Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print September 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018754
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/1/123    most recent
biolreprod.103.018754v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by White, C. A.
Right arrow Articles by Robertson, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by White, C. A.
Right arrow Articles by Robertson, S. A.
Agricola
Right arrow Articles by White, C. A.
Right arrow Articles by Robertson, S. A.
BIOLOGY OF REPRODUCTION 70, 123–131 (2004)
DOI: 10.1095/biolreprod.103.018754
© 2004 by the Society for the Study of Reproduction, Inc.

Immunology

Effect of Interleukin-10 Null Mutation on Maternal Immune Response and Reproductive Outcome in Mice1

Christine A. White, Martina Johansson, Claire T. Roberts, Alistair J. Ramsay, and Sarah A. Robertson2

Department of Obstetrics and Gynaecology and Reproductive Medicine Unit,3 University of Adelaide, Adelaide 5005, Australia Louisiana State University/Tulane Gene Therapy Consortium,4 Louisiana State University Health Sciences Centre, New Orleans, Louisiana 70112


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-10 (IL-10) is an anti-inflammatory and immune-deviating cytokine expressed in the endometrium and placenta. IL-10 null mutant (IL-10-/-) mice have been employed to examine the role of IL-10 in regulating immune events in early pregnancy and its significance in implantation and pregnancy success. The inflammatory response elicited in endometrial tissue by insemination was amplified in IL-10-/- mice, with a 66% increase in leukocytes in the endometrial stroma on Day 3 of pregnancy. Despite this, no evidence of abnormal type 1/type 2 skewing was seen in T-lymphocytes from lymph nodes draining the uterus. On Day 18 of gestation, IL-10-/- females mated with IL-10-/- males had 15% more implantation sites and 27% more viable fetuses than pregnant wild-type (IL-10+/+) mice. Placental weight was unaffected, but fetal weight and the fetal:placental weight ratio were higher in IL-10-/- pregnancies. Similar data were obtained in allogeneic pregnancies when IL-10-/- females were mated with major-histocompatibility complex (MHC) disparate IL-10-/- males. Pups delivered by IL-10-/- mothers had increased birth weight and followed an altered growth trajectory, with growth impairment evident from early postnatal life into adulthood, which was reflected in alterations in body composition at 14 wk of age. This study shows that although IL-10 is not essential for maternal immune tolerance or successful pregnancy irrespective of MHC disparity in the fetus, maternal IL-10 is a determinant of growth trajectory in progeny in utero and after birth.

cytokines, female reproductive tract, immunology, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-10 (IL-10) is a pleiotropic cytokine secreted by leukocytes and somatic cells with well-characterized anti-inflammatory and immune-deviating properties [1]. IL-10 inhibits proliferation and cytokine synthesis in type 1 T lymphocytes and can induce nonresponsiveness or anergy. Conversely, IL-10 favors differentiation and function of lymphocyte subsets with pivotal roles in immune tolerance [2], namely the newly discovered T regulatory (Tr1) cells [3] and T helper 3 (Th3) cells [4]. The ability of IL-10 to skew immune outcomes is indirect and is mediated through programming antigen-presenting cells to preferentially support expansion of alternative types of T cells [1]. Moreover, IL-10 terminates inflammatory responses and limits inflammation-induced tissue pathology by inhibiting synthesis of tumor necrosis factor {alpha} (TNF{alpha}), interleukin-1, and a large array of other pro-inflammatory cytokines and chemokines in monocytes/macrophages [1].

Inhibition of type 1 immunity at the feto-maternal interface and systemically in the mother is associated with successful pregnancy outcome in mice and women [5], and several lines of evidence implicate IL-10 as a potentially important immune-deviating cytokine in pregnancy. IL-10 is expressed in the uterus, oviducts, and ovaries of cycling mice and during pregnancy in the uterine myometrium and cervix [6]. Decidual cells express IL-10 across the duration of pregnancy in mice [7], and placental synthesis occurs in early and midpregnancy [8]. Pregnancy-specific glycoprotein 18 synthesized by trophoblast giant cells in the placental junctional zone in mice induces IL-10 in resident macrophages [9]. In human placenta, IL-10 expression is prominent in the first and second trimesters [10] where both cytotrophoblast cells [11] and placental leukocytes [12] are implicated in its synthesis. IL-10 induces expression of human leukocyte antigen (HLA)-G [13] and inhibits matrix metalloproteinase (MMP)-9 activity [14]. IL-10 may therefore act to modulate placental trophoblast differentiation and invasion, as well as to suppress potentially harmful maternal immune reactivity.

Support for a physiological role for IL-10 in pregnancy comes from women suffering gestational pathologies where immune aberrations are implicated. Elevated levels of IL-10 in amniotic fluid at midtrimester have been associated with small-for-gestational-age first pregnancies [15], and increased expression of IL-10 in term placenta has been observed in women with preeclampsia [16]. IL-10 is among the cytokines aberrantly expressed in decidual T-lymphocyte populations of women with unexplained recurrent miscarriage [17], and decreased IL-10 expression is associated with preterm labor [18, 19].

The physiological significance of IL-10 in pregnancy has been examined by administration of either recombinant IL-10 or neutralizing anti-IL-10 monoclonal antibodies (mAb) to pregnant mice [20, 21]. Treatment with recombinant IL-10 abrogated high resorption rates in CBA/J x DBA/2 pregnancies, where defective placental IL-10 production is characteristic. Conversely, an 80% increase in resorption rate was observed after anti-IL-10 treatment [20]. Antibody-mediated depletion of maternal IL-10 in mice with low risk of fetal resorption did not affect pregnancy parameters, but transient growth impairment was observed around 4 wk of age in offspring [21].

IL-10 deficient (IL-10-/-) mice have been generated by targeted mutation of the IL-10 gene in murine embryonic stem cells [22]. These mice have functional B and T lymphocytes and normal immune responses to T cell-dependent immunization, but inflammatory responses are exaggerated, with chronic enterocolitis developing in response to normal enteric antigens [22, 23] and amplified type 1 responses to bacterial and parasitic infections [24, 25]. Null mutation in the IL-10 gene has been found to be compatible with successful pregnancy in mice [22, 26]. However, a study of appropriate design and sufficient power to formally investigate the possibility of compromised reproductive outcome has not been reported. Moreover, the effect of both fetal and maternal IL-10 deficiency in allogeneic pregnancy has not been investigated. The purpose of the current study was to define the physiological consequences of maternal and fetal IL-10 null mutation for implantation, fetal and placental development, and postnatal viability and growth. Since IL-10 is implicated in terminating inflammatory cascades and inhibiting type 1 immunity, parameters of the inflammatory and immune responses to insemination were also examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

IL-10 null mice were generated by targeted mutation of the IL-10 gene in 129/Ola embryonic stem cells propagated on a C57BL/6 background (IL-10-/- B6; H-2b) as previously described [25]. Control C57BL/6 mice (IL-10+/+ B6; H-2b) and Balb/c (H-2d) males were obtained from the University of Adelaide Central Animal House. Male IL-10-/- mice carrying H-2d were prepared by four cycles of backcrossing through Balb/c males, with individual offspring analyzed by polymerase chain reaction (PCR) for IL-10 and major histocompatibility complex (MHC) genotype (IL-10-/- Balb/c.B6). All mice were housed under specific pathogen-free conditions at the University of Adelaide Medical School Animal House on a 12L:12D cycle and were administered food and water ad libitum. IL-10-/- mice received broad-spectrum antibiotics (Oxymav 100: 100 g/kg oxytetracycline hydrochloride, Mavlab, Slack's Creek, Australia) in autoclaved drinking water twice weekly at a concentration of 2 mg/ml to prevent colitis. IL-10+/+ mice were also given antibiotics for 2 wk prior to and over the duration of experiments. All investigations were approved by the University of Adelaide Animal Ethics Committee and carried out in accordance with the Guiding Principals for the Care and Use of Research Animals endorsed by the Society for the Study of Reproduction.

Estrous Cycles, Breeding Experiments, and Body Composition Analysis

For analysis of estrous cycles, vaginal smears were prepared from adult (6–8 wk) virgin IL-10-/- B6 and IL-10+/+ B6 mice at 0900–1000 h and examined by phase contrast microscopy daily for 21 days. Mice were allocated to one of four stages of the cycle on the basis of the cellular composition: proestrus (>50% intact, live epithelial cells); estrus (100% cornified epithelial cells); metestrus (~50% leukocytes and ~50% cornified epithelial cells); or diestrus (>70% leukocytes, ± cornified or intact epithelial cells). The first day of observation of a smear indicative of estrus was taken as Day 1 of the cycle, and the next estrus as Day 1 of the following cycle.

In all breeding studies, 1–3 females (IL-10-/- B6 or IL-10+/+ B6, 8–12 wk old) were housed with a proven fertile male. For syngeneic pregnancies, males were IL-10-/- B6 or IL-10+/+ B6. For allogeneic pregnancies, males were IL-10+/+ Balb/c (H-2d) or IL-10-/- Balb/c.B6 (H-2d). The day of vaginal plug detection was designated Day 1 of pregnancy, when females were removed from the male and housed in groups of two to five.

For analysis of late gestation pregnancy parameters, adult virgin IL-10-/- and IL-10+/+ B6 females were mated and killed by cervical dislocation at 1000–1200 h on Day 18 of gestation. The intact uterus of each female was removed and total, viable, and resorbing implantation sites were counted. Each viable fetus was dissected from the amniotic sac and umbilical cord, and fetuses and placentae were weighed. In some experiments crown-rump length was also measured.

For analysis of term parameters, adult virgin IL-10-/- and IL-10+/+ B6 females were mated and pregnant females were checked at 0900 h and 1800 h from Day 18 of pregnancy until birth, when gestation length was determined to the half day and litter size recorded. Pups were weighed 12–24 h after birth and at 8 day, 3 wk, and at various intervals thereafter depending on the experiment. Pup death between birth and 8 day was recorded as postnatal loss, and between 8 day and 3 wk as preweaning loss. At 3 wk, pups were weaned and housed in groups of 10–12 of the same gender. Body composition of adult progeny was assessed at 14 wk of age, when mice were killed by cervical dislocation, and internal organs and tissues were dissected and weighed individually.

PCR and Generation of IL-10-/- Balb/c.B6 (H-2d) Mice

IL-10 and H-2 genomic status was assessed by PCR of DNA extracted from blood or tail tissue of adult mice. PCR primers diagnostic for the IL-10 mutation and the neomycin insertion cassette were as previously reported [25]. Four sets of H-2 genotyping primers were designed against d and b haplotypes of H-2K and H-2D. H-2Kd primers were (forward) 5'-GAGCCTGAGGACCGCACAGA and (reverse) 3'-CGAACATCCGCTGGAAC; H-2Kb primers were (forward) 5'-GGACCTGAGGACCCTGCTCG and (reverse) 3'-GGCCCTGAGTCTCTCTGCTT; H-2Dd primers were (forward) 5'-GCCTCCTTCATCCACCAAGA and (reverse) 3'-TTCACTCCAATGTCTCTAGG, and H-2Db primers were (forward) 5'-GCCTCCTCCGTCCACTGACT and (reverse) 3'-GCTGTGAGAGTTCAAGGAAG. The integrity of extracted DNA was confirmed by PCR with primers for ß-actin DNA: (forward) 5'-CGTGGGCCGCCCTAGGCACCA and (reverse) 3'-TTGGCCTTAGGGTTCAGAGGG. The PCR amplification employed reagents supplied in a Taq DNA polymerase kit (Biotech International, Bentley, Australia). After initial denaturation (5 min at 94°C), 40 amplification cycles were performed (denaturation for 1 min at 94°C, annealing for 1 min at 64°C, and extension for 2 min at 72°C), followed by a final extension for 7 min at 72°C. Reaction products were analyzed by agarose gel electrophoresis and the size of PCR products were confirmed by comparison with molecular weight markers (Bresagen, Adelaide, Australia). Individual offspring used to generate and propagate IL-10-/- Balb/c.B6 mice were PCR genotyped to establish H-2 status as positive for H-2Dd and H-2Kd, and negative for H-2Db and H-2Kb.

Immunohistochemistry

To determine the effect of IL-10 deficiency on uterine leukocyte parameters during early pregnancy, IL-10-/- and IL-10+/+ B6 females mated with Balb/c males were killed by cervical dislocation between 0900 and 1200 h at estrus, or on Days 1, 3, or 4 of pregnancy. Uterine tissue was embedded in OCT compound (Tissue Tek; Sakura, Tokyo, Japan); snap frozen in liquid nitrogen cooled isopentane (BDH, VWR International, Poole, UK); and stored at -70°C until use. Sections (7 µm) were fixed in 96% ethanol, blocked with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO); and immunolabeled with rat anti-mouse CD45 ({alpha}CD45), specifically reactive with all leukocytes. Reactivity was detected by horseradish peroxidase (HRP)-conjugated rabbit anti-rat immunoglobulin (DakoCytomation, Glostrup, Denmark), and diaminobenzidine (DAB) (Sigma), and sections were counterstained in hematoxylin.

Quantification of positive staining density was carried out on all {alpha}CD45 stained slides using video image analysis (VIA) software (VideoPro, Leading Edge, Adelaide, Australia). The area of positive DAB staining in the endometrial stroma, excluding the lumen, luminal epithelium, and glandular epithelial cells (expressed as percent positivity after normalization to the area of total staining) was determined as the average of values from 10 separate low power fields. Coefficients of variation (0.45% intraassay and 7.8% interassay) in measurement precision were determined using a standard field of stained tissue.

Flow Cytometry for Intracellular Cytokines

Cells from the para-aortic lymph nodes (PALN) were excised from estrous mice or from mated mice killed on Day 4 of pregnancy. Single cell suspensions were prepared from PALNs using a manually operated glass homogenizer, washed in RPMI 1640 (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal calf serum (RPMI-FCS), and counted using a hemocytometer. To measure production of intracellular cytokines, PALN lymphocytes (2 x 106 cells/ml) were stimulated in vitro for 6 h at 37°C in 5% CO2 in RPMI-FCS with polyclonal activators: phorbol myristate acetate (PMA; Sigma; 50 ng/ml), and calcium ionophore (Sigma; 1 µg/ml) and monensin (2 µM; Calbiochem, La Jolla, CA) was added to enhance the expression and intracellular retention of cytokines. One hundred microliter aliquots of 106 cells were treated with anti-Fc-{gamma}IIR antibody (Pharmingen, San Diego, CA) for 5 min to block nonspecific binding. Thereafter fluorescein- (FITC) and/or phycoerythrin- (PE) labeled mAbs from Pharmingen reactive with CD3 were added for 30 min prior to fixation in 5% paraformaldehyde in PBS. The cells were then kept for up to 1 wk at 4°C until intracellular cytokine staining, which was carried out according to recommendations by Pharmingen as previously described [27, 28]. FACS buffer containing 0.1% saponin (Sigma), 1 ml/tube, was added to permeabilize cells for 15 min. The cells were washed and FITC- and/or PE-labeled mAbs (Pharmingen) or unlabeled rat anti-mouse antibodies (clones: IL-4, 11B11; IFN{gamma}, AN18 and R4–6A2; IL-5, TRFK-5) were added to the cells in the presence of 0.1% saponin and incubated for 30–45 min on ice. Where unlabeled mAb was used, a FITC-labeled secondary antibody (Silenus, Melbourne, Australia) was then added to the cells and incubated for 30 min. Cells were then washed and resuspended in 300 µl FACS buffer. Lymphocytes were analyzed using forward and side scatter to exclude other leukocytes and dead cells. Data analysis was conducted using CellQuest software (Becton Dickinson), and the proportion of CD3+ T-lymphocytes expressing IL-4, IFN{gamma}, or IL-5 was determined.

Statistical Analysis

All statistical analysis was carried out using SPSS 9.0 software (Chicago, IL). Immunohistochemical data were evaluated by Kruskal-Wallis one-way ANOVA and Mann Whitney Rank Sum test. Flow cytometry data and parameters of pregnancy expressed as mean weights, numbers, or days were compared by parametric tests after confirmation of normal distribution by linear Q-Q plot. One-way ANOVA and Bonferroni t-test were used when more than two treatment groups were compared, and independent samples t-test was used to evaluate effect of genotype. Postnatal growth data were examined by repeated measures ANOVA to test for an effect of genotype over time, then by independent samples t-test to examine individual time points. Data expressed as proportions were compared by chi-square analysis. Differences between groups were considered significant when the probability P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of IL-10 Deficiency on the Uterine Inflammatory Response

To determine the effect of IL-10 deficiency on the dynamics of the inflammatory response characteristic of early pregnancy, uterine tissue was recovered from adult virgin IL-10+/+ and IL-10-/- B6 females at estrus, or on Days 1, 3, or 4 of pregnancy after mating with IL-10+/+ Balb/c males and analyzed by immunohistochemistry. Leukocytes were detected in sections of fresh frozen uterine tissue after immuno-labeling with {alpha}CD45. Qualitative evaluation indicated that leukocyte abundance was similar in null mutants and wild-type mice at estrous and on Day 1 of pregnancy, when large numbers of inflammatory cells invade the endometrial stroma in response to activation by factors in seminal plasma [29, 30]. However, on Days 3 and 4 of pregnancy, when the inflammatory response normally subsides in preparation for implantation, leukocytes were abnormally abundant in tissue from IL-10 deficient mice. Quantification of labeled cells by VIA showed that mean CD45 positivity was 66% higher in endometrial tissue of IL-10-/- uteri than in IL-10+/+ uteri on Day 3 of pregnancy (Fig. 1). The morphology of stained cells together with additional experiments using lineage-specific antibodies showed that absence of IL-10 was associated with retention of neutrophils and eosinophils, in addition to the populations of macrophages and dendritic cells normally residing in the endometrial stroma subjacent to the luminal epithelium on Day 3. In sections from three of eight IL-10-/- mice, neutrophils were detected migrating through the luminal epithelium (Fig. 2), whereas this is not seen in IL-10+/+ uteri beyond 48 h after insemination [29].



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. The effect of IL-10 null mutation on abundance of CD45+ cells in uterine tissues. Immunohistochemical staining with anti-CD45 mAb in uterine tissue recovered 1 and 3 days after natural mating with IL-10+/+ Balb/c males was quantified by VIA. Symbols represent data from individual mice, and median values for treatment groups are scored. Data were compared by Kruskal-Wallis one-way ANOVA and Mann-Whitney Rank Sum test. Data sets labeled on the x-axis with different lowercase letters denote statistical significance between treatment groups (P < 0.01)



View larger version (150K):
[in this window]
[in a new window]
  		FIG. 2. The effect of IL-10 null mutation on abundance of CD45+ cells in uterine tissues. Sections of uterine tissue from IL-10+/+ (A) and IL-10-/- (B) mice recovered 3 day after natural mating with IL-10+/+ Balb/c males were stained immunohistochemically with {alpha}CD45 mAb to detect all leukocytes, including neutrophils and eosinophils (small densely stained cells [arrow]) and macrophages (arrow heads). Arrows in (B) indicate neutrophils migrating through the luminal epithelium. Ep = Epithelium. Magnification x50

Effect of IL-10 Deficiency on Maternal T-Lymphocyte Responses

The inflammatory response elicited by mating causes activation and proliferation of T-lymphocytes in the lymph nodes draining the uterus [31]. To investigate the effect of IL-10 deficiency on the immune response elicited after exposure to semen during early pregnancy, PALN were dissected from IL-10-/- and IL-10+/+ B6 mice at estrus and on Day 4 after natural mating with IL-10+/+ Balb/c males. The number of cells recovered after preparation of single-cell suspensions was enumerated as a measure of lymph node hypertrophy (Fig. 3). The number of cells recovered from Day 4 pregnant mice was greater than from virgin mice (1.6-fold increase, P = 0.03). PALN on Day 4 of pregnancy in IL-10-/- mice contained more cells than those from IL-10+/+ mice (P = 0.04), but the fold increase over virgin numbers was comparable in both genotypes.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. The effect of IL-10 null mutation on lymph node hypertrophy and cytokine expression in T-lymphocytes after mating. Para-aortic lymph nodes were dissected from IL-10-/- and IL-10+/+ B6 mice at estrus and on Day 4 after natural mating with IL-10+/+ Balb/c males. Cell number (A) is given as a box plot with median values scored and mean values defined by dashed line; n = 10–20 mice per group. The 25th and 75th percentiles are defined by the box, 10th and 90th percentiles shown as whiskers, and outliers as symbols. Data were compared by one-way ANOVA and Bonferroni t-test. Data sets labeled on the x-axis with different lowercase letters denote statistical significance between treatment groups (P < 0.05). B) The relative proportion of cytokine producing CD3+ T-lymphocytes from PALN of IL-10-/- and IL-10+/+ B6 mice on Day 4 after natural mating was determined by flow cytometry to detect intracellular cytokines. The proportion of cells with detectable cytokine in PALN from virgin mice was used to set the baseline level (100%). Data are mean ± SEM number of IL-4, IFN{gamma}, and IL-5 positive cells expressed relative to the mean number of cytokine positive cells in PALN from virgin mice (100%). Numbers in parentheses are the proportion of mice in which cytokine was detected in lymph node cells. No effect of genotype on relative cytokine expression was shown when data were compared by independent samples t-test

Expression of cytokines in CD3+ T-lymphocytes was evaluated by intracellular cytokine flow cytometry. Cytokine synthesis was evident in less than 1% of stimulated CD3+ lymphocytes from virgin mice, and this population was used to set the baseline level of cytokine production. Increases in the proportion of cells expressing IL-4, IFN{gamma}, and to a lesser extent IL-5 cytokine production were seen on Day 4 of pregnancy in IL-10+/+ mice, with 83%, 50%, and 50% of the mated mice yielding cells with synthesis of IL-4, IFN{gamma}, and IL-5, respectively, above the threshold value in virgin mice (Fig. 3B). There was no effect of IL-10 genotype on the proportion of mice showing expression of type 1 cytokine IFN{gamma}, or type 2 cytokines IL-4 and IL-5, or on the mean percentage of CD3+ cells expressing each cytokine (Fig. 3B).

Effect of IL-10 Deficiency on Estrous Cycling and Syngeneic Pregnancy Outcome

To determine the effect of IL-10 deficiency on estrous cycling, the morphology of cells in vaginal smears made from adult virgin IL-10+/+ and IL-10-/- B6 mice were examined daily for 21 days. Neither the proportion of mice exhibiting normal cycling behavior (defined as three or more full cycles in the 21-day period), nor the length of the estrous cycle, were influenced by IL-10 genotype. Mean ± SD cycle length in IL-10-/- females was 5.5 ± 0.2 days (n = 12), compared with 5.6 ± 0.3 days in IL-10+/+ B6 mice (n = 11).

To examine the role of IL-10 in pregnancy outcome, adult virgin IL-10+/+ and IL-10-/- B6 females were mated with males of the same genotype and killed at Day 18 of pregnancy. There was no effect of IL-10 deficiency on the interval between housing with a male and the observation of a vaginal plug, the proportion of females mated, or the proportion of plugged females pregnant at Day 18. IL-10-/- pregnancies had 15% more implantation sites on average than IL-10+/+ pregnancies (P = 0.01) and 27% more viable fetuses (P = 0.001; Table 1), with a reduction in resorption rate from 19% to 11% (P = 0.03). Mean fetal weight was 6% greater in IL-10-/- pregnancies than in IL-10+/+ pregnancies (P < 0.001). Placental weights were similar in the two genotypes, but the fetal:placental weight ratio was 8% greater in IL-10-/- pregnancies (P < 0.001, Table 1). Thus IL-10 deficiency did not impede pregnancy success and moderately increased the number and size of fetuses.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effect of maternal and fetal IL-10 deficiency on fetal and placental parameters at Day 18 of syngeneic pregnancy

To further examine the effect of IL-10 deficiency on pregnancy, term and postnatal outcomes were evaluated in adult virgin IL-10+/+ and IL-10-/- B6 females mated with males of the same genotype. There was no effect of IL-10 deficiency on mating parameters or the proportion of plugged females producing viable litters (Table 2). The timing of parturition and the proportion of pregnant animals that successfully delivered pups were not affected by genotype. Although the mean litter size at birth was 14% larger in IL-10 null mutant mice; unlike the previous data set, this increase was not statistically significant. Litter sizes at Day 8 and 3 wk (weaning) were also comparable in IL-10-/- and IL-10+/+ pregnancies, with similar low rates of pup losses between these time points (Table 2). Thus IL-10 deficiency did not compromise delivery of healthy pups nor influence their viability in the neonatal period.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Effect of maternal and fetal IL-10 deficiency on syngeneic pregnancy outcome at term

To evaluate the effect of previous pregnancy, smaller groups of postpartum IL-10+/+ and IL-10-/- B6 mice (n = 13 and 15, respectively) were mated with males of the same strain and genotype, and pregnancies were allowed to proceed to term. There was no effect of IL-10 deficiency on mating or parameters of second pregnancy outcome, including gestation length or litter size at birth or weaning (data not shown).

Effect of IL-10 Deficiency on Allogeneic Pregnancy Outcome

To examine the effect of IL-10 deficiency on pregnancy outcome in the event of maternal and fetal antigenic disparity, pregnancy outcome was measured following mating with males expressing allogeneic MHC antigens. Initially, adult virgin IL-10-/- and IL-10+/+ B6 (H-2b) females were mated with IL-10+/+ Balb/c (H-2d) males, and pregnancies were allowed to proceed to term. There was no effect of maternal IL-10 deficiency and MHC haplotype disparity on mating interval, the proportion of females mated, the proportion of plugged females producing a viable litter, or gestation length. Litter sizes at birth and weaning, as well as postnatal and preweaning mortality, were similar irrespective of maternal IL-10 genotype (data not shown). Thus maternal IL-10 deficiency did not compromise pregnancy even in the event of fetal MHC disparity.

To determine the effect of both maternal and fetal IL-10 deficiency in allogeneic pregnancy, it was necessary to generate IL-10 null mutant males on an MHC background distinct from the H-2b of B6. Male IL-10-/- mice carrying H-2d (IL-10-/- Balb/c.B6) were prepared by 4 cycles of backcrossing through Balb/c males, with individual offspring selected for further rounds of breeding following analysis by PCR for IL-10 and MHC genotype. IL-10-/- Balb/c.B6 (H-2d) males were then mated with virgin IL-10-/- and IL-10+/+ B6 females (H-2b) to generate IL-10-/- and IL-10+/+ mothers carrying MHC disparate IL-10-/- and IL-10 +/- fetuses, respectively. There was no effect of IL-10 deficiency on the interval between housing with IL-10-/- Balb/c.B6 males and the observation of a vaginal plug, the proportion of females mated, or the proportion of plugged females pregnant at Day 18 (data not shown). The numbers of total and viable implantation sites were not affected by IL-10 status. Mean fetal weight was comparable in IL-10-/- pregnancies, and mean placental weight was 6% smaller in IL-10-/- pregnancies (P = 0.015), leading to a 6% larger fetal:placental weight ratio in IL-10-/- pregnancies (mean ± SD = 9.5 ± 1.8 [n = 86] in IL-10-/- versus 9.0 ± 2.0 [n = 91] in IL-10+/+), although this difference was not statistically significant. Thus IL-10 deficiency in both the mother and fetuses did not impede pregnancy success even when fetuses were MHC disparate.

Effect of Maternal IL-10 Deficiency on Growth Trajectory and Adult Body Composition in Progeny

Growth trajectory in utero is a determinant of growth trajectory after birth. The effect of maternal IL-10 deficiency on subsequent growth trajectory of pups was evaluated. In progeny born after syngeneic pregnancy (IL-10+/+ and IL-10-/- B6 mothers mated with males of the same genotype), growth trajectory differed depending on IL-10 status. IL-10-/- progeny were 3% heavier than cytokine replete pups at 12–24 h after birth (P = 0.004), comparable at 8 day of age, and then 9% and 12% lighter in males and females, respectively, at 3 wk of age (both P < 0.01; Table 2). In pups born from second pregnancies, an effect of IL-10 status on growth trajectory in male but not female pups was noted. This was statistically significant at 8 day and particularly evident from 4 wk of age, when male IL-10-/- pups were 10% smaller than male IL-10+/+ pups (mean ± SEM = 14.7 ± 2.2 g [n = 51] versus 16.3 ± 1.8 g [n = 41], respectively; P < 0.001). However, since both mothers and progeny were IL-10 deficient in both experiments, the extent to which the difference can be ascribed to IL-10 deficiency in utero is not clear, and potential effects of low-grade colitis on nutrient uptake in pups cannot be disregarded.

To more specifically address the effect of maternal IL-10 deficiency in utero on postnatal growth trajectory, IL-10+/+ and IL-10+/- progeny gestated in IL-10+/+ and IL-10-/- B6 mothers both mated with IL-10+/+ Balb/c males were examined. Pups born to IL-10 deficient females weighed 3% more than those of replete females at 12–24 h after birth (mean ± SD = 1527 ± 172 mg [n = 109] in IL-10+/- pups and 1478 ± 132 mg [n = 86] in IL-10+/+ pups; P = 0.02), but weighed 9% less by 8 day (P = 0.000). There was considerable growth impairment seen in IL-10+/- males and females, with both weighing 5%–10% less than IL-10+/+ pups from 8 day through to 14 wk (Fig. 4).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 4. The effect of maternal IL-10 deficiency on postnatal growth in male (A) and female (B) progeny of allogeneic matings (mean ± SEM). Values shown at 0 wk (12 h after birth) and 1 wk (8 day after birth) are combined male and female weights. Numbers in parentheses are numbers of animals (n) in each group. Data were compared by repeated measures ANOVA showing a significant effect of genotype over time (P < 0.05). Data at each time point were then compared by independent samples t-test (*P < 0.05)

Altered fetal growth trajectory can be associated with changes in body morphometry in adulthood. To detect any effect of IL-10 deficiency on postnatal development, evident as disproportionate growth of body components relative to total body weight, IL-10+/+ and IL-10+/- male and female progeny from allogeneic pregnancies were dissected at 14 wk of age and body composition was analyzed. In males, maternal IL-10 deficiency resulted in a 10% decrease in total body weight compared with controls (P < 0.001). This was accompanied by an 8% increase in relative brain weight (P = 0.001), a 51% increase in retroperitoneal fat weight (P = 0.007), a 27% increase in abdominal fat weight (P = 0.035), and a 27% decrease in spleen weight (P < 0.001) as a proportion of body weight (Fig. 5A). The proportional weights of the lungs, heart, liver, kidneys, perirenal fat, quadriceps muscle, and testes were unaffected.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 5. The effect of maternal IL-10 null mutation on body composition in male (A) and female (B) progeny at 14 wk of age. Mice (n = 20 males and n = 20 females per genotype) are the progeny of IL-10+/+ and IL-10-/- B6 females mated with IL-10+/+ Balb/c males. Data are the percent change in mean organ weight normalized to total body weight in progeny of IL-10-/- females, relative to values from progeny of IL-10+/+ females. Data were confirmed as normally distributed by linear Q-Q plot, then compared by independent samples t-test (***P < 0.001; **P < 0.01; *P < 0.05).

In females, maternal IL-10 deficiency resulted in a 6% decrease in total body weight compared with controls (P = 0.011). There was a concomitant 7% increase in spleen weight (P = 0.036) and a 10% increase in quadriceps muscle weight (P = 0.016) as a proportion of body weight (Fig. 5B). In females, the proportional weights of the brain, lungs, heart, liver, kidneys, perirenal fat, retroperitoneal fat, and abdominal fat were not significantly affected.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The immune-deviating and anti-inflammatory properties of IL-10 implicate this cytokine in regulation of the immune response to pregnancy. The prominence of IL-10 in gestational tissues from mouse [7, 8] and human [10, 14] and data from the abortion-prone CBA x DBA mouse model [20] are consistent with such a role. However, administration of neutralizing antibodies to IL-10 [21] and initial observations in IL-10 null mutant mice [22, 26] indicate that pregnancy can proceed in the absence of this cytokine. The current study aimed to more precisely characterize the physiological significance of IL-10 in pregnancy. We have confirmed that null mutation of the IL-10 gene does not impair embryo implantation and development to term even in allogeneic pregnancy when both mother and fetus are cytokine deficient. However, subtle effects of IL-10 mutation are evident in the intrauterine and postnatal growth trajectory of progeny. These effects may involve cells or effector molecules of the immune axis since IL-10 deficiency alters uterine inflammatory parameters, as evidenced by impaired resolution of the physiological response to semen early in pregnancy.

Altered postnatal growth trajectory was a consistent finding in each of three experiments where pups gestated by IL-10 deficient mothers were followed after birth and occurred irrespective of whether there was fetal-maternal MHC disparity and irrespective of the IL-10 status of the pups. The latter result excludes the possibility that growth restriction is a consequence of impaired gut development or function due to IL-10 mutation, and it supports the argument that the altered intrauterine environment in IL-10 deficient females perturbs postnatal growth. Growth restriction was most evident at 3–4 wk of age when the mean weights of both male and female progeny delivered by IL-10 mothers were reduced by approximately 10%. Our findings concur with previous observations in IL-10 null mutant mice, where mean body weight was seen to be approximately 30% less in groups of mutant mice studied from 3 to 11 wk of age [22], although failure to distinguish between genders restricts the value of this report. This caveat may explain the difference in magnitude of growth restriction between the current and previous studies. Growth was also transiently compromised in pups born after maternal treatment of pregnant mice with IL-10 neutralizing antibodies [24]. In this experiment, impairment was greatest at 3–4 wk after birth but corrected by 6 wk of age.

Adult mice gestated in an IL-10 deficient uterus were not only smaller, but exhibited altered body morphometry. In common with several other experiments linking perturbation of the intrauterine environment with altered growth in adult life, male progeny appeared to be more severely affected. In the current study, males showed relative sparing of brain development at the expense of the liver and lymphoid organs, with increased abdominal adiposity. This pattern of altered body structure is reminiscent of that seen in small-for-gestational-age human babies, where a form of metabolic dysregulation known as "thrifty" phenotype is programmed in utero and predisposes to adult obesity and late-onset illnesses including cardiovascular disease, hypertension, and diabetes [32, 33]. The major distinction between the current observations and those in humans and in previously described mouse models is the direction of growth perturbation—the "thrifty" phenotype is characterized by decreased weight at birth followed by "catch-up" growth in infancy. Babies who remain small during infancy, however, also have increased risk of metabolic syndrome as adults [33, 34].

In contrast to the impaired growth of pups after birth, measurements at Day 18 and within 24 h of birth show that fetal growth in utero was accelerated in IL-10 null mutant mothers. This was not a reflection of smaller litters, as mean litter size was generally larger in IL-10 deficient mice, and this reached significance in syngeneic pregnancies. Changes in the structure of the placenta in IL-10 null mutant pregnancies have been identified as the likely cause of enhanced fetal growth [35]. Histochemical analyses show that structural correlates of placental function are altered in the absence of IL-10, consistent with enhanced opportunity for maternal-fetal nutrient and metabolite exchange [35]. Specifically, the labyrinth or exchange region of placentae from IL-10-/- mice is larger, with a greater proportion of maternal blood space and increased trophoblast surface area for nutrient transfer. IL-10 is thus implicated as a regulator of placental morphogenesis, acting to retard expansion of the placental labyrinth and to modify the architecture of the maternal blood sinuses. This may be achieved through effects on trophoblast invasiveness and/or transformation of endothelial cells in the placental bed. In vitro experiments show that human cytotrophoblasts express IL-10 receptor and secrete IL-10, which has a dose-dependent inhibitory effect on synthesis of MMP-9 [14, 36], consistent with the postulate that IL-10 deficiency may promote trophoblast invasion through enhancing matrix degradation at the trophoblast-decidual interface. IL-10 may also influence placental vasculature via regulation of nitric oxide (NO) mediated vasodilation [37]. In a rat model of fetal growth restriction and demise, an inhibitory effect of IL-10 on elaboration of NO has been demonstrated [38]. Other pro-inflammatory molecules regulated by IL-10, such as TNF{alpha}, are implicated in trophoblast cell proliferation and differentiation [39], so several possible roles for IL-10 in placental morphogenesis exist.

In mucosal tissues IL-10 is recognized principally as an immune deviating cytokine, acting to promote development of regulatory T-lymphocyte subsets (Tr1 cells) implicated in limiting induction of type 1 immunity and promoting immune tolerance [40]. Our findings of uncompromised pregnancy outcome in the absence of IL-10, even in the event of both maternal and fetal cytokine deficiency and despite maternal and fetal MHC disparity, confirm and extend previous findings [22, 26] and provide conclusive evidence that IL-10 is not required for functional maternal tolerance of allogeneic pregnancy. This result is perhaps not surprising in view of the fact that IL-10 appears not to be essential for mucosal tolerance in tissues other than the gut. IL-10 null mutant mice demonstrate autoimmune pathology limited to the intestinal tract with no evidence of tolerance failure in other mucosae [22]. This contrasts with the multiorgan disease induced by null mutation in transforming growth factor ß1 (TGFß1)[41]. Deficiency in IL-10 also failed to result in skewing of T-lymphocyte phenotypes in PALN during the early stages of the maternal immune response to pregnancy, with no evidence of aberrant expansion of type 1 cells. These data may imply that IL-10 driven Tr1 cells have little physiological role in maternal tolerance to pregnancy. Although Tr1 cells do share some features with the unusual subsets of T-cells found in the implantation site [42], decidual lymphocytes known to be required for successful pregnancy outcome are characterized by their secretion of TGFß [43] and bear a greater resemblance to Th3 cells, the induction of which are dependent on TGFß but not IL-10 [4].

The second major immune function attributed to IL-10 is termination of inflammatory responses, a role likely to be of particular importance in containing events that if left unchecked can lead to tissue pathology following infection or other forms of inflammatory insult. Exposure to semen at mating causes a characteristic inflammatory reaction in the uterus of mice [30] and also in the cervix of women [44]. Consistent with a role in regulating the dynamics of the inflammatory response to semen, IL-10 synthesis has been shown to be induced by seminal plasma in human cervical keratinocytes [45] and is also expressed during early pregnancy in uterine epithelial cells of mice [6]. The experiments reported herein show that IL-10 is required to achieve the timely demise of this postmating inflammatory response. The effect of IL-10 deficiency was most notable on Day 3 of pregnancy when macrophages and granulocytes were markedly more abundant in mutant than control mice. Although the decline of this response is temporally linked with uterine receptivity, the data presented herein show that the extent or nature of perturbation affected by IL-10 deficiency is insufficient to impede normal embryo implantation. The remainder of pregnancy is characterized by a relative dampening of inflammatory events, interrupted at term when declining progesterone again activates the inflammatory cascade that orchestrates parturition. Although it might have been expected that IL-10 deficiency would advance inflammatory processes linked with birth, our data show that the timing of parturition and the survival of pups were unaltered by the absence of this cytokine. Moreover, uterine repair and ability to accommodate a new pregnancy was not impaired, since postpartum matings were similarly successful regardless of IL-10 status.

In women, pre-eclampsia has been linked with exaggerated inflammatory parameters [46], and it is intriguing that IL-10 appears to be up-regulated in placental tissues in this disease [16] as well as in the amniotic fluid of women carrying a small-for-gestational-age fetus [15]. Although these human data do not inform on causal linkages, in view of the observed effect of IL-10 status on fetal growth, it is reasonable to speculate that increased expression of IL-10 in gestational tissues during pathologies of pregnancy acts to limit placental development and hence fetal growth. However, polymorphisms in the IL-10 gene promoter are not associated with placental pathologies [47, 48], so increased IL-10 in pre-eclampsia may be a consequence, rather than a cause, of the condition.

Together, these data confirm that the processes of immune tolerance required to accommodate the embryo are not dependent on IL-10. In contrast, IL-10 is identified as a key regulator of physiological events associated with pregnancy, particularly fetal and placental growth. Our findings highlight the significance of the cytokine environment in utero for programming fetal and postnatal growth trajectory and identify IL-10 as a candidate target in the design of novel therapies for pathologies of pregnancy associated with fetal growth aberrations.


   ACKNOWLEDGMENTS
 
The technical assistance of Ms. Katherine Pensa is gratefully acknowledged.


   FOOTNOTES
 
1 Supported by project and fellowship grants from the NHMRC (Australia) and a project grant from the Pest Animal Control CRC (Australia). Back

2 Correspondence: Sarah A. Robertson, Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA 5005 Australia. FAX: 618 8303 4099; sarah.robertson{at}adelaide.edu.au Back

Received: 28 April 2003.

First decision: 24 May 2003.

Accepted: 29 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001 19:683-765[CrossRef][Medline]
  2. Enk AH, Saloga J, Becker D, Mohamadzadeh M, Knop J. Induction of hapten-specific tolerance by interleukin 10 in vivo. J Exp Med 1994 179:1397-1402[Abstract/Free Full Text]
  3. Asseman C, Powrie F. Interleukin 10 is a growth factor for a population of regulatory T cells. Gut 1998 42:157-158[Free Full Text]
  4. Weiner HL. Oral tolerance: immune mechanisms and the generation of Th3-type TGF-beta-secreting regulatory cells. Microbes Infect 2001 3:947-954[CrossRef][Medline]
  5. Raghupathy R. Th1-type immunity is incompatible with successful pregnancy. Immunol Today 1997 18:478-482[CrossRef][Medline]
  6. Sallinen K, Verajankorva E, Pollanen P. Expression of antigens involved in the presentation of lipid antigens and induction of clonal anergy in the female reproductive tract. J Reprod Immunol 2000 46:91-101[CrossRef][Medline]
  7. Lin H, Mosmann TR, Guilbert L, Tuntipopipat S, Wegmann TG. Synthesis of T helper 2-type cytokines at the maternal-fetal interface. J Immunol 1993 151:4562-4573[Abstract]
  8. Chaouat G, Cayol V, Mairovitz V, Dubanchet S. Localisation of the Th2 cytokines IL-3, IL-4, IL-10 at the murine feto-maternal interface during pregnancy. In: Gupta SK (ed.), Reproductive Immunology. New Delhi: Narosa; 1999: 61–70.
  9. Wessells J, Wessner D, Parsells R, White K, Finkenzeller D, Zimmermann W, Dveksler G. Pregnancy specific glycoprotein 18 induces IL-10 expression in murine macrophages. Eur J Immunol 2000 30:1830-1840[CrossRef][Medline]
  10. Hanna N, Hanna I, Hleb M, Wagner E, Dougherty J, Balkundi D, Padbury J, Sharma S. Gestational age-dependent expression of IL-10 and its receptor in human placental tissues and isolated cytotrophoblasts. J Immunol 2000 164:5721-5728[Abstract/Free Full Text]
  11. Roth I, Corry DB, Locksley RM, Abrams JS, Litton MJ, Fisher SJ. Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10. J Exp Med 1996 184:539-548[Abstract/Free Full Text]
  12. Sacks GP, Clover LM, Bainbridge DR, Redman CW, Sargent IL. Flow cytometric measurement of intracellular Th1 and Th2 cytokine production by human villous and extravillous cytotrophoblast. Placenta 2001 22:550-559[CrossRef][Medline]
  13. Moreau P, Adrian-Cabestre F, Menier C, Guiard V, Gourand L, Dausset J, Carosella ED, Paul P. IL-10 selectively induces HLA-G expression in human trophoblasts and monocytes. Int Immunol 1999 11:803-811[Abstract/Free Full Text]
  14. Roth I, Fisher SJ. IL-10 is an autocrine inhibitor of human placental cytotrophoblast MMP-9 production and invasion. Dev Biol 1999 205:194-204[CrossRef][Medline]
  15. Heyborne KD, McGregor JA, Henry G, Witkin SS, Abrams JS. Interleukin-10 in amniotic fluid at midtrimester: immune activation and suppression in relation to fetal growth. Am J Obstet Gynecol 1994 171:55-59[Medline]
  16. Rinehart BK, Terrone DA, Lagoo-Deenadayalan S, Barber WH, Hale EA, Martin JN Jr, Bennett WA. Expression of the placental cytokines tumor necrosis factor alpha, interleukin 1beta, and interleukin 10 is increased in preeclampsia. Am J Obstet Gynecol 1999 181:915-920[CrossRef][Medline]
  17. Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S. Defective production of both leukemia inhibitory factor and type 2 T-helper cytokines by decidual T cells in unexplained recurrent abortions. Nat Med 1998 4:1020-1024[CrossRef][Medline]
  18. Greig PC, Herbert WN, Robinette BL, Teot LA. Amniotic fluid interleukin-10 concentrations increase through pregnancy and are elevated in patients with preterm labor associated with intrauterine infection. Am J Obstet Gynecol 1995 173:1223-1227[CrossRef][Medline]
  19. Sharma S, Plevyak M, Hanna N. Regulation of IL-10 in human gestational tissues: attenuated expression at term and in missed abortions and preterm labour [abstract]. Placenta 2000 21:A38
  20. Chaouat G, Assal Meliani A, Martal J, Raghupathy R, Elliot J, Mosmann T, Wegmann TG. IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in IL-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-tau. J Immunol 1995 154:4261-4268[Abstract]
  21. Rijhsinghani AG, Thompson K, Tygrette L, Bhatia SK. Inhibition of interleukin-10 during pregnancy results in neonatal growth retardation. Am J Reprod Immunol 1997 37:232-235
  22. Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 1993 75:263-274[CrossRef][Medline]
  23. Rennick D, Davidson N, Berg D. Interleukin-10 gene knock-out mice: a model of chronic inflammation. Clin Immunol Immunopathol 1995 76:S174-178[CrossRef][Medline]
  24. Yang X, Gartner J, Zhu L, Wang S, Brunham RC. IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following chlamydia trachomatis lung infection. J Immunol 1999 162:1010-1017[Abstract/Free Full Text]
  25. Gazzinelli RT, Wysocka M, Hieny S, Scharton Kersten T, Cheever A, Kuhn R, Muller W, Trinchieri G, Sher A. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4+ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J Immunol 1996 157:798-805[Abstract]
  26. Svensson L, Arvola M, Sallstrom MA, Holmdahl R, Mattsson R. The Th2 cytokines IL-4 and IL-10 are not crucial for the completion of allogeneic pregnancy in mice. J Reprod Immunol 2001 51:3-7[CrossRef][Medline]
  27. Sander B, Andersson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 1991 119:65-93[CrossRef][Medline]
  28. Carter LL, Swain SL. Single cell analyses of cytokine production. Curr Opin Immunol 1997 9:177-182[CrossRef][Medline]
  29. Robertson SA, Mau VJ, Tremellen KP, Seamark RF. Role of high molecular weight seminal vesicle proteins in eliciting the uterine inflammatory response to semen in mice. J Reprod Fertil 1996 107:265-277[Abstract/Free Full Text]
  30. Tremellen KP, Seamark RF, Robertson SA. Seminal transforming growth factor beta1 stimulates granulocyte-macrophage colony-stimulating factor production and inflammatory cell recruitment in the murine uterus. Biol Reprod 1998 58:1217-1225[Abstract/Free Full Text]
  31. Johansson MA, Robertson SA. CD69 upregulation and cytokine production in para-aortic lymph nodes following insemination in mice. Am J Reprod Immunol 2001; 46:37-38
  32. Godfrey KM, Robinson S, Hales CN, Barker DJ, Osmond C, Taylor KP. Nutrition in pregnancy and the concentrations of proinsulin, 32–33 split proinsulin, insulin, and C-peptide in cord plasma. Diabet Med 1996 13:868-873[CrossRef][Medline]
  33. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993 341:938-941[CrossRef][Medline]
  34. Robinson JS, Moore VM, Owens JA, McMillen IC. Origins of fetal growth restriction. Eur J Obstet Gynecol Reprod Biol 2000 92:13-19[CrossRef][Medline]
  35. Roberts CT, White CA, Weimer N, Ramsay A, Robertson SA. Altered placental development in interleukin-10 null mutant mice. Placenta 2003; 24: (Suppl A) S94-99
  36. Szony BJ, Bata-Csorgo Z, Bartfai G, Kemeny L, Dobozy A, Kovacs L. Interleukin-10 receptors are expressed by basement membrane anchored, alpha(6) integrin(+) cytotrophoblast cells in early human placenta. Mol Hum Reprod 1999 5:1059-1065[Abstract/Free Full Text]
  37. Myatt L, Brewer AS, Langdon G, Brockman DE. Attenuation of the vasoconstrictor effects of thromboxane and endothelin by nitric oxide in the human fetal-placental circulation. Am J Obstet Gynecol 1992 166:224-230[Medline]
  38. Rivera DL, Olister SM, Liu X, Thompson JH, Zhang XJ, Pennline K, Azuero R, Clark DA, Miller MJ. Interleukin-10 attenuates experimental fetal growth restriction and demise. FASEB J 1998 12:189-197[Abstract/Free Full Text]
  39. Phillips TA, Ni J, Hunt JS. Death-inducing tumour necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentae, cytotrophoblasts, placental macrophages and placental cell lines. Placenta 2001 22:663-672[CrossRef][Medline]
  40. Groux H, Powrie F. Regulatory T cells and inflammatory bowel disease. Immunol Today 1999 20:442-446[CrossRef][Medline]
  41. Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, Doetschman T. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 1992 359:693-699[CrossRef][Medline]
  42. Robertson SA. Control of the immunological environment of the uterus. Rev Reprod 2000 5:164-174[Abstract]
  43. Arck PC, Ferrick DA, Steele Norwood D, Egan PJ, Croitoru K, Carding SR, Dietl J, Clark DA. Murine T cell determination of pregnancy outcome. Cell Immunol 1999 196:71-79[CrossRef][Medline]
  44. Robertson SA, Sharkey DJ, Tremellen KP, Danielsson KG. Semen elicits immunological changes in the human cervix. J Soc Gynecol Invest 2001 9:228A
  45. Denison FC, Grant VE, Calder AA, Kelly RW. Seminal plasma components stimulate interleukin-8 and interleukin-10 release. Mol Hum Reprod 1999 5:220-226[Abstract/Free Full Text]
  46. Sacks G, Sargent I, Redman C. Innate immunity in pregnancy. Immunol Today 2000 21:200-201[Medline]
  47. Babbage SJ, Arkwright PD, Vince GS, Perrey C, Pravica V, Quenby S, Bates M, Hutchinson IV. Cytokine promoter gene polymorphisms and idiopathic recurrent pregnancy loss. J Reprod Immunol 2001 51:21-27[CrossRef][Medline]
  48. Karhukorpi J, Laitinen T, Karttunen R, Tiilikainen AS. The functionally important IL-10 promoter polymorphism (-1082G–>A) is not a major genetic regulator in recurrent spontaneous abortions. Mol Hum Reprod 2001 7:201-203[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
S. A. Robertson, A. S. Care, and R. J. Skinner
Interleukin 10 Regulates Inflammatory Cytokine Synthesis to Protect Against Lipopolysaccharide-Induced Abortion and Fetal Growth Restriction in Mice
Biol Reprod, May 1, 2007; 76(5): 738 - 748.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. A. Robertson, R. J. Skinner, and A. S. Care
Essential Role for IL-10 in Resistance to Lipopolysaccharide-Induced Preterm Labor in Mice
J. Immunol., October 1, 2006; 177(7): 4888 - 4896.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
M. Kimatrai, O. Blanco, R. Munoz-Fernandez, I. Tirado, F. Martin, A. C. Abadia-Molina, and E. G. Olivares
Contractile Activity of Human Decidual Stromal Cells. II. Effect of Interleukin-10
J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6126 - 6130.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. P. Murphy, L. D. Fast, N. N. Hanna, and S. Sharma
Uterine NK Cells Mediate Inflammation-Induced Fetal Demise in IL-10-Null Mice
J. Immunol., September 15, 2005; 175(6): 4084 - 4090.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Sjoblom, C. T. Roberts, M. Wikland, and S. A. Robertson
Granulocyte-Macrophage Colony-Stimulating Factor Alleviates Adverse Consequences of Embryo Culture on Fetal Growth Trajectory and Placental Morphogenesis
Endocrinology, May 1, 2005; 146(5): 2142 - 2153.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
A. C. Zenclussen, K. Gerlof, M. L. Zenclussen, A. Sollwedel, A. Z. Bertoja, T. Ritter, K. Kotsch, J. Leber, and H.-D. Volk
Abnormal T-Cell Reactivity against Paternal Antigens in Spontaneous Abortion: Adoptive Transfer of Pregnancy-Induced CD4+CD25+ T Regulatory Cells Prevents Fetal Rejection in a Murine Abortion Model
Am. J. Pathol., March 1, 2005; 166(3): 811 - 822.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/1/123    most recent
biolreprod.103.018754v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal