an internal standard. Tissues stimulated with lipopolysaccharide alone were compared with those stimulated with lipopolysaccharide and interleukin-10 and used as controls. The bands resolved on 1.5% ethidium bromide-stained agarose gels were quantitated by densitometry using Alpha-Ease software (AlphaInnotech Corporation, San Leandro, CA).

Release of immunoreactive matrix metalloproteinases into the culture medium was quantitated using enzyme-linked immunosorbent assay, which involved multiplesite two-step sandwich immunoassay with oligoclonal antibodies (several monoclonal antibodies directed against different epitopes of each specific protein of interest). Those were done in our laboratory with commercially available kits (Amersham-Pharmacia, Piscataway, NJ) according to manufacturer’s instructions. Standard curves were developed using duplicate samples of known quantities of recombinant proteins. Sample concentrations were determined by relating absorbance to standard curve by linear regression analysis. Colorimetric absorption was read at 450 nm with a microplate reader. Controls consisted of assay buffer, plain culture media, lipopolysaccharide, and interleukin-10-containing media incubated in tissue culture wells without tissues.

A mixed model analysis of variance was used to compare mean protein and messenger RNA levels in the eight subjects for three groups, lipopolysaccharide stimulation, interleukin-10 stimulation, and unstimulated control. Mixed models were necessary to adjust the standard errors and degrees of freedom for the test statistics. These adjustments were necessary because of the correlations between groups arising from the fact that the samples were sections taken from the same eight individuals, which were then stimulated by two methods and used as an unstimulated control. The mixed model approach uses the correct degrees of freedom for the tests between groups with this correlation taken into account. Further, because we were performing many tests of significance, we also looked at the statistical significance using the Scheffe multiple comparison procedure, which adjusts the P values obtained from the differences between the mean protein and messenger RNA levels between groups. All these analyses were performed using SAS version 8.1 (SAS/STAT Software Release 8.1, SAS Institute, Cary, NC).

	RESULTS

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Reverse transcriptase PCR showed induction of matrix metalloproteinase 9 mRNA in response to infectious stimulus (lipopolysaccharide stimulation), whereas no definitive difference was seen in expression of matrix metalloproteinase 2 in human fetal membranes compared with controls (data not shown). Lipopolysaccharide stimulation was used as a proxy for infection and a matrix metalloproteinase stimulant because we reported the effect of infection and lipopolysaccharide on fetal membrane gelatinase production.⁷

Our result was further verified by quantitative PCR. Data are summarized in Table 1. Quantitative PCR showed that mean matrix metalloproteinase 9 messenger RNA expression was statistically significantly higher with stimulation of lipopolysaccharide (55.14 transcripts) as compared with both interleukin-10 stimulation (10 transcripts; P < .001) and unstimulated control tissues (0.83 transcripts; P < .001) with no difference between the interleukin-10 and unstimulated control tissue (P = .077). These differences remained statistically significant upon adjustment for multiple comparisons (P_adj < .001, P_adj < .005, P_adj = .19, respectively). Matrix metalloproteinase 2 messenger RNA expression showed a statistically significant higher mean expression in the interleukin-10 as compared with the unstimulated control group (2.9 x 10⁶; P = .002, P_adj = .007), and a higher mean expression between lipopolysaccharide (4.37 x 10⁶ transcripts) and unstimulated control tissue (2.8 x 10⁵ transcripts; P = .032), which was not statistically significant upon Scheffe adjustment (P_adj = .082). There was no statistically significant difference between the interleukin-10 and lipopolysaccharide group (P = .350).

Mean protein and messenger RNA expression was increased in the lipopolysaccharide group compared with both other groups in all cases. The addition of interleukin-10 significantly reduced the mean messenger RNA expression and mean protein levels for metalloproteinase 9 and metalloproteinase 2. There was no statistical difference in mean messenger RNA expression and mean protein levels between the interleukin-10 and unstimulated tissues.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We confirmed our earlier reports of inducibility of matrix metalloproteinase 9 gene in human fetal membranes during infection.⁷ Lipopolysaccharide stimulation in this culture system mimics in vivo infection.¹¹ Several lines of evidence indicate that matrix metalloproteinase 9 strongly might affect labor and membrane rupture: matrix metalloproteinase 9 gene is induced in amniochorion during labor, PROM, and infection⁷; matrix metalloproteinase 9 levels are increased in the amniotic fluid as gestation progresses and during preterm and term active labor^10,12,13; gelatinolytic activity corresponding to latent and active forms of matrix metalloproteinase 9 is increased in AF from pregnancies after PROM^3,6; a significant increase in matrix metalloproteinase 9 levels is seen in AF of women with microbial invasions of the intra-amniotic cavity with and without rupture of membranes^5,6; active, inhibitor-free forms of matrix metalloproteinase 9 are elevated significantly during PROM compared with other conditions.³

In this report, we proposed interleukin-10 as a potential regulator of matrix metalloproteinase 9 gene expression and protein release. During simulated in vitro infection, interleukin-10 transcriptionally regulated matrix metalloproteinase 9 messenger RNA expression and protein production. We reported similar regulatory effects of interleukin-10 on inflammatory cytokine production (tumor necrosis factor, interleukin-8, and interleukin-6).^14–16 All those interleukin-10-regulated genes have a B motif in their gene promoter region, and the binding of transcriptional activator protein, nuclear factor B, is required for gene induction. Interleukin-10 is known to inhibit activation and nuclear translocation of nuclear factor- B by blocking its activator kinase and gene transcription.¹⁷ The matrix metalloproteinase 9 gene promoter region has a B motif and is activated by nuclear factor- B binding,^18,19 which can be regulated by interleukin-10.

We reported on constitutive expression of matrix metalloproteinase 2 in fetal membranes.⁷ In view of that pattern of expression, matrix metalloproteinase 2 is likely involved in tissue remodeling throughout pregnancy. Matrix metalloproteinase 2 messenger RNA and protein levels increase after lipopolysaccharide treatment. Interleukin-10 significantly reduced matrix metalloproteinase 2 protein production after lipopolysaccharide stimulation. Those data did not surprise us because the matrix metalloproteinase 2 gene promoter region resembles that of a housekeeping gene in that it has minimal regulatory sites and appears to be designed for constitutive expression.¹⁹ The matrix metalloproteinase 2 promoter region does not contain B motif or sites for transcriptional activator proteins except for AP-2, which is required for constitutive expression and can be bound by p53, a proapoptotic antigen.²⁰ Interleukin-10 will not directly regulate matrix metalloproteinase 2 gene because of lack of binding sites for interleukin-10-regulated transactivator proteins such as nuclear factor- B. The increase of matrix metalloproteinase 2 expression is most likely through increased p53 protein in the cell primarily caused by lipopolysaccharide-associated toxicity.²¹ The regulatory mechanism exerted by interleukin-10 on matrix metalloproteinase 2 protein production needs to be investigated.

Active matrix metalloproteinase 9 is more potent than matrix metalloproteinase 2 in breaking the Type IV collagen of the basement membrane. Matrix metalloproteinase 9 can be activated by proteases, including matrix metalloproteinase 2, and prompt regulation (induction and activation) might be required to stop extracellular matrix degradation leading to PROM.²² Matrix metalloproteinase 9 appears to be induced in abnormal conditions such as PROM, and appears to be inhibited totally by interleukin-10 at the transcriptional level. Conversely, matrix metalloproteinase 2 is constitutively produced and its production is affected only minimally by interleukin-10 costimulation, which suggests a potential therapeutic role for interleukin-10, in that it would inhibit matrix metalloproteinase 9 production and potentially its deleterious effects on the membrane while allowing matrix metalloproteinase 2 to continue its physiologic effect on membrane remodeling.

Recent study in rhesus macaques²³ found that interleukin-10 administered into the amniotic cavity has a half-life of therapeutic concentrations for longer than 1 weeks. In association with a favorable side effect profile, that makes it an ideal candidate for PROM prophylaxis. Before that can be implemented, we need a sensitive and specific method of identifying women at risk of PROM. The lack of an established animal model also makes it a difficult question.

	Footnotes

 
PII S0029-7844(01)01441-7

Received June 22, 2000. Received in revised form March 16, 2001. Accepted March 23, 2001.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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