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【摘要】
Preterm birth is a major contributor of adverse perinatal outcome. Clinical data suggest that an inflammatory response is important in the process leading to preterm labor. By using a recently introduced mouse model of localized intrauterine lipopolysaccharide-induced inflammation, the effect of interleukin (IL)-18 gene disruption and/or IL-18 neutralization as well as combined IL-1/ß gene disruption on inflammation-induced fetal loss was investigated. The frequency of preterm fetal loss was significantly higher in IL-18 knockout mice (58.9%) and in mice administered IL-18-binding protein (59.7%) compared to wild-type controls (34.7%). The rate of fetal loss was not affected by IL-1/ß gene deficiency (38.7%). Decreased IL-18 protein expression combined with elevated IL-12 protein expression in uterine tissue of IL-18 knockout mice and IL-18-binding protein-treated animals was noticed. These data demonstrate that preterm pregnancy loss in response to intrauterine inflammation was enhanced by disruption of the IL-18 gene and/or IL-18 neutralization, events that may relate to exaggerated Th1 responses because of an increased IL-12/IL-18 ratio.
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Preterm birth affects 5 to 12% of all pregnancies and remains a major world-wide contributor of neonatal morbidity, mortality, and long-term neurological handicap.1,2 Up to 30% of cases of spontaneous preterm birth are associated with intrauterine microbial colonization or histological evidence of chorioamnionitis. Approximately half of the cases of preterm labor and preterm prelabor rupture of membranes have intra-amniotic inflammation.3,4 The presence of intrauterine inflammation is not only a cause of preterm birth but is also associated with an increase in neonatal morbidity and mortality.5 Despite widespread use of preventive strategies, the rate of preterm birth has not decreased, with the survival of preterm infants having improved throughout the past several decades. As a consequence, the prevalence of long-term handicap attributed to preterm birth is increasing.6 Therefore, it is critical to elucidate the mechanisms and mediators that contribute to intrauterine inflammation-promoted preterm parturition and the child outcome.
Expression patterns of cytokines and other immunomodulatory proteins in the fetal membranes and decidua suggest that inflammatory activation occurs modestly with term labor but much more robustly in preterm delivery, particularly in the presence of intrauterine infection. Enhanced chemokine expression, particularly evident in deliveries involving an infected amniotic cavity, is presumably responsible for recruiting infiltrating leukocytes into the membranes, thereby amplifying the inflammatory process and hastening membrane rupture and delivery. In both humans and rodents, increased expression of proinflammatory cytokines and chemokines during infection-induced preterm labor has been recognized as a critical element of the hypothesized signaling cascade.5,7,8 Several members of the proinflammatory interleukin (IL)-1 family of cytokines (eg, IL-1, IL-1ß, and IL-18) are supposed to play a key role in this process.8-11 IL-1 levels are elevated in the amniotic fluid of pregnancies complicated by intra-amniotic infection.9 IL-1ß has been found to be increased in a nonhuman primate model of infection-related preterm labor and to be capable of initiating preterm labor by up-regulating prostaglandin production, leading to myometrial contractions.12 Moreover, administration of an IL-1 receptor antagonist inhibits IL-1-induced preterm labor in mice.13 Recent findings also suggest that polymorphisms in the IL-1 gene locus might be related to spontaneous preterm birth and abortion.14,15 However, the effect of IL-1 combined with IL-1ß on preterm birth has not been studied.
IL-18 is a novel cytokine of the IL-1 family that is similar to IL-1 in terms of structure, processing, receptor complex, signal transduction pathway, and proinflammatory properties. Both IL-1ß and IL-18 are synthesized as inactive precursors that require cleavage by IL-1ß converting enzyme (caspase-1) to become fully bioactive.16 In addition, it was suggested that IL-18 shares functional similarities with IL-12, and both cytokines induce the production of interferon (IFN)-.17
In the present study, we used a recently developed mouse model of localized, lipopolysaccharide (LPS)-induced intrauterine inflammation18 to test the hypothesis that the deletion of the proinflammatory cytokine IL-1/ß or IL-18 might reduce the incidence of preterm delivery. We adapted the model to C57BL/6 mice and tested both high and low doses of LPS exposure. The objectives of this study were to evaluate the effect of IL-18 gene disruption (IL-18C/C), IL-18 neutralization via IL-18-binding protein (IL-18BP), and combined IL-1/ß gene disruption (IL-1ßC/C) in dams and their fetuses on inflammation-induced preterm birth and fetal loss and to investigate the related mechanisms and mediators that contribute to preterm parturition.
【关键词】 disruption interleukin- interleukin- increases vulnerability delivery mortality intrauterine inflammation
Materials and Methods
Animals
Time-mated pregnant C57BL/6 wild-type mice (originally obtained from Charles River Laboratories, Sulzfeld, Germany) and C57BL/6 mice lacking the gene for IL-1819 or IL-1ß20 were bred under the same conditions at Experimental Biomedicine (Göteborg University, Göteborg, Sweden). The original C57BL/6 wild-type mice came from the same breeding colonies (founding strain) as the original C57BL/6 mice used for making the knockout mice. Both IL-1ß- and IL-18-deficient mice were backcrossed to the C57BL/6 line for at least 10 generations before the experiments were started. For the experiments, IL-18C/C females were impregnated by IL-18C/C males, and IL-18+/+ females were impregnated by IL-18+/+ males. To ensure that the above IL-18C/C animals did not differ in their response to LPS compared to IL-18C/C animals derived from heterozygote breeding pairs, control experiments were performed in which homozygous (IL-18C/C) and heterozygous (IL-18+/C) mutant mice and wild-type (IL-18+/+) littermate females were generated from heterozygous pairs. All animal experimentation was approved by the Ethical Committee of Göteborg (no. 290-2004).
Genotyping
The genotype of the IL-18C/C and IL-1ßC/C mice was determined by polymerase chain reaction (PCR) of genomic DNA obtained from both pregnant dam and pup tails.
DNA Preparation
The tail was digested with 400 µl of lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 100 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L NaCl, 1% sodium dodecyl sulfate) containing 1 mg/ml proteinase K (Roche, Mannheim, Germany). After incubation at 60??C overnight, 200 µl of 5 mol/L potassium acetate was added to the lysate, which was then thoroughly mixed and centrifuged at 10,000 x g for 20 minutes. The supernatant was transferred into a clean tube, and 800 µl of 100% ethanol was added and mixed. After incubation at C20??C for 30 minutes, the DNA was pelleted by centrifugation at 10,000 x g for 25 minutes at 4??C. The pellet was washed once with 500 µl of 75% ethanol. After removing all of the liquid, the pellet was left to dry at room temperature. The pellet of DNA was dissolved with 100 µl of sterile water, and DNA concentration was determined.
PCR Amplification
IL-18 Wild-Type (Wt) and Knockout (KO)
Each PCR reaction (25 µl) contained 1 µl of genomic DNA, 0.2 mmol/L dNTP, 2.5 µl 10x PCR buffer (250 mmol/L Tris-HCl, pH 8,3, 375 mmol/L KCl, 15 mmol/L MgCl2; Sigma, Stockholm, Sweden), 1 U of TaqDNA polymerase (Sigma), 1 µmol/L of IL-18 common and IL-18 Wt primers, and 0.5 µmol/L of IL-18 mutant primer. The PCR cycles were 94??C for 30 seconds, 67??C for 1 minute 30 seconds, and 74??C for 1 minute throughout 40 cycles. The following primers were used: IL-18 common, 5'-TTGCTGCACCTAGAGGTATGTACTGAC-3'; IL-18 Wt, 5'-TAATGGGTGGTCTTCTCATCTCTGTGT-3'; and IL-18 mutant, 5'-ATCGCCTTCTATCGCCTTCTTGACGAG-3'.
The IL-18 common and Wt primers detected the wild-type allele, which was 300 bp, whereas the IL-18 common and IL-18 mutant primers detected the mutated allele, which was 350 bp. Mice homozygous for the wild-type allele of IL-18 (IL-18 Wt) produced a single PCR band of 300 bp, mice homozygous for the mutated allele of IL-18 (IL-18 KO) produced a single PCR band of 350 bp, and mice heterozygous for the mutated allele of IL-18 (IL-18 het) produced double PCR bands of both 300 bp and 350 bp.
IL-1 and IL-1ß Wt and KO
Each PCR (25 µl) contained 2 to 4 µl of genomic DNA, 0.8 mmol/L dNTP, 2.5 µl of 10x PCR buffer (250 mmol/L Tris-HCl, pH 8,3, 375 mmol/L KCl, 15 mmol/L MgCl2; Sigma), 2.5 U of TaqDNA polymerase (Sigma), and 1 µmol/L of each primer. The PCR cycles were 94??C for 30 seconds, 63??C for 30 seconds, and 72??C for 1 minute 30 seconds throughout 40 cycles. The following primers were used: IL-1ß common, 5'-GCGAATGTGTCACTATCTGCCACC-3'; IL-1ß Wt, 5'-GGTCAGTGTGTGGGTTGCCTTATC-3'; IL-1ß mutant, 5'-GAGGTGCTGTTTCTGGTCTTCACC-3'; IL-1 common, 5'-CTTGGCCATACTGCAAAGGTCATG-3'; and IL-1 Wt, 5'-CTTCTGCCTGACGAGCTTCATCAG-3'.
The IL-1 common and Wt, IL-1 common and IL-1ß mutant, IL-1ß common and Wt, IL-1ß common, and IL-1ß mutant primers detected the wild-type or mutated allele for IL-1 or IL-1ß, respectively, which were all at 1700 bp. Because the PCR product bands for the different genotypes of IL-1ß Wt/KO and IL-1 Wt/KO were all of similar size, samples from the same animal had to be repeatedly run and each PCR reaction had to be performed in separate tubes. All primers were from CyberGene AB, Huddinge, Sweden. The PCR products were separated on a 1.5% agarose/0.5x TBE gel containing ethidium bromide. A 100-bp ladder was used to verify the size of the PCR products. The gels were visualized using a LAS 1000 cooled charge-coupled device camera (Fujifilm, Tokyo, Japan).
Induction of Preterm Birth in C57BL/6 Mice
LPS (Escherichia coli 055:B5; Sigma) was injected into the uteri of pregnant mice as described elsewhere.18 Briefly, LPS was injected at a time corresponding to 79% of average gestation (ie, gestation day 15; C57BL/6 mice normally deliver pups on days 19 to 20 of gestation), which is an appropriate time to mimic late onset of human chorioamnionitis.6,21,22 Animals were anesthetized with isoflurane (5% for induction, 3.5% for maintenance) in nitrous oxide/oxygen (1:1) in an induction chamber. Laparotomy of the mouse lower abdomen was performed. The mouse uterus was exposed, and the number of fetuses in each horn was counted. LPS (250 µg/mouse or 125 µg/mouse) or saline of the same volume (100 µl) was injected into the right uterine horn at a site between the lower two gestational sacs most proximal to the cervix. After the uterus was returned to the abdomen, the fascia and the skin were closed, and animals recovered in individual cages. The entire procedure lasted for less than 5 minutes per mouse. For mice treated with mouse IL-18-binding protein (IL-18BP, 122-BP; R&D Systems, Inc., Abingdon, UK), IL-18BP (15 µg/mouse, 0.5 mg/kg body weight) was administrated intraperitoneally at the same time as LPS administration. Animals were observed every 4 to 6 hours for 48 hours for any signs of morbidity (piloerection, decreased movement), vaginal bleeding, and/or preterm delivery (at least one pup present in the cage or in the lower vagina within 48 hours of surgery). The number of live-born or dead pups delivered was recorded.
At 48 hours after LPS injection, all animals were laparotomized again, and the number of total fetuses (dead or alive) left in the uterus horn were counted. Fetal deaths were identified by white discoloration, markedly smaller fetal size, and lack of blood flow in the umbilical cord. Uterine tissues from each group of animals including tissue from normal 17-day gestation pregnant mice were harvested and processed for immunoblotting and immunohistochemistry staining.
Immunoblotting
After harvesting, uterine tissue was washed in sterile saline, snap-frozen, and stored at C80??C. Tissue samples were homogenized by sonication in ice-cold 50 mmol/L Tris-HCl (pH 7.3) containing 5 mmol/L ethylenediaminetetraacetic acid and 0.5% protease inhibitor cocktail (P8340, Sigma), and samples were aliquoted and stored at C80??C. The protein concentration was determined according to Whitaker and Granum,23 adapted for microplates. Samples were mixed with an equal volume of concentrated (3x) sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer and heated (96??C) for 5 minutes. Individual samples were run on 4 to 20% Tris-glycine gels (Novex, San Diego, CA) and transferred to reinforced nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After blocking with 30 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, and 0.1% Tween 20 (TBS-T) containing 5% fat-free milk powder for 1 hour at room temperature, the membranes were incubated with primary antibodies: goat anti-IL-18 (M-19; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-actin (A2066, Sigma) at room temperature for 1 hour followed by an appropriate peroxidase-labeled, secondary antibody (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. Immunoreactive species were visualized using the Super Signal Western Dura substrate (Pierce, Rockford, IL) and a LAS 1000-cooled charge-coupled device camera (Fujifilm). Immunoreactive bands were quantified using the Image Gauge software (Fujifilm).
Immunohistochemistry
Uterine tissue was washed in sterile saline and immersion-fixed in 5% buffered formaldehyde (Histofix; Histolab, Göteborg, Sweden) for 24 hours. After dehydration with graded ethanol and xylene, uterine tissues were paraffin-embedded. Uterus was cut into 6-µm transverse sections across the lumen. Parallel sections were used for various stains, as described below. Antigen recovery was performed by heating the sections in 10 mmol/L boiling sodium citrate buffer (pH 6.0) for 10 minutes. Nonspecific binding was blocked for 30 minutes with 4% horse or goat serum (depending on the species used to raise the secondary antibody) in phosphate-buffered saline. The following primary antibodies were used: goat anti-IL-12A p35 (M-19, sc-9350; Santa Cruz Biotechnology), goat anti-IL-18 (M-19, sc-6179; Santa Cruz Biotechnology), and goat anti-mouse IL-18R (AF856; R&D Systems, Minneapolis, MN). After incubating the primary antibodies for 60 minutes at room temperature, the appropriate, biotinylated secondary antibodies (all from Vector Laboratories) were added for 60 minutes at room temperature. Endogenous peroxidase activity was blocked with 3% H2O2 for 5 minutes. Visualization was performed using Vectastain ABC Elite with 0.5 mg/ml 3,3'-diaminobenzidine enhanced with 15 mg/ml ammonium nickel sulfate, 2 mg/ml ß-D glucose, 0.4 mg/ml ammonium chloride, and 0.01 mg/ml ß-glucose oxidase (all from Sigma). Control experiments were performed by omission of the primary antibodies, substitution by goat serum (G9023; Sigma) for IL-12, and preabsorption of primary antibody with an excess (10x and 100x) of blocking peptide (sc-6179 P; Santa Cruz Biotechnology) for IL-18.
Statistics
Fisher??s exact test was used for comparing the differences between groups with respect to the number of fetuses delivered preterm and the number of nonviable fetuses. Spearman??s rho method was used to test for trends of the starting time for the preterm delivery. Student??s unpaired t-test was used to compare the average IL-18 immunoreactivity after densitometric quantification of individual samples after immunoblotting.
Results
Intrauterine Inflammation Induced Preterm Fetal Loss in a Dose-Dependent Manner in C57BL/6 Mice
Intrauterine injection of LPS (250 or 125 µg) evoked preterm birth in 100% (10 of 10) or 80% (8 of 10), respectively, of C57BL/6 dams within 26 hours (Figure 1A) . All pups were nonviable, consistent with E15 to E16 of gestation. In contrast, none of the 10 mice injected with vehicle (saline) delivered preterm. The number of fetuses delivered preterm and the number of nonviable fetuses (dead fetuses retained in the uterine horn) were 51 of 70 (72.8%) and 14 of 19 (73.7%), respectively, in the group administered 250 µg of LPS, as compared to 25 of 75 (34.7%) and 27 of 49 (55.1%), respectively, in the group administered 125 µg of LPS (Figure 1, B and C) . Lower doses of LPS (62.5 µg/mouse or 15 µg/mouse), however, did not cause preterm delivery but evoked intrauterine fetal death (14 of 23, 60.8%) as evaluated at 48 hours after LPS administration.
Figure 1. The dose-dependent response of intrauterine LPS on preterm delivery (PTD) in mice. A: Preterm delivery rate. B: Incidence of fetuses delivered preterm (number of fetuses delivered preterm of total number of fetuses). C: Incidence of nonviable fetuses (number of dead fetuses retained in the uterus of total number of fetuses remaining in the uterus at 48 hours after intrauterine injection of LPS). LPS 250 (LPS 250 µg/mouse), LPS 125 (LPS 125 µg/mouse). ***P < 0.001 using Fisher??s exact test.
IL-1ß Gene Disruption Did Not Affect Preterm Delivery
To investigate the role of IL-1 in preterm parturition, mice lacking both IL-1 and IL-1ß (IL-1ßC/C) were used. The genotyping of IL-1ßC/C mice is shown in Figure 2 . IL-1ßC/C mice injected with saline did not display any sign of preterm delivery. The number of fetuses delivered preterm and the number of nonviable fetuses at 48 hours after LPS administration (LPS 250 µg) did not significantly differ in IL-1ßC/C mice compared to wild-type mice. The number of preterm fetuses and nonviable fetuses was 24 of 33 (72.7%) and 51 of 70 (72.8%) (P = 1.00, Figure 3A ), respectively, in IL-1ßC/C mice compared to nine of nine (100%) and 14 of 19 (73.7%) (P = 0.14, Figure 3B ), respectively, in wild-type mice. To exclude the possibility that an excessively high dose of LPS might conceal a dependency on the IL-1ß genotype, we decreased the LPS dosage to 125 µg/mouse. Again, there was no significant difference in the number of fetuses delivered preterm or the number of nonviable fetuses in IL-1ßC/C mice compared with the wild-type mice. The number of fetuses delivered preterm was 12 of 31 (38.7%) and 26 of 75 (34.7%) in IL-1ßC/C mice versus wild-type mice (P = 0.66, Figure 3C ), respectively. The number of nonviable fetuses was 11 of 19 (57.9%) and 27 of 49 (55.1%) in IL-1ßC/C mice versus wild-type mice (P = 1.0, Figure 3D ), respectively.
Figure 2. Representative genotyping results of the PCR analysis from IL-18 (C/C, +/C, +/+) and IL-1ß (+/+, C/C) mouse tail DNA. The top panel shows representative PCR products of IL-1ß and the bottom panel shows IL-18 genotypes. The molecular marker (M) for verifying the size of the PCR products was a 100-bp ladder. For IL-1ßC/C, the PCR products were only observed in the mutated allele for both IL-1 and IL-1ß whereas absence of both IL-1 and IL-1ß products indicated wild-type alleles. For IL-18C/C, the mutant allele-specific products were amplified by the primer sets of IL-18 common and IL-18 mutant, 350 bp. The wild-type allele-specific products were detected from the wild-type allele-specific primer sets IL-18 common and wild-type, 300 bp. Mice heterozygous for the mutated allele of IL-18 (IL-18 het) had products of both 300 bp and 350 bp.
Figure 3. Effect of IL-18 and IL-1ß on the rate of preterm delivery (PTD). The incidence of fetuses delivered preterm (A, C) and incidence of nonviable fetuses (B, D) at 48 hours after LPS administration are shown. A and B: LPS dosage 250 µg/mouse for wild-type mice (Wt) and IL-1ßC/C mice. C and D: LPS dosage 125 µg/mouse for wild-type mice (LPS 125, n = 10), IL-1ßC/C mice (n = 5), IL-18BP co-treated wild-type mice (IL-18BP, n = 19), and IL-18C/C mice (n = 10). **P < 0.01, ***P < 0.001 using Fisher??s exact test.
IL-18 Deficiency Increased Preterm Delivery and Fetal Mortality
Next, to test the hypothesis that deletion of IL-18, a novel member of the IL-1 family of proinflammatory cytokines, reduces preterm delivery after intrauterine LPS administration, we compared IL-18 gene-deficient and wild-type mice. The mutated IL-18 allele was detected in all IL-18C/C mice as shown in Figure 2 . Intrauterine saline injection did not induce preterm delivery in IL-18C/C mice as expected. But unexpectedly, when IL-18C/C mice were injected with 125 µg/mouse LPS, we found a significantly increased number of fetuses that were delivered preterm and an increased number of nonviable fetuses compared with wild-type mice. The number of fetuses delivered preterm was 43 of 73 (58.9%) in IL-18C/C mice versus 26 of 75 (34.7%) in wild-type mice (P = 0.003, Figure 3C ), and the number of nonviable fetuses was 30 of 30 (100%) versus 27 of 49 (55.1%) in IL-18C/C mice versus wild-type mice (P < 0.0001, Figure 3D ). Moreover, IL-18C/C mice delivered more rapidly in response to LPS than wild-type mice (P = 0.0011, Figure 4 ). In IL-18C/C mice, 100% delivered within 14 hours and 60% delivered within 8 hours of LPS injection. The same dose of LPS in wild-type mice induced preterm delivery in only 50% of dams at 14 hours, with no preterm births at 8 hours. The remaining wild-type mice (30%) delivered by 20 to 26 hours after LPS administration and 20% of wild-type mice did not deliver preterm. In the IL-18C/C dams, fetuses that were not delivered preterm were all found dead in the uterine horn when examined at 48 hours after LPS administration (Figure 2D) .
Figure 4. Latency (h) between LPS and preterm delivery (PTD) in wild-type, IL-18C/C, and IL-1ßC/C mice. Using the same dose of LPS (125 µg/mouse), IL-18C/C mice delivered more rapidly in response to LPS than wild-type mice; however, there was no significant difference between IL-1ßC/C and wild-type mice regardless of LPS dose. **P < 0.01 using Spearman??s rho method. LPS 250 (LPS 250 µg/mouse), LPS 125 (LPS 125 µg/mouse).
Control experiments in homozygous (IL-18C/C) and wild-type (IL-18+/+) littermate females generated from heterozygous pairs confirmed the findings above. With 125 µg/mouse LPS injected into the uteri, the number of fetuses delivered preterm was 34 of 56 (60.7%) in IL-18C/C mice versus 20 of 59 (33.8%) in wild-type mice (P = 0.005), and the number of nonviable fetuses was 22 of 22 (100%) versus 23 of 39 (58.9%) (P = 0.002) in IL-18C/C mice versus wild-type mice. IL-18C/C mice also delivered more rapidly in response to LPS than wild-type mice (P = 0.001). These results indicate that the different reaction to LPS between the IL-18C/C and IL-18+/+ mice is not dependent on whether the animals are derived from heterozygote or homozygote breeding pairs.
To further confirm the role of IL-18 in preterm delivery, IL-18BP, a naturally occurring and specific inhibitor of IL-18, was administered intraperitoneally. It was found that IL-18BP-treated animals were more vulnerable to LPS-induced preterm delivery than the nontreated mice. The number of fetuses born preterm (Figure 3C) was significantly increased in the IL-18BP-treated animals (89 of 149, 59.7%) compared with the non-IL-18BP-treated wild-type mice (26 of 75, 34.7%), and the number of nonviable fetuses was 37 of 60 (61.7%) versus 24 of 49 (55.1%) in IL-18BP-treated versus non-IL-18BP-treated wild-type mice (NS) (Figure 3D) .
Expression and Localization of IL-18, IL-12, and IL-18R Protein in Mouse Uterine Tissue
To characterize the expression of IL-18, IL-12, and IL-18R, we investigated immunoexpression of these proteins in homogenates and tissue sections. Immunoblotting demonstrated the specificity of the polyclonal antibody against mouse IL-18. The antibody displayed a distinct band with an apparent molecular mass of 24 kd, corresponding to the size of pro-IL-18 (Figure 5) . As expected, the uterine tissue from IL-18C/C mice did not yield a band at 24 kd, whereas there was a distinct band in the wild-type uterine tissue at 17 days of gestation. IL-18 was increased at 48 hours after LPS administration, and immunoreactivity significantly decreased after co-administration of IL-18BP (Figure 5) .
Figure 5. Immunoblots demonstrating IL-18 protein expression in uterine tissue homogenates. The top panel shows representative immunoblots of IL-18 protein expression and actin (as a control for equal loading), demonstrating low levels of IL-18 in the IL-18BP-treated mouse and an absence of IL-18 immunoreactivity in the IL-18C/C mouse. The bottom panel shows the average IL-18 immunoreactivity ?? SEM after densitometric quantification of individual samples (n = 4 in each group). *P < 0.05 using Student??s t-test. Cont, wild-type mouse uterine tissue at 17 days of gestation. LPS, wild-type mouse uterine tissue after LPS treatment; IL-18BP, wild-type mouse uterine tissue after LPS and IL-18BP co-treatment; IL-18C/C, IL-18C/C mouse uterine tissue after LPS treatment. LPS dosage was 125 µg/mouse in all treated groups.
Immunohistochemical staining of parallel uterine tissue sections revealed moderate to intense IL-18 staining in the uterine decidua at 17 days of gestation, while weaker staining was observed in the myometrium (Figure 6B) . IL-18 immunoreactivity increased in the decidua at 48 hours after LPS administration in wild-type mice (Figure 6E) and decreased after IL-18BP co-administration (Figure 6H) . As expected, no appreciable staining in the tissue from the IL-18C/C mouse was found (Figure 6K) . IL-1ßC/C mice treated with LPS displayed a moderate IL-18 staining not different from LPS controls (Figure 6N) .
Figure 6. IL-12, IL-18, and IL-18R immunoreactivity in uterine tissue sections. Representative immunohistochemistry staining by using the IL-12 (A, D, G, J, M), IL-18 (B, E, H, K, N), and IL-18R (C, F, I, L, O) antibodies based on different mouse strains and treatments. Cont, normal wild-type mouse at 17 days of gestation (ACC); LPS 125, wild-type mouse with LPS treatment (DCF); IL-18BP, wild-type mouse with LPS and IL-18BP co-treatment (GCI); IL-18C/C, IL-18C/C mice with LPS treatment (JCL); IL-1ß-, IL-1ß mice with LPS treatment (MCO). LPS dosage was 125 µg/mouse in all of the groups. Original magnifications, x10.
Because there is a strong interaction between IL-12 and IL-18 with respect to IFN- production, we examined the IL-12 immunoreactivity in parallel uterine sections. Very weak IL-12 immunoreactivity was detected in uterine tissue of normal control (Figure 6A) and LPS-treated (Figure 6D) wild-type mice. Although the IL-18BP co-treatment mice (Figure 6G) , LPS-treated IL-18C/C mice (Figure 6J) , and IL-1ßC/C mice (Figure 6M) displayed a moderately increased IL-12 staining in the decidua compared with control animals. IL-18R immunoreactivity (Figure 6, C, F, I, L, and O) was found primarily in decidua and to some extent in the smooth muscle cells of controls but the expression was not affected by LPS administration in wild-type, gene-deficient, or IL-18BP-treated mice.
Discussion
In the present study, we found that LPS induced preterm fetal loss in C57BL/6 mice in a dose-dependent manner, which agrees with the previous report in heat-killed E. coli-induced preterm delivery in mice.24 The preterm pregnancy loss in response to intrauterine LPS administration in C57BL/6 mice was not affected by the combined disruption of the IL-1 and ß genes though, unexpectedly, enhanced by the disruption of the IL-18 gene and/or IL-18 neutralization. The enhanced preterm delivery rate was associated with decreased IL-18 in combination with increased IL-12 protein expression in the uterus. In contrast, increased expression of IL-12 alone, as found in the uterine tissue of IL-1 ßC/C mice, was not accompanied by an increased rate of preterm delivery.
Clinical studies have demonstrated that IL-1 levels increase in both amniotic fluid and gestational tissues toward parturition and are significantly elevated during labor.25,26 Furthermore, infusion of IL-1 and tumor necrosis factor (TNF)- into the amniotic cavity of rabbits,27 or systemic administration of IL-1ß to mice,13 induced preterm delivery. In the present study, we found that the preterm pregnancy loss in response to intrauterine LPS administration in C57BL/6 mice was not affected by the combined disruption of the IL-1 and ß genes, suggesting that IL-1 may not play an essential role in the pathophysiology of labor during infection in this model. This is in agreement with previous studies that demonstrate that IL-1ß knockout28 and IL-1 receptor knockout29 mice are not more prone to preterm pregnancy loss than wild-type controls.
IL-18 is an 18-kd proinflammatory cytokine structurally and functionally related to IL-1ß.30 IL-18BP decreases endogenous IL-18 activity by abolishing IL-18 induction of IFN-.31 Four isoforms of IL-18BP have been described in humans and two in mice.32 Isoform a, used in the present study, has the highest affinity for IL-18.33 We found that both IL-18 gene disruption and IL-18BP co-treatment enhanced the sensitivity to LPS-induced preterm delivery. Whether it is the mother or the fetus that plays the most important role in the effect of IL-18 needs to be further addressed in future studies.
In addition to the high sensitivity of IL-18C/C mice to LPS-induced preterm delivery, we found that no single fetus was alive 48 hours after LPS administration in IL-18C/C mice. This is probably attributable to the high susceptibility of the IL-18C/C mice to LPS-induced inflammation. Kohmura and colleagues34 suggested that although LPS injected into mothers could pass through placenta to fetuses, LPS-induced intrauterine fetal death depends mainly on maternal sensitivity to LPS. It is suggested that IL-18 is important in the host defense against severe infections via induction of other cytokines, effector cells, and molecules.35 The survival time of IL-18/IL-12 double-knockout mice was significantly shorter than that of IL-12 single-knockout mice in response to Mycobacterium tuberculosis infections, suggesting the possible contribution of IL-18 in host protection.36 Thus, we speculate that the absence of IL-18 may increase the mouse vulnerability against LPS-induced inflammation. However, the underlying mechanisms of IL-18 deletion-provoked intrauterine fetal death need to be further investigated.
There are a limited number of studies regarding the expression and localization of IL-12, IL-18, and IL-18R in gestational tissues.37,38 In the present study, we found that IL-12, IL-18, and IL-18R were expressed in the decidua and smooth muscle cells, in agreement with previous reports. LPS increased the level of IL-18 in the mouse uterine tissue as found by both immunohistochemistry and immunoblotting compared with vehicle controls. Interestingly, in the IL-18C/C mouse and IL-18BP-treated mouse uterine tissue in which absence or decreased levels of IL-18 were noted, we observed an increased level of IL-12 not seen in wild-type controls. This is consistent with reports from humans that showed the presence of IL-12 immunoexpression in intrauterine tissues in connection with preterm delivery.39,40
Because IL-18 shares functional similarities with IL-12,30 it is possible that IL-12 is increased in the absence of IL-18 as part of a compensatory response. However, a study from Kinjo and colleagues36 showed that serum IL-12 levels were significantly lower in IL-18-deficient mice than in wild-type mice after infection with M. tuberculosis. This suggests that the balance between IL-18 and IL-12 may not be simply associated with compensation. Other closely related cytokines such as IL-15,41 IL-12 family new members IL-23 and IL-2717 may be involved in the host response as well.
In the uterus of IL-1ßC/C mice exposed to LPS, we found increased levels of IL-12 whereas IL-18 was unchanged. Taken together (Table 1) , increased levels of IL-18 (as seen in wild-type mice) or IL-12 (as found in IL-1ßC/C mice) was associated with unaltered response to LPS. In contrast, increased IL-12 combined with decreased IL-18 was associated with increased susceptibility to LPS. These results are consistent with the view that the IL-12/IL-18 balance determines Th1/Th2 responses.37,39,42 A high IL-12 level combined with low IL-18 level increases Th1 predominance and would ultimately increase the proinflammatory response in preterm delivery. Indeed, such a delicate relationship between IL-18 and IL-12 has also been proposed to be important in severe preeclampsia.43
Table 1. Scheme of Possible LPS-Induced Preterm Delivery Mechanisms Related to the IL-18/IL-12 Balance
Hypothetically, a proper balance between these two cytokines is necessary to both contest infection and protect the host from immune-based injury. IL-18 is likely to have different effects alone or if in combination with high levels of IL-12 in preterm delivery and pregnancy. Zourbas and colleagues44 have undertaken a systematic study of the expression of IL-12, IL-18, and other cytokines in the uterine tissues throughout pregnancy. They found that these cytokines have specific location and distinct sequential expression patterns, suggesting that they have important regulatory functions. Absence or excessively high levels of one or both of these cytokines may have immediate (implantation failures, abortion) or delayed (preeclampsia) consequences.38,42,45,46
The different reactions in IL-1ßC/C and IL-18C/C mice to LPS-induced preterm delivery might also relate to TNF-. LPS induces TNF-, which is strongly involved in preterm labor and pregnancy loss (for a summary, see a recent review by Romero et al47 ). Indeed, an elevated level of TNF- has been noted in the uterine wall and fetal membranes in mouse models of heat-killed E. coli-induced preterm delivery.24,48 Previous work from Sakao and colleagues49 showed that LPS challenge increased serum TNF- level in IL-18C/C mice compared with those of wild-type mice, thus indicating that IL-18 is a negative regulator for TNF- during sepsis. On the other hand, Reznikov and colleagues28 observed reductions of TNF- in IL-1ßC/C mice compared to IL-1ß+/+ mice. We speculate that the different reaction of IL-1ß and IL-18 deficiency to the LPS-induced preterm delivery might relate to different levels of and response to TNF- after LPS in these mice. However, the precise role of TNF- in LPS-induced preterm delivery in these mice remains to be defined.
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作者单位：From the Department of Physiology,* Perinatal Center, and the Departments of Microbiology and Rheumatology and Inflammation Research,¶ Göteborg University, Göteborg, Sweden; the Department of Pediatrics, the Department of Obstetrics and Gynecology, Perinatal Center, Sahlgrenska Academ