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BOR - Papers in Press, published online ahead of print October 19, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044891
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BIOLOGY OF REPRODUCTION 74, 236–245 (2006)
DOI: 10.1095/biolreprod.105.044891
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Timing of Neutrophil Activation and Expression of Proinflammatory Markers Do Not Support a Role for Neutrophils in Cervical Ripening in the Mouse1

Brenda C. Timmons, and Mala S. Mahendroo 2 

Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9032

ABSTRACT

The mechanisms that facilitate remodeling of the cervix in preparation for and during parturition remain poorly understood. In the current study, we have evaluated the timing of inflammatory cell migration in cervix through comparisons between wild-type mice and steroid 5alpha-reductase type 1 null mice (Srd5a1–/–), which fail to undergo cervical ripening due to insufficient local progesterone metabolism. The timing of migration and distribution of macrophages, monocytes, and neutrophils were examined using cervices from wild-type and Srd5a1–/– mice before Day 15 (d15) and during cervical ripening (late d18), and postpartum (d19). Neutrophil numbers were quantitated by cell counts and activity was estimated by measurement of myeloperoxidase activity. The mRNA and/or protein expression of neutrophil chemoattractants, CXCL2 and CXCL1, and other proinflammatory and adhesion molecules, including IL1A, IL1B, TNF, CCL11, CCL5, CCL3, ITGAM, and ICAM1, were measured in cervices collected before, during, and after birth. The effect of neutrophil depletion on parturition was tested. Tissue macrophages, myeloperoxidase activity, and expression of proinflammatory molecules are not increased within the cervix until after birth. Neutrophil numbers do not change after birth and neutrophil depletion before term has no effect on timing or success of parturition. These results suggest that cervical ripening does not require neutrophils. Moreover, neutrophil activation and a general inflammatory response are not initiated within the cervix until shortly after parturition. The timing of inflammatory cell migration and activation in pregnant cervix suggest a role for these cells in postpartum remodeling of the cervix rather than in the initiation of cervical ripening at parturition.

cervical ripening, cervix, cytokines, immunology, inflammatory cells, parturition, pregnancy, 5{alpha}-reductase type 1

INTRODUCTION

Parturition is a multifactorial process for which the molecular mechanisms remain poorly understood. Successful labor and delivery requires both coordinated uterine contractions and extensive remodeling of the cervix. In human, sheep, guinea pig, and rat pregnancy, cervical remodeling can be divided into two phases [1, 2]. The first phase, termed cervical softening, is a gradual process that begins in the first trimester of human pregnancy and by Day 12 (d12) in the rat [1, 2]. The second phase, termed cervical ripening, begins in the day(s) before onset of parturition [2]. Because 85% of the cervix is connective tissue [3], remodeling requires extensive alterations of the connective-tissue matrix. The changes in connective tissue during pregnancy and parturition in part reflect alterations in glycosaminoglycan (GAG) and proteoglycan compositions of the cervix [4–6] and increased collagen turnover [2, 7].

In addition to the influence of proteoglycans on cervical collagen structure, several studies have reported a decline in collagen concentration as a result of increased activity of collagenases and other proteolytic enzymes [8–11]. These proteases are present in cervical fibroblasts [10, 11] as well as polymorphonuclear leukocytes and macrophages [8, 9]. Infiltration of inflammatory cells into the cervical tissue is one of the main histological features observed in the cervix immediately after birth or in the late stages of normal cervical ripening [9, 12, 13]. Infections of the lower reproductive tract in human subjects is frequently associated with premature cervical ripening and labor [14–17].

These observations have led to the currently accepted model of cervical ripening, in which the influx of inflammatory cells is a major regulatory event in the initiation of cervical ripening during normal parturition. Inflammatory cells, such as neutrophils, macrophages, and eosinophils, are proposed to play an important role in the synthesis of cytokines and proteolytic enzymes that regulate the ripening process [8, 9, 12, 18, 19]. These cells are recruited to affected tissue from circulating blood. Inflammatory stimuli in the tissue result in increased expression of chemokines [e.g., IL8 and TNF (TNFalpha)] and adhesion molecules (e.g., ICAM1 and selectins) on endothelial surfaces. Passing inflammatory cells adhere via surface ligands, such as ITGAM (CD11B) [20]. Adherence is followed by extravasation across the vessel wall into the tissue stroma. The leukocytes respond to chemoattractant concentration gradients, which help navigate the cells through the tissue to their destination [20, 21].

Although there is evidence for a role of inflammatory cells in infection-induced preterm labor in women [22], as well as animal models of infection-induced preterm labor [14, 23, 24], an unequivocal role for infiltration of inflammatory cells in the initiation of cervical ripening in normal pregnancy remains controversial. Several studies failed to detect an increase in collagenase activity in term cervix of rats, guinea pigs, and sheep [25–27]. Biomechanical changes of cervical ripening in the rat were inconsistent with an increase in collagenase activity [25]. Messenger RNA and immunolocalization of proinflammatory cytokines in cervix of women indicated low to undetectable levels of cervical leukocytes until immediately after birth, when there was a dramatic increase in expression of interleukins IL-1B, IL-6, and IL-8 [13, 28]. Sakamoto et al. report a lack of correlation between interleukin 8 levels in cervical tissue and cervical ripening as assessed by Bishop score and fetal fibronectin levels [29].

The majority of studies conducted in tissues from women and various animal models indicate that inflammatory cells, chemoattractants, and adhesion molecules are increased in the cervix after initiation of cervical ripening and dilatation or immediately after birth [29–31]. Although the influx of inflammatory cells in the final stages of labor is proposed to correlate with changes occurring at the initiation of cervical ripening, little evidence exists for inflammatory cell activation in cervical samples obtained from term pregnancies before the onset of labor [28–31]. Other human studies have investigated expression of inflammatory mediators (IL-8, MMP8, MMP9, IL-1B) in the lower uterine segment with the assumption that this tissue mimics cellular activation events that occur in the cervix [19, 32, 33]. The cellular composition of the lower uterine segment, however, is quite different from that of the cervix [34]. Taken together, these studies highlight the need for further investigations into the role and function of inflammatory cells in the initiation and progression of normal cervical ripening.

In this study, migration and function of inflammatory cells within the wild-type cervix before, during, and after the ripening process is investigated. Additionally, the Srd5a1–/– mouse is used to study how local progesterone affects the distribution and activation of inflammatory cells in the cervix and determine the role of these cells in cervical ripening. A null mutation in the gene encoding this enzyme causes an accumulation of progesterone within the cervix, resulting in a lack of cervical ripening and defective parturition in female mice [35]. These studies provide new insights into the mechanisms controlling cervical ripening at parturition.

MATERIALS AND METHODS

Mice

Mice used in these studies were of the following strains: C57BL6/129 SvEv, C57BL6, and NIH Swiss. Steroid 5{alpha}-reductase type 1 deficient mice (Srd5a1tmMahe/Srd5a1tmMahe) were generated and genotyped as described previously [35]. These mice have been designated as Srd5a1–/– in this article. Srd5a1 null females were mated to Srd5a1 null males in all timed matings. Timed matings were carried out by housing one male with four females in a cage from 800 to 1300 h. Females were checked at midday for vaginal plugs. Plug day was counted as d0 and birth occurred in the early hours on d19. Unless specified as not in labor or in labor, wild-type d19 mothers have completed parturition. However, Srd5a1–/– mice fail to give birth on d19 and are thus still pregnant at this time point. All studies were conducted in accordance with the standards of humane animal care described in the NIH Guide for the Care and Use of Laboratory Animals using protocols approved by an institutional animal care and research advisory committee.

Immunohistochemistry

Freshly excised cervices were embedded in OCT compound (Tissue Tek; Bayer Corp., Elkhart, IN) and frozen immediately in liquid nitrogen. Air-dried tissue sections (thickness, 5 µm) were fixed for 10 min in acetone. Nonspecific binding was blocked using 1.5% normal donkey serum for 20 min. Sections were incubated for 30 min at 25°C with monoclonal antibody rat anti-mouse neutrophil 7/4 at a working dilution of 0.01 mg/ml (Serotec, Raleigh, NC) or rat anti-mouse BM8 (1:800 of a 0.5 µg/ml stock; BACEM Biosciences, Inc., King of Prussia, PA). The mAb 7/4 recognizes myeloid lineage leukocytes, including neutrophils and monocytes, but not macrophages [36, 37]. BM8 is a pan-macrophage marker that recognizes the F 4/80 antigen found on cell membranes and in the cytosol of mononuclear phagocytes [18]. Biotinylated donkey anti-rat (1:200; Jackson Laboratories, Westgrove, PA) and alkaline phosphatase-conjugated avidin-biotin complex (Vector Laboratories, Burlingame, CA) were applied in sequence followed with Vector Red substrate (Vector Laboratories). Tissues were counterstained in hematoxylin for 10 sec. The primary antibody was replaced with rat IgG2a (Caltag Laboratories, Burlingame, CA) as a negative control. Four to six animals from two mouse strains (C57BL6/129SvEv and NIH Swiss) were tested for each time point and genotype.

Myeloperoxidase Assay

Tissue-associated myeloperoxidase (MPO) activity was determined by modification of the methods of Krawisz et al. [38]. Tissues were homogenized in ice-cold 20 mM K2PO4 (pH 7.4; 1:20 w/v). Homogenates were washed two times in ice-cold 20 mM K2PO4 and resuspended 1:6 in 0.5% hexadecyltrimethylammonium bromide (HTAB; Sigma, St. Louis, MO), 10 mM EDTA in 50 mM K2PO4 (pH 6). The samples were sonicated 5 x 1 sec on ice, freeze-thawed three times, and incubated for 20 min at 4°C. After a final centrifugation (15 000 x g, 20 min, 4°C), the supernatants were tested for MPO activity. The supernatants were combined (1:4) with substrate (0.2 mg/ml O-dianisidine, 0.0005% H2O2 in 50 mM K2PO4) and measured in triplicate. The change in absorbance at 450 nm over 5 min was measured. One unit of activity is defined as a change in the absorbance of 1 optical density unit/min at 25°C. Results are expressed as units of MPO per microgram of total protein as determined by bichonic acid assay (Pierce Chemical, Rockford, IL). Three mice of the strain C57BL6/129SvEv were used for each genotype and time point.

Cell Counts

Neutrophils and eosinophils were counted from single-cell suspensions of cervices from C57BL6/129SvEv mice. Freshly harvested cervix tissue was minced and incubated in 1 mg/ml Collagenase B (Roche, Indianapolis, IN) in 10 ml 25 mM HEPES (Invitrogen, Carlsbad, CA), 1x Hanks buffered salt solution (HBSS) (Invitrogen, Carlsbad, CA) for 1.5 h at 37°C. Samples were pipetted with a 10-ml pipette to facilitate dispersal of cells during incubation, passed through a 70-µm filter, and centrifuged 10 min, 3000 x g, 4°C. Pellets were resuspended in HEPES-buffered HBSS and total cell numbers determined using a hematocytometer. Cytospin preparations were made with 106 cells per slide and stained with Diff-Quik (Baxter Scientific, McGraw, IL). Entire slides were counted at high magnification. Neutrophils and eosinophils were identified based on cell morphology. Both cell types have a dark, multilobed nucleus. Eosinophils were distinguished from neutrophils by bright pink-staining cytoplasmic granules. Three animals were used for each time point and results averaged.

RNA Isolation and Quantitative Real-Time PCR

Total RNA was extracted from frozen mouse tissue using RNA Stat 60 (Tel-test B, Friendswood, TX). Individual cervices from 3–5 mice were used for each genotype and time point. Total RNA was treated with DNaseI (DNA free; Ambion, Austin, TX) to remove any genomic DNA contamination. CDNA was synthesized using a TaqMan cDNA synthesis kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using SYBR Green and a PRISM 7900HT Sequence Detection System (Applied Biosystems). Aliquots (20 ng) of total cDNA were used for each PCR reaction and were performed in triplicate. Each gene was normalized to the expression of the housekeeping gene cyclophilin and relative expression was calculated using the d19 cervix as the external calibrator in the ddCt method (step 1; User Bulletin 2; Applied Biosystems). The data also give an estimate of the relative abundance of various mRNA transcripts relative to one another. To do this, the relative abundance of each gene (normalized to cyclophilin) was expressed relative to that of Il1a in the d19 cervix as the external calibrator in the ddCt method. Il1a was chosen because it is a gene that is well expressed in the d19 cervix. Based on these values, we could estimate the relative abundance of each gene relative to the expression of Il1a. This correction factor could then be used to change the relative expression values in step 1 so that the numbers will also give an estimate of the relative gene abundance. Therefore, the d19 value for Il1a content was assigned an arbitrary value of 100, and the expression profile of each transcript was plotted after calibration of the respective d19 values against the value for Il1a. This method provides only a semiquantitative estimate of transcript abundance in a given tissue. Cervical tissues from both C57BL6/129SvEv and NIH Swiss strains were used in these studies, and representative data from the NIH Swiss mice are reported in Figure 6.


Figure 6
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  		FIG. 6. The majority of proinflammatory markers are increased in the cervix after parturition. Relative expression determined by quantitative real-time PCR. Tissues were collected from mice on Gestation Day 15, late on d18, d19 not in labor (NIL), after the birth of the first pup (IL), and 2 and 10 h after the birth of the first pup (PP). Top panel: Time course of expression in cervical tissue from 3–5 NIH Swiss mice per time point. Data presented is the mean ± SEM. Lower panel: Time course of expression in fetal membranes from 3–5 NIH Swiss mice per time point. Data presented is the mean ± SEM

Neutrophil Depletion

RB6-8C5 is a rat anti-mouse monoclonal antibody directed against Ly6G (GR1), an antigen on the surface of mature murine neutrophils [39–41]. The antibody is weakly recognized by monocytes [37, 42]. Pooled ascites (a gift from Dr. Borna Mehrad, University of Texas Southwestern Medical Center) from nude mice injected with RB6-8C5 hybridoma was administered i.p. at a concentration known to maintain neutrophil depletion [41, 43]. Injections were given the morning of d14, d16, and d18 of gestation, and mice were monitored for their ability to deliver their pups. Both C57BL6 and C57BL6/129SvEv strains were used in this study.

ELISA

Frozen tissues from 3–6 C57BL6/129SvEv animals for each time point and genotype were homogenized in 0.01 M PBS with 10% proteinase inhibitor (Sigma). The samples were then sonicated on ice for 2 x 5 sec and centrifuged at 15000 x g, 4°C, for 20 min. Cytokines were detected in supernatants by commercially available ELISA kits (R&D Systems, Minneapolis, MN) used according to manufacturer's instructions. Absorbance was read at 450 nm using the Safire 2 microplate reader (Tecan, San Jose, CA). Tissue cytokine levels were expressed relative to total protein as determined by the bichonic acid assay (Pierce Chemical).

Statistics

Differences in expression between two groups (e.g., comparison of wild-type and Srd5a1–/– samples) were determined using the Student t-test for normally distributed data or the Mann-Whitney rank sum for data not normally distributed. Differences among three or more groups were determined using Kruskal-Wallis one-way analysis of variance.

RESULTS

Immunohistochemical Analysis of Macrophages and Neutrophil/Monocytes

To evaluate qualitative changes in the number and distribution of macrophages and neutrophils in the cervix during parturition, cervical tissues were obtained from preparturient mice before (Gestational Day 15) and during (late d18) cervical ripening as well as shortly after birth (early d19, 2–4 h postpartum). Immunohistochemistry with monoclonal antibody BM8 revealed abundant resident macrophages in the cervical stroma of wild-type (WT) mice on Gestation Day 15 (Fig. 1A). The distribution of these cells did not change by late d18 (d18.75) before parturition, whereas the amount of staining for BM8 increased by 4 h postpartum relative to d15, suggesting an increased number of macrophages in the tissue (Fig. 1, B and C). Compared with WT cervix, immunostaining with the BM8 antibody indicates there was no obvious difference in macrophage number or distribution in the steroid Srd5a1–/– cervix at Gestation Days 15 or 18.75 (data not shown).


Figure 1
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  		FIG. 1. Macrophage numbers do not increase in the cervix late in gestation. Frozen cervical sections were reacted with monoclonal antibody BM8 to visualize macrophages (red). Photomicrographs represent sections from the cervix as outlined in the INSET of A; vagina (V), cervical stroma (S), cervical os (OS), and epithelia (E). Wild-type (WT) Gestation Day 15 (A); WT d18.75 (B); WT, d19, 4 h postpartum (C). Bar = 200 µm

In contrast with these results, the number and localization of neutrophils and/or monocytes were altered in cervical tissue during ripening (Fig. 2). On d15 in the WT mouse, staining with the antibody 7/4 revealed that 7/4 positive cells (neutrophils/monocytes) were localized to the epithelium and subepithelium (Fig. 2A). By late d18, these cells were redistributed into the cervical stroma (Fig. 2B). Like WT animals on d15, 7/4 positive cells were localized to the subepithelium in cervical tissues from Srd5a1–/– mice, although the overall number of 7/4-positive cells was diminished relative to d15 WT (Fig. 2C). On d18, an increased number of 7/4-positive cells within the stroma was not observed in the Srd5a1–/– cervix (Fig. 2D). The defect in recruitment of 7/4-positive cells may arise from the abnormal elevation in tissue progesterone in these animals. To test this hypothesis, Srd5a1–/– mice at Gestation Day 18 and WT females at Gestation Day 15 were administered the progesterone receptor antagonist, ZK98299 (onapristone; Schering Corp., Kenilworth, NJ) by subcutaneous injection and sacrificed 13 h after treatment. Depletion of progesterone action by ZK98299 treatment resulted in premature recruitment of 7/4-positive cells to the cervical stroma in the Gestation Day 15 WT mouse (Fig. 2E). Furthermore, treatment of d18 Srd5a1–/– mice with ZK98299 resulted in rescue of migration of 7/4-positive cells to the stroma (although to a much lesser extent than WT) (Fig. 2F). These results suggested that progesterone in part regulates changes in the number and distribution of 7/4-positive cells during cervical ripening.


Figure 2
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  		FIG. 2. The 7/4-positive cells are recruited to the cervical stroma by late d18 in wild-type (WT) but not Srd5a1–/– mice. Cervical sections were reacted with monoclonal antibody 7/4 to visualize neutrophils and monocytes (red). WT Gestation Day 15 (A); WT d18.75 (B); Srd5a1–/– (KO) d15 (C); Srd5a1–/– d18.75 (D); WT d15 treated for 13 h with ZK98299 (ZK) (E); Srd5a1–/– d18.75 treated for 13 h with ZK98299 (F). Bar = 200µm. The data are representative of 3–6 different animals per panel

MPO Activity in Cervical Tissues in Pre- and Postparturient Mice

The 7/4 antibody recognizes both neutrophils and monocytes but not macrophages or eosinophils [36, 44]. Because MPO is an enzyme made in the primary granules of neutrophils (and to a much lesser extent in monocytes), its activity is used to quantify neutrophil infiltration into tissue [45]. To determine if the 7/4-positive immune cells on late d18 were neutrophils, MPO activity was measured in cervical tissue extracts (Fig. 3). MPO activity was not detectable in pregnant cervices from WT mice at Gestation Day 15 or Day 18.75 or Srd5a1–/– mice at d18.75. In contrast, MPO activity was detectable shortly after birth in the d19 2- to 4-h postpartum cervix at levels similar to nonpregnant mice at estrus. MPO activity was most pronounced in nonpregnant mice at metestrus, consistent with the fact that polymorphonuclear leukocytes are most concentrated at this stage of the estrous cycle [46, 47].


Figure 3
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  		FIG. 3. MPO activity is undetectable in the pregnant cervix. MPO assays were performed on tissue extracts from pregnant WT (d15 and d18.75); WT d19, 2–4 h postpartum and Srd5a1–/– (d18.75). Cervices from nonpregnant animals in estrus and metestrus were also examined. Three to five animals were tested for each time point and genotype. Results were normalized to total protein. Data represent mean ± SEM. *P< 0.05 compared with all other time points. **P< 0.05 compared with pregnant cervices

Quantification of Neutrophils and Eeosinophils in Cervical Tissues

The paradoxical increase in 7/4-positive cells within the cervical stroma during ripening yet absence of MPO activity warranted further investigation. Cell counts were performed on single-cell suspensions of cervical tissue (Fig. 4). Both neutrophils and eosinophils were assessed by their characteristic cell morphology. Unexpectedly, the number of neutrophils was similar in cervical tissues from WT mice on d15, late d18, or Postpartum Day 19. Shortly after birth (d19), there was a significant increase in the number of eosinophils. The number of both neutrophils and eosinophils was decreased in Srd5a1–/– animals at late d18 compared with WT cervix at the same time point.


Figure 4
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  		FIG. 4. Neutrophil numbers do not increase in the cervix during parturition. Single-cell suspensions were made from whole cervix. Cytospin preparations were made with 106 cells per slide and stained with Diff-Quik. Slides were examined under high magnification, and eosinophils and neutrophils were counted based on cell morphology. Open and solid bars represent number of neutrophils and eosinophils per 106 cells, respectively. Data represents mean ± SEM of three cervices, and two slides were counted for each cervix. *P ≤ 0.05 compared with other time points

The Effect of Neutrophil Depletion on Parturition

The absence of MPO activity and lack of increase in neutrophil numbers at a time point when cervical ripening has initiated (late Gestation Day 18) brings into question the role of neutrophils in cervical ripening in the mouse. To evaluate the requirement of this cell type in the initiation of cervical ripening, neutrophils were depleted from WT mice by treatment with the anti-neutrophil monoclonal antibody RB6-8C5 [41]. Intraperitoneal injections at a concentration known to deplete neutrophils in mice were administered starting at d14 of gestation, and depletion was maintained by further injections on d16 and d18. All 11 animals treated with RB6-8C5 delivered in a timely manner on d19 of gestation. To verify neutrophil depletion in the RB6-8C5-treated cervices, frozen sections were obtained from these cervices on d19 postpartum or on late d18. Immunohistochemistry was carried out using the monoclonal antibody 7/4. This antibody was used rather than the RB6-8C5 antibody, which did not work well for immunohistochemistry (Fig. 5). Comparison of stained sections from untreated cervices on Postpartum Day 19 to those of Postpartum Day 19 RB6-8C5 treated animals confirmed a significant depletion of 7/4-positive cells in the treated group (Fig. 5, compare A versus B). Immunohistochemistry using mAb 7/4 on sections from RB6-8C5-treated mice collected before parturition on d18 also confirmed depletion of 7/4-positive cells at this gestational time point (Fig. 5C).


Figure 5
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  		FIG. 5. Mice treated with RB6-8C5 are depleted of neutrophils as assessed by immunostaining with mAb, 7/4. A) Cervix from untreated mouse on d19, 4 h postpartum. B) Cervix from RB6-8C5-treated mouse on d19, 2–4 h postpartum. C) Cervix from RB6-8C5-treated mouse late on gestation d18. Bar = 200 µm

Expression of Immune-Modulating Genes in Cervix and Fetal Membranes

Extravasation of inflammatory cells from blood vessels into the tissue and their migration and activation within the tissue is regulated by adhesion molecules and cytokines (including chemokines and their receptors). Expression of these immune-modulating genes was assessed in the cervix with the goal of determining whether changes in gene expression occur before, during, or after birth. To address this question, cervical tissues were obtained from mice late on d18, d19 before birth (not in labor, NIL); d19 during labor (IL), d19, 2-h postpartum; and d19, 10-h postpartum. The timing and abundance of cervical expression was compared with expression in fetal membranes in which inflammatory responses modulate function during pregnancy/parturition [13, 48, 49]. Quantitative real-time PCR was used to evaluate mRNA expression. Each gene was normalized to the expression of cyclophilin, and data are presented as gene expression relative to expression of the gene in the d19 cervix. To estimate the abundance of transcripts for one gene relative to all genes measured, data have been corrected based on the relative abundance of each gene to that of Il1a in d19 cervix. It should be pointed out that the relative abundance of each gene is only an estimate because the efficiency of amplification can vary for primer sets from one gene to another.

The mRNA levels of Il1a, Tnf, Cxcl2, Ccl11, Icam1, Itgam, and Ccl5 were measured in cervices (Fig. 6). No change in expression of Il1a, Tnf, Cxcl2, or Icam1 was evident until 2 h after birth, at which time expression increased several fold. Transcripts for the eosinophil chemoattractant, Ccl11, were increased before onset of labor, and the overall estimated abundance of this transcript was greater than of the other genes measured.

The timing of expression and composition of proinflammatory markers in fetal membranes differed from the cervix. The expression of Il1b, Ccl3, Tnf, and Cxcl2 in fetal membranes was already increased by late d18 and expression levels were further increased closer to labor (Fig. 6). The expression of Il1a, Ccl11, Itgam, and Ccl5 was low or undetectable in fetal membranes and did not change from Gestation Day 15 to 18 (data not shown). Proinflammatory gene expression presented in Figure 6 using tissues from NIH Swiss mice were similar to our findings in the C57BL6/129SvEv mice (data not shown), indicating that these expression patterns are not unique to a single mouse strain.

In addition to gene-expression studies, protein expression of the neutrophil chemoattractant CXCL1 (KC), monocyte chemoattractant CCL2 (MCP1/JE), and IL1A, was measured by ELISAs using protein extracts of cervices from C57BL6/129SvEv WT mice at Gestation Day 15; d18.75; d19, 2–4 h postpartum; and d18.75 Srd5a1–/– mice (Fig. 7). Although the expression of the mouse IL8 homologue (CXCL1) was detectable at all time points in the cervix, temporal changes in CXCL1 protein levels were not observed. These results are consistent with the lack of significant change in neutrophil numbers at these times. There was, however, a significant increase in monocyte chemoattractant protein 1 (CCL2) within 2–4 h after birth. Consistent with the expression pattern of cervical Il1a transcripts (Fig. 6 and data not shown), IL1A protein expression was low to undetectable in Srd5a1–/– mice and in WT mice at d15 and d18.75, yet IL1A was increased 30-fold within 2–4 h postpartum in WT cervices.


Figure 7
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  		FIG. 7. ELISAs of tissue extracts to determine the protein expression of CXCL1, CCL2, and IL1A in the cervix. WT cervices at Gestation Day 15; d18.75; and d19, 2–4 h postpartum and Srd5a1–/– cervices at gestation d18.75 were analyzed. While protein expression of CXCL1 does not vary between time points and genotype, CCL2 and IL1A are increased after parturition in WT mice. Data represents mean ± SEM of 3–6 samples. *P ≤ 0.05 compared with WT d18.75

DISCUSSION

In the present study, we show that cervical ripening in the mouse does not require activation of a typical inflammatory response. This conclusion is supported by the fact that tissue macrophages, eosinophils, myeloperoxidase activity, and the expression of proinflammatory molecules are not increased within the cervix until shortly after birth on d19. Furthermore, neutrophil numbers do not change even after birth and neutrophil depletion before term has no effect on timing or success of parturition.

Conflicting Evidence for a Role of Inflammatory Cells in Cervical Ripening

A substantial body of literature exists linking intrauterine infection/inflammation with subsequent preterm birth, as well as inflammatory responses facilitating remodeling/rupture of the fetal membranes during term parturition [14, 22, 50–52]. Nevertheless, evidence for the role of an inflammatory response in mediating normal cervical ripening is clouded by variability from study to study in the tissue site from which the biopsy is collected and the timing of collection. Biopsy specimens from women are from a small area of the cervix that may not be representative of the whole cervix. Often, biopsies are collected immediately following delivery and investigators correlate this data to what is occurring during the onset of labor [29, 31, 53, 54]. Other studies have used myometrial lower uterine segment biopsies to study cervical ripening even though the cellular compositions of the two tissues are quite different [19, 32, 33]. Recently, numerous studies in the rat, mouse, human, and sheep have cast doubt on the requirement of inflammatory cells and the proteases they produce on remodeling of the cervical extracellular matrix at term [25, 27, 34, 55]. The findings from this investigation using an animal model to systematically analyze the timing of an inflammatory response do not support a role of inflammatory mediators during initiation of cervical ripening.

Migration of Inflammatory Cells Occurs Before Parturition

In the current study, we evaluated the temporal relationship between inflammatory cell migration into the cervix, activation of these cells, and cervical ripening through comparison of WT cervices before cervical ripening (Gestation Day 15), during cervical ripening (late Gestation Day 18), and shortly after birth (Gestation Day 19). Immunohistochemical staining for macrophages identified a population of resident cells within the cervix that does not change in number or distribution by Gestation Day 18.75 (Fig. 1). The qualitative increase in macrophage number observed after birth on d19 suggests the arrival or maturation of new macrophages distinct from the residential macrophages detected during pregnancy. The number and distribution of macrophages in the cervical ripening defective Srd5a1–/– mouse is similar to WT before parturition, suggesting that the resident macrophages may not play a crucial role in cervical remodeling; however, further studies are required to confirm that macrophage function is normal in the Srd5a1–/– mouse.

In contrast with macrophages, the number of cells recognized by the mAb 7/4 in the cervical stroma increased on Gestation Day 18, before the onset of parturition. The increased numbers of cells within the stroma could be due to a redistribution of cells within the cervix or more likely to migration of new cells into the tissue. The mAb 7/4 recognizes myeloid lineage leukocytes, including neutrophils and monocytes but not macrophages [36, 37]. The invasion of these cells into the cervical stroma is negatively regulated by progesterone, as evidenced by the absence of migration of 7/4-positive cells in the Srd5a1–/– mouse and the ability of the progesterone receptor antagonist to partially restore migration in these mice. In further support of progesterone's regulatory role, premature migration of 7/4-positive cells into cervical stroma is observed in WT mice treated with progesterone receptor antagonist on Gestation Day 15 (Fig. 2, E and F).

Activation of Inflammatory Cells Within the Cervix Occurs after Parturition

The large influx of cells staining with the 7/4 antibody suggests increased numbers of neutrophils within the cervical stroma late in pregnancy, as previously reported in the rodent and other species [9, 30, 56, 57]; however, MPO activity is not detectable in cervices at Gestation Days 15 or 18 (Fig. 3) nor is there an increase in the number of neutrophils based on quantitative cell counts (Fig. 4). Within 2–4 h after birth, there is a significant increase in MPO activity in WT d19 cervices similar to levels detected in nonpregnant cervix at estrus. This result is consistent with the fact that mice enter into the estrous phase within hours after delivery [46, 47]. Despite measurable MPO activity in Postpartum Day 19 cervix, there is no change in neutrophil numbers based on quantitative cell counts.

There was a significant increase in the number of eosinophils by d19, consistent with an increase in transcripts encoding the eosinophil chemoattractant, Ccl11, on Gestation Day 18; however, IL-5 null mice that are deficient in eosinophils are reported to have normal parturition and postpartum repair of the reproductive tract, suggesting that these cells are not required for normal cervical function during parturition [58]. Eosinophils secrete eosinophil peroxidase (EPO), which is similar in function to MPO; however the substrate used in the MPO assays is recognized only weakly by the EPO enzyme [59]. Thus, the increase in MPO activity in the Postpartum Day 19 cervix is most likely not a result of the increased infiltration of eosinophils. In agreement with our findings, MPO activity cannot be detected in sheep cervix until parturition is initiated [60], and MPO activity is reduced in circulating neutrophils of pregnant women from 12 wk of pregnancy until 6 wk postpartum [61].

Taken together, these data suggest that the majority of 7/4 positive cells on late d18 are not neutrophils and those neutrophils that are present are not activated. The latter statement is supported by studies in human and bovine pregnancy that show elevations in estradiol and progesterone result in inhibition of neutrophil activation, resulting in decreased superoxide anion generation, degranulation, phagocytosis, and MPO activity [62–66]. While serum progesterone levels decline before onset of parturition in the mouse, tissue progesterone concentrations decline more slowly and sufficient progesterone may be present to inhibit neutrophil function similar to the human. Moreover, studies in humans suggest there is a lag between the withdrawal of progesterone and the increase in MPO levels in the postpartum female [61]. Inconsistencies between the 7/4-immunostaining, quantitative neutrophil cell counts, and MPO-activity data led us to address the question as to whether neutrophils are required for cervical ripening. Neutrophil depletion failed to alter the timing or success of parturition. Normal parturition in the absence of neutrophils suggests that these granulocytes do not play a crucial role in the onset of cervical ripening. This conclusion is supported by the findings of Yamanaka et al., in which mice that fail to make granulocytes due to null mutations in the CCAAT/enhancer binding protein (a transcription factor that regulates granulopoiesis), reproduce normally [67].

Cervical Proinflammatory Responses Are Activated Shortly after Birth

Migration of inflammatory cells from blood into tissue, movement to the site of action within tissues, and immune-cell activation are regulated by adhesion molecules and proinflammatory cytokines/chemokines. Within the WT cervix, expression of adhesion and most proinflammatory molecules tested did not increase until shortly after birth, which is consistent with the increase in macrophages, eosinophils, and presence of functional neutrophils after birth on d19. In comparison, an inflammatory response is evident in fetal membranes before onset of parturition. By gestation d18.75, Il1b, Ccl3, Tnf, Cxcl2, Il8rb, and Icam1 increased in the fetal membranes (Fig. 6 and data not shown). The timing of the inflammatory response in fetal membranes is consistent with a role of this response in facilitating the reduction in tensile strength and ruptures of the membranes upon initiation of uterine contractions and exemplifies the point that the distinct compartments/tissues of pregnancy are regulated in an independent yet coordinated fashion. Our studies in the mouse, together with those reported in human reproductive tissues, suggest varied regulation and function of proinflammatory markers within specific regions of the reproductive tract (fetal membranes, cervix, uterus) [13, 28].

Insights from the Cervical Ripening-Defective 5{alpha} Reductase Type 1 Deficient Mouse

The failure of migration of 7/4-positive cells 1 day before birth in the Srd5a1–/– cervix and subsequent partial rescue of migration upon administration of a progesterone receptor antagonist indicate that distribution of these cells is regulated by progesterone. In the absence of SRD5A1, cervical tissue progesterone levels remain elevated, resulting in a block of migration of 7/4-positive cells. As a result, there is a decrease in the numbers of neutrophils and eosinophils and a decrease in MPO activity. Furthermore, a decline in mRNA expression of neutrophil and eosinophil chemoattractants as well as a decline in expression of cell adhesion molecules and cytokines, Il1a, Ccl5, Icam1, was observed in the Srd5a1–/– cervix on the morning of d19 (data not shown). The altered inflammatory response observed in the cervix is not a global defect because the temporal pattern of gene expression in the fetal membranes of the Srd5a1–/– mice is relatively normal, with increased expression of Il1b, Cxcl2, Tnf, Ccl3, and Il8rb on late d18 as compared with d15 (data not shown). Taken together, these data suggest that-7/4 positive cells are required for production of proinflammatory molecules or, alternatively, that lack of 7/4-positive cell recruitment is secondary to progesterone-mediated inhibition of proinflammatory cytokine synthesis by nonleukocyte cells. Additionally, these data suggest that cytokine/chemokine production by fetal membranes does not influence cervical function.

This work provides evidence that inflammatory cell migration but not activation occurs in the cervix before onset of cervical ripening. In contrast with other studies in the mouse, we find no change in macrophage number and distribution within the cervix until after birth [18]. This difference may be due to the experimental methods used (e.g., frozen sections versus formalin-fixed paraffin-embedded sections) or, alternatively, due to the use of different mouse strains (C3/HeN versus C57BL6/129SvEv and NIH Swiss). Because the mAb 7/4 is known to bind monocytes and neutrophils and neutrophil cell counts do not vary, while MPO activity increases after birth, we propose that the majority of 7/4-positive cells in the d18 cervix are monocytes. Upon migration into the cervical stroma, these cells remain inactive until birth, at which time they differentiate into tissue macrophages. Consistent with this idea is the increased staining of macrophages and the increase in CCL2 protein (monocyte chemotactic protein 1) on d19, which together promote macrophage activation and an increase in cell adhesion molecules, such as ITGAM. Future studies using specific antibodies and fluorescent-activated cell sorting will be required to verify the hypothesis that these cells are monocytes that mature into macrophages postpartum as well as to identify the signal that recruits these cells into the cervix by d18 of pregnancy.

These novel findings raise the question as to why inflammatory cells are recruited to the cervix if they are not required for secreting proteases that degrade the extracellular matrix during the final stages of cervical remodeling. We propose that the cells are recruited to the cervix in preparation for the extensive remodeling that occurs after birth as the cervix must remove the large amounts of hyaluronan and other extracellular matrix proteoglycans that are increased in the cervix during cervical ripening as well as remove denatured collagen. This process must occur in a rapid and efficient fashion to prevent infiltration of pathogens into the intrauterine environment.

A second question that arises from this work is the mechanism by which cervical ripening occurs if inflammatory cells do not mediate this process. Changes in cervical collagen structure and tensile strength during pregnancy and parturition are reported to be influenced by changes in GAG and proteoglycan composition, similar to collagen integrity in skin and cartilage. The GAG, hyaluronan, is increased in the cervix during late pregnancy in numerous species [4, 56, 68–70]. The accumulations of hyaluronan and water molecules in the interstices between collagen fibrils may promote dispersion or prevent aggregation of collagen fibrils, thus facilitating the reduced tensile strength of the cervix. Both large and small proteoglycans have been described in cervical tissue during pregnancy [71, 72]. Mice deficient in the small proteoglycan, decorin, have fragile skin with reduced tensile strength due to changes in orientation of collagen fibrils [73]. Further investigations are required to ascertain the potential role of small and large proteoglycans in mediating loss of collagen integrity during cervical ripening. Finally, cervical stromal cells have been described to have collagenase and protease activities in vitro; thus, these cells may be a source of extracellular matrix-degrading enzymes required during cervical remodeling [11, 74].

In summary, these studies suggest that normal cervical ripening does not require a typical inflammatory response involving neutrophil activation in the mouse. We speculate that the mechanisms that initiate premature cervical ripening in infection-induced preterm labor must differ from normal labor and this difference must be considered in devising therapies for prevention of preterm labor that are not caused by infection. Future studies will be required to determine if these processes are conserved in the human.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Ann Word for helpful discussions and critical reading of the manuscript. The authors thank Dr. Borna Mehrad for providing the RB6-8C5 antibody and for helpful discussions during the course of this work. We thank Dr. Judith Head for providing technical advice for immunohistochemistry and for critical reading of the manuscript. We also thank Dr. David Russell for critical reading of the manuscript. The excellent technical assistance of John Shelton and Kelly Straach is gratefully acknowledged.

FOOTNOTES

1 Supported by NIH RO1 HD043154–03 (M.S.M.). Back

2 Correspondence. FAX: 214 648 9242; mala.mahendroo{at}utsouthwestern.edu Back

Received: 1 July 2005.

First decision: 17 August 2005.

Accepted: 19 October 2005.

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