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Macrophage Trafficking in the Uterus and Cervix Precedes Parturition in the Mouse

^a Center for Perinatal Biology, Departments of Physiology, ^b Pathology, ^c and Anatomy, ^d Loma Linda University School of Medicine, Loma Linda University Medical Center, Loma Linda, California 92350

	ABSTRACT

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Immune activation is implicated in the etiology of preterm labor, but little is known about macrophage number or distribution in the uterus or cervix at term. This study tested the hypothesis that macrophages migrate into the reproductive tract before the onset of parturition. Paraffin-embedded sections from the mid-uterine horn and cervix of C3/HeN mice on Days 15 and 18 of pregnancy, the day of birth (Day 19), and 1 day postpartum were stained with a pan-macrophage marker to analyze cell numbers and distribution. During pregnancy, uterine macrophages were dispersed in endometrium, usually associated with vasculature and subluminal epithelium. In myometrium, macrophages were clustered in stromal connective tissue; near term and postpartum, cells appeared to surround the muscle bundles. Total macrophage numbers were increased on Day 15 relative to those in nonpregnant controls, declined before birth, and increased postpartum. In the cervix, macrophages congregated in subepithelium, often perivascular or near ganglia. Macrophage numbers in the cervix peaked on Day 18, then declined to nonpregnant levels by the day after birth. Thus, macrophage numbers in the uterus were inversely related to those in the cervix. These findings raise the possibility that macrophages and their products may be involved in uterine contractility and cervical remodeling during the processes of parturition.

	INTRODUCTION

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Preterm labor is correlated with enhanced risk for fetal morbidity and mortality [1, 2]. Over the last decade, a significant percentage of preterm births has been associated with enhanced immune cell activity within the womb [3, 4]. It is suspected that increased inflammatory cytokine and local prostaglandin production in the amniotic fluid and decidua stimulate uterine contractility and initiate premature labor in mice and humans [5]. Whether immune cells that are resident in the reproductive tract participate in the process of normal parturition remains to be determined.

Among leukocytes that may contribute to recruitment and amplification of inflammatory reactions, the macrophage is the predominant immune cell that resides in the uterus. Macrophages account for upward of 10% of cells in the virgin murine uterus and upward of 20% of cells in the pregnant uterus [6, 7]. Macrophages produce a variety of factors, including prostaglandins and cytokines that are known to regulate uterine contractile activity [8, 9]. Whether resident macrophages have dominion over uterine contractile activity is not known, but there are indications that more macrophages are present within the uterus as gestation progresses [10]. Increased numbers of macrophages were found in cell suspensions from pooled uteri harvested during mid-pregnancy as compared to early gestation. Estimates of macrophage numbers in uterine tissue sections (comparisons between the first and last week of gestation) also suggest a rise as pregnancy progresses [11]. However, actual numbers of macrophages in the uterus in situ, during individual days immediately before or after birth, have not been assessed. Even less is known about macrophages in the cervix. At term, macrophage inflammatory protein-1, a chemotactic signal for myeloid cell immigration and activation, is elevated in the cervix [12, 13]. This is consistent with the suggestion that macrophage numbers may increase at term. Moreover, enhanced concentrations of specific cytokines, i.e., interleukin (IL)-1 and IL-8, in the cervical stroma and mucosa at term are associated with the softening and dilation of the tissue [14]. It is recognized that such cytokines are produced by macrophages [15]. Whether production of these cytokines in the peripartum cervix are elevated as a consequence of increased numbers of resident macrophages before completion of pregnancy is not known. Therefore, the aim of the present study was to test the hypothesis that macrophage numbers in both the uterus and cervix are increased before the onset of normal parturition.

	MATERIALS AND METHODS

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Animal Model

Time-dated-pregnant C3/HeN mice were obtained on Day 12 of pregnancy from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Animals were housed individually in plastic cages in a 12L:12D cycle (lights-on 0600 h); food and water were provided ad libitum. Mice were killed by cervical dislocation on gestational Day 15 or 18, on the day of birth (postpartum; pups were typically born before 0400 h of Day 19 postmating), or 1 day postpartum (n = 11, 6, 4, and 9 per group, respectively). To avoid any possible confound resulting from variation of cell number with respect to time of day, mice were killed in the morning between 0700 h and 0800 h. Briefly, the uterus was excised, and, following an incision along the antimesometrial border, fetuses and placental disks were removed. Two strips from the mid-uterine horn regions (between points of implantation, excluding metrial gland or mesometrium) and the cervix from individuals in each group were immersed in 10% neutral buffered formalin for 24 h, embedded in paraffin, and processed for immunocytochemistry as described below. Similarly, tissue from nonpregnant mice served as control (n = 9 per group). Nonpregnant controls were group housed in the vivarium and at the breeder, and they were the same age post puberty. Nearly all nonpregnant mice were in estrus or proestrus as evidenced by inspection of vaginal discharge and uterine morphology. However, uterine macrophage numbers do not appear to vary with respect to phase of the estrous cycle [10]. This research protocol was reviewed and approved by the Institutional Animal Care and Use Committee.

Tissue Processing and Macrophage Counts

Paraffin-embedded uterine strips were cross sectioned (6 µm, parallel to the circular smooth muscle) and immunostained with an automated "sandwich" technique using the Tekmate 1000 (Biotek Solutions, Santa Barbara, CA). Briefly, sections were mounted on positively charged glass slides, microwaved for 6 min in citrate buffer to unmask antigenicity, and then incubated with primary antibody (BM8; Bachem Biosciences, King of Prussia, PA). BM8 is an affinity-purified rat IgG2a antibody specific for a 125-kDa protein on cell membranes and in the cytosol of mononuclear phagocytes. Biotinylated goat anti-mouse IgG was used as the secondary antibody (Vector Laboratories, Burlingame, CA). Nonimmunized goat serum (15 mM NaN₃) was the source of blocking antibody (DAKO, Carpenteria, CA). Tissue sections processed in the absence of primary antibody displayed no appreciable nonspecific stain, demonstrating the specificity of the immunocytochemical protocol (data not shown). Splenic macrophages served as a positive control (data not shown). Cellular staining after the BM8 antibody procedure was similar to that previously described for the F4/80 monoclonal antibody [16]. Distribution and cell numbers in uterine tissue were also comparable to those in a previous report [7]; in our hands, the BM8 stain produced a darker and more intense contrast of cell bodies and processes than when the F4/80 antibody was used. Other sections processed with periodic acid-Schiff stain to identify resident natural killer cells showed no (uterus) or few (cervix) of this lymphocyte subtype; these findings were comparable to those in a previous report [17].

Uterine macrophages were visually identified by brightfield microscopy (BH-2 microscope; Olympus Optics, Tokyo, Japan). A macrophage was defined as a distinct soma with a brown chromagen deposit (BM8+) surrounding a blue hemotoxylin-labeled nucleus. Brown stain in pseudopodia and cellular fragments, those not associated with a specific nucleus, was noted but excluded from the sum. Cell numbers were counted according to stereological procedures that have been previously described [18, 19]. Briefly, macrophages were counted within every field of view for the uterus (analysis with x40 lens every half x10 field), or every third visual field for the cervix (x20 lens), that was systematically moved from left to right and from top to bottom across the entire tissue section. Total macrophage number was the average sum of all BM8+ cells counted in a total of 4 tissue sections per mouse (2 sections per duplicate uterine strips per mouse). Since hypertrophy and hyperplasia of the reproductive tract vary with respect to stage of pregnancy, BM8+ cell counts were normalized to cell nucleus area. Measurements of cell nucleus area in tissue sections were accomplished by digital imaging of hematoxylin stain (Image Pro Plus; Media Cybernetics, Silver Spring, MD). No change in average nucleus size was noted in mice among the pregnant and postpartum groups (data not shown). This methodology permitted assessment of macrophage density at different stages of pregnancy in comparison to that in the nonpregnant uterus, where the nucleus area was increased because of enhanced cell density as compared to that in the peripartum groups. Peripartum refers to the period from 1 day before to 1 day after birth, represented by the Day 18, postpartum, and 1 day postpartum experimental groups.

Statistical Analysis

Data were evaluated by one-way ANOVA (SPSS, Chicago, IL). When main effects were statistically significant, individual comparisons were made using Bonferroni's test (P < 0.05 was considered significant). If Levene's test for homogeneity of variance was significant, then nonparametric analysis using the Kruskal-Wallis test with multiple comparisons was performed.

	RESULTS

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Morphology and Macrophages in Uterus and Cervix

Morphology of the nonpregnant and pregnant uterus was similar to that previous reported in the mouse [7]. In general, macrophages were particularly associated with glandular and vascular tissue in the endometrium, while in the myometrium, these cells were evident in the connective tissue between muscle bundles. In the nonpregnant uterus, stain was confined to the soma; labeled cells were diffusely present in the densely packed tissue. By Day 15 of pregnancy, uterine hypertrophy was evident, and macrophages were congregated in three distinct subcompartments in the pregnant uterus. Labeled cells were aligned along the subepithelial region of the endometrium at the interface with the uterine lumen (Fig. 1A). In addition, macrophages were prevalent in the endometrial stroma, often near blood vessels or around glandular tissue. Finally, in myometrium, macrophages were clustered in connective tissue or in stromal areas between longitudinal muscle fascicles and circular muscle bundles (Fig. 1B). On Day 18, a diminished macrophage density was apparent in the endometrium, while myometrial macrophage distribution appeared comparable to that at Day 15. Stain not associated with a specific cell body or nucleus was more apparent between muscle bundles, particularly in the longitudinal myometrium, during the peripartum period as compared to Day 15 or in nonpregnant groups.

View larger version (137K): [in this window] [in a new window]   FIG. 1. Photomicrograph of macrophages in the peripartum murine uterus and cervix. Macrophages (arrows) stained dark brown in paraffin-embedded tissue sections (6 µm) that were counterstained a light blue with hematoxylin. A) Macrophage infiltration into the luminal epithelium (epi), endometrium (endo), and stroma between circular myometrial fascicles (myo) of the 1 day postpartum uterus. B) Macrophages were evident in the connective tissue and stroma surrounding longitudinal myometrial bundles of the 1 day postpartum uterus. C) Tissue section of cervix at Day 15 of pregnancy shows macrophages along subepithelium of the mucosa (mucosa) and perimetrium (peri). Inset depicts a single macrophage with extended pseudopodia at high magnification. D) Macrophages adjacent to a cluster of neural ganglion cells (g). Bars = 50 µm (A–C) and 10 µm (D).

The cervix was composed of mucosa, myometrium, and perimetrium (Fig. 1C). During pregnancy, labeled cells were diffusely distributed in each area but were more concentrated along the submucosal epithelium. Perimetrial infiltration of stained cells was associated with nerve bundles and ganglia (Fig. 1D). Polymorphonuclear cells (neutrophils, defined by cellular morphology) often accompanied macrophages in regions that displayed edema. Edema was characterized by the appearance of increased interstitial fluid. Moderate edema was noted at Day 15; on Day 18, edema extended through all tissue layers of the cervix. In the nonpregnant cervix, macrophages were predominately found in the mucosa and myometrium and were most often associated with foci of neutrophils along the luminal epithelium and lamina propria.

Macrophage Number in the Uterus and Cervix

Macrophage numbers in the pregnant uterus varied during the days preceding birth and postpartum (Fig. 2, left). Total macrophage cell numbers, normalized to nucleus area, were significantly increased on Day 15 of pregnancy in comparison to those in the nonpregnant uterus. By Day 18, 1 day before birth, macrophage numbers declined to the value obtained in nonpregnant controls. A diminished macrophage density was apparent in the endometrium on Day 18, while myometrial macrophage density appeared comparable to that on Day 15. On the day after birth, there was an increase in labeled cells as compared to the value in the uterus of nonpregnant mice.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Mean number of macrophages (± SE) in the peripartum uterus and cervix of the mouse relative to that in the nonpregnant group. Labeled cells were counted on Day 15 or 18 of pregnancy (d15 or d18), on the day of delivery (postpartum, PP = Day 19), or 1 day after birth (PPd1); nonpregnant (NP) uteri served as control. Mean numbers of macrophages in NP mice were 476 cells/mm2 nucleus area in uterus and 243 cells/mm2 nucleus area in cervix. See Materials and Methods for details of the cell-counting procedure. Statistical differences are indicated by "a" versus NP, "b" versus d15, "c" versus PP, and "d" versus PPd1; P < 0.05, ANOVA and Kruskal-Wallis with multiple comparisons

Macrophage numbers in the cervix also varied with respect to stage of pregnancy (Fig. 2, right). Macrophage density appeared increased in the myometrium and perimetrium on Day 18, but decreased in the mucosa. The peak number of labeled cells in the cervix was found on Day 18. Macrophage numbers remained elevated in the postpartum group within 12 h of birth. By 1 day postpartum, macrophage infiltration of tissue was markedly decreased and was rarely present in the perivascular regions of the peripartum uterus. Macrophage numbers in the 1 day postpartum group decreased to the value obtained in the cervix of nonpregnant mice. Infiltration by neutrophils increased postpartum.

	DISCUSSION

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At term, two seemingly dissimilar phenomena take place; the uterus prepares for and experiences enhanced contractility while the cervix relaxes, softens, and dilates. Immune cells may participate in both processes [13, 20]. The goal of the present study was to determine whether migration of macrophages into the uterus and cervix occurs during the peripartum period—an integral component of a possible role for this cell in the process of parturition. Results in this report fail to support the hypothesis that an increase in macrophage numbers occurs in the uterus at term. Findings, however, do support the hypothesis that there is an increased number of macrophages in the cervix in anticipation of delivery.

As described by Hunt, the myometrium is a "favored location" of macrophages during pregnancy [7]. Present findings suggest a decline in uterine macrophage numbers during the days preceding term as compared to accumulation of these cells earlier in pregnancy [10, 11]. After this decline in the resident macrophage population, numbers increase to reflect migration back into the uterus during the postpartum period. This migration may be along the loose matrix of cells associated with the lamina propria in the endometrium. Whether proliferation of macrophages contributes to this postpartum increase in number remains to be determined. In any case, the return of macrophages into the uterus might be an important component in the restoration of uterine tissue to the nonpregnant state [21].

In contrast, macrophage numbers in the cervix increase at term, peaking on the day before delivery. Infiltration of the mucosa, as well as myometrial and perimetrial regions of the cervix, is apparent 3 days before delivery. As term approaches, macrophages are increasingly associated with the myometrium, and particularly ganglia or nerve bundles in the perimetrium. Recent data have suggested that primary afferent nerves in the cervix produce secretoneurin [22], a neuropeptide that induces monocyte and macrophage chemotaxis [23]. As the first reported findings of macrophage numbers in the cervix at term, the present results raise the possibility that macrophages may migrate into the cervix, perhaps from the uterus, before the day of delivery in the mouse. Further investigation is needed to test this possibility.

While studies have associated increased inflammatory cytokines in the uterus and amniotic fluid with the initiation of premature and possibly normal labor [24, 25], our findings suggest that the number of macrophages resident in the uterus declines at term. Three days earlier, a peak in macrophage numbers in the uterus on Day 15 coincides with a diminished uterine capacity for powerful and synchronous contractions [26]. These results may be attributed to elevated production of an inducible form of nitric oxide synthase (iNOS), presumably macrophage derived, that inhibits myometrial contractile activity [27]. Decreased uterine macrophage numbers during the peripartum period (Day 18 and postpartum groups) correlate with the decrease in iNOS production that has been observed to occur near term [28] and the appearance of high-amplitude contractions by the murine uterus. Thus, these data suggest that macrophage migration out of the uterus may be associated with disappearance of local factors that promote uterine quiescence, a preparation for labor and delivery. Alternatively, trafficking of remaining resident macrophages within the uterus from endometrium to myometrium may facilitate contractile capabilities, a possibility that will require further investigation.

Unlike the situation with the uterus, increased cytokine concentrations in the cervix at term appear to result from an increased macrophage population. Tanaka et al. [13] in 1998 reported IL-1 and IL-8 concentrations to be correlated with one another and to increase exponentially in the cervix as gestation progresses to term in the human. These cytokines were elevated during premature delivery and were significantly correlated with the degree of cervical dilation and the tocolytic index. Recently, IL-1 and IL-8 concentrations were correlated with increased hyaluronic acid levels and matrix metalloproteinase concentrations in the cervix at term [29, 30]. Hyaluronic acid is a product of glycosaminoglycan remodeling and is associated with increased tissue water content [31], while matrix metalloproteinases are necessary for collagen breakdown in the cervix [32]. As in the uterus, iNOS is present in the cervix; however, in the rat, its production is increased at term [27]. The correlation between increased immune cell-associated products and enhanced number of macrophages raises the possibility that this immune cell may participate in processes of cervical relaxation, remodeling, and ripening that are necessary for parturition.

In summary, macrophage trafficking in the reproductive tract may underlie changes in cytokine concentrations and iNOS production at the conclusion of pregnancy. Timing the onset of active labor depends on a local, though dynamic, relationship between uterine contractile activity and cervical ripening. The present findings suggest that trafficking of macrophages and changes in immune cell products may contribute to the capacity of the myometrium to produce coordinated and powerful contractions, as well as the ripening of the cervix, in preparation for the initiation of parturition.

	ACKNOWLEDGMENTS

 
The authors thank Mary North, Conrad Daniels, and technicians in the Laboratory of Clinical Pathology for technical training and support during immunocytochemical procedures. We also thank Ms. Jocelyn C. Bennett for help in aspects of data acquisition and analyses.

	FOOTNOTES

 
¹ Correspondence. FAX: 909 824 4029; syellon{at}som.llu.edu

Accepted: May 27, 1999.

Received: April 7, 1999.

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				K. Tsuboi, A. Iwane, S. Nakazawa, Y. Sugimoto, and A. Ichikawa Role of Prostaglandin H2 Synthase 2 in Murine Parturition: Study on Ovariectomy-Induced Parturition in Prostaglandin F Receptor-Deficient Mice Biol Reprod, July 1, 2003; 69(1): 195 - 201. [Abstract] [Full Text] [PDF]

				A. M. Mackler, E. M. Barber, O. Takikawa, and J. W. Pollard Indoleamine 2,3-Dioxygenase Is Regulated by IFN-{gamma} in the Mouse Placenta During Listeria monocytogenes Infection J. Immunol., January 15, 2003; 170(2): 823 - 830. [Abstract] [Full Text] [PDF]

				A. Kruse, N. Martens, U. Fernekorn, R. Hallmann, and E. C. Butcher Alterations in the Expression of Homing-Associated Molecules at the Maternal/Fetal Interface During the Course of Pregnancy Biol Reprod, February 1, 2002; 66(2): 333 - 345. [Abstract] [Full Text] [PDF]

				A.M. Mackler, L.M. Green, P.J. McMillan, and S.M. Yellon Distribution and Activation of Uterine Mononuclear Phagocytes in Peripartum Endometrium and Myometrium of the Mouse Biol Reprod, May 1, 2000; 62(5): 1193 - 1200. [Abstract] [Full Text]

				A. M. Mackler, C. A. Ducsay, J. D. Veldhuis, and S. M. Yellon Maturation of Spontaneous and Agonist-Induced Uterine Contractions in the Peripartum Mouse Uterus Biol Reprod, October 1, 1999; 61(4): 873 - 878. [Abstract] [Full Text]

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