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Regular Article |
a Institut für Immunologie und Transfusionsmedizin, Universität zu Lübeck, 23538 Lübeck, Germany
b Lehrstuhl für Experimentelle Medizin I, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
c Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, female reproductive tract, immunology, placenta, pregnancy, trophoblast, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leukocyte recruitment is controlled primarily at the level of leukocyte-endothelial cell (EC) interactions and is mediated by specialized cell adhesion molecules on the surface of circulating cells and their counterreceptors or ligands on the endothelium. Integrins are heterodimeric glycoproteins that are widely expressed by diverse cell types. They are important to leukocyte adhesion because of their diversity and differential expression but also because of their ability to respond to intracellular signals with rapid and dramatic changes in adhesive function. The 
4 integrins and the ß2 integrins have been implicated in specific leukocyte-endothelium interactions. The 4ß7 integrin, for example, is a lymphocyte homing receptor for the mucosal vascular addressin MAdCAM-1 (mucosal addressin cell adhesion molecule 1), which is expressed by high endothelial venules (HEV) in mucosal lymphoid tissues and by postcapillary venules in the intestinal lamina propria in adult mice. The 4ß1 integrin binds to the vascular cell adhesion molecule 1 (VCAM-1), which can be induced in diverse sites of (nonmucosal) inflammation (reviewed in [8, 9]). The 4ß7 integrin can also bind VCAM-1, but its interaction may require a higher state of integrin activation than that needed for binding to MAdCAM-1 [10]. The leukocyte ß2 integrins LFA-1 (lymphocyte function associated antigen-1) and, more recently, Mac-1 (macrophage receptor 1) are constitutive receptors for their vascular ligands intercellular adhesion molecule (ICAM)-1 and ICAM-2, which are widely expressed on endothelium. ICAM-1 is further upregulated on the endothelium during inflammation [11].
The L-, P-, and E-selectins are C-type lectins whose known physiologic roles are limited to leukocyte-EC and leukocyte-leukocyte interactions primarily or exclusively occurring within the bloodstream. These selectins are specialized to support the inital contact of leukocytes with venules at sites of extravasation and can also mediate the subsequent rolling and potentially slowing of rolling of interacting cells. L-selectin is a dominant component of constitutive lymphocyte homing via HEV of peripheral lymph nodes, but it can also participate in lymphocyte homing to the organized mucosal lymphoid tissues [8, 9]. P-selectin is expressed by endothelium and activated platelets and plays an important role in platelet interactions with leukocytes. It is thought to allow leukocytes to roll over aggregations of platelets on vessel walls [12]. E-selectin is exclusively expressed by endothelium and can be induced by proinflammatory cytokines and endotoxin [13]. It binds neutrophils, monocytes, and lymphocytes and plays an important role in leukocyte contact and rolling in certain inflammatory models [14].
The maternal decidua is a tissue undergoing rapid morphological and functional changes, suggesting that these changes may influence the nature of local immune responses at the maternal/fetal interface at different stages of gestation. Even though mechanisms of selective leukocyte-EC recognition and recruitment are known to play an important role in regulating the nature of immune responses in different tissues throughout the body, little attention has been focused on the mechanisms that mediate leukocyte recruitment into the placenta. In addition, an understanding of how these mechanisms are modulated during the course of pregnancy is required. Thus, in this study we sought to identify the homing mechanisms at the maternal/fetal interface from initial trophoblast invasion to development of the mature placenta by defining the vessels that mediate leukocyte recruitment into the decidua and assessing their expression and the function of vascular ligands known to regulate the specificity of leukocyte trafficking. We also characterized the expression of trafficking receptors by maternal leukocytes in the placenta, which may provide insight into the selectivity of maternal leukocyte recruitment mechanisms during development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female BALB/c mice were bred at the VA Medical Center Research Animal Facilities (Palo Alto, CA) or were obtained from the Charles River Breeding Laboratories (Wilmington, MA). Male C57BL/6J mice were obtained from the Charles River Breeding Laboratories. BALB/c females were paired with C57BL/6J males for 1 night. Evidence of mating was determined by vaginal plug appearance the following morning. Plugged females, designated Day 1 pregnant, were caged separately. Pregnant uteri were collected on Days 9, 12, 14, and 20 of pregnancy. Six animals were evaluated for each of the 4 days of pregnancy. Short-term homing studies were performed at Day 9 of pregnancy. Two animals were evaluated for each cell line, time period, and monoclonal antibody (mAb) used. All procedures involving animals were approved by the Stanford University Institutional and German Animal Care and Use Committee and were conducted in accordance with the Guide for Care and Use of Agricultural Animals in Teaching and Research.
Antibodies and Cell Lines
The primary rat anti-mouse mAbs used in this study are summarized in Table 1.
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Anti-human CD44 mAb Hermes-1 (American Type Culture Collection, Rockville, MD) was used as a rat IgG2a isotype-control mAb. The mAbs were purified and concentrated from serum-free culture supernatant by ammonium sulfate precipitation. Rat anti-mouse cytokeratin, B220, Gr-1, Mac-1, and Thy1.2 mAbs were conjugated with fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO). R-Phycoerythrin (PE)-conjugated affinity-purified mouse anti-rat IgG Fab2 polyclonal antibody (Chromaprobe, Mountain View, CA) or biotin-spacer (SP)-conjugated affinity-purified donkey anti-rat IgG (H+L) (Jackson Immuno Research Laboratories, West Grove, PA) was used as the second stage. All antibodies were titered and used at saturation for staining.
The spontaneous AKR/Cum CD8 lymphoma TK1 cell line and the C57L pre-B cell lymphoma line L1-2 [21, 29, 30] were cultured in RPMI 1640 medium supplemented with 10 M Hepes, 100 U/ml penicillin/streptomycin, and 10% fetal calf serum.
Tissue Preparation and Immunofluorescence Histology
Pregnant uteri were collected and rapidly frozen in Tissue-TEK ornithine carbamoyltransferase (Miles, Naperville, IL). Cryostat sections (6 µm) were air-dried overnight, fixed in acetone, and stained using a 2-step immunofluorescence technique. To assist with orientation and selection, every 10th tissue section was stained with hematoxylin and eosin. To facilitate confident identification of maternal versus fetal vessels, in most experiments mice were injected with luconyl blue [31] i.v. 5 min prior to being killed. Luconyl blue is retained in the vascular lumen and is readily visualized in frozen and routine histologic sections as bright blue particles in the maternal vessels. For immunofluorescence staining, tissue sections were incubated with 10% normal mouse serum in PBS, pH 7.1, for 15 min and then exposed for 30 min to individual primary mAbs diluted in PBS. Samples were washed with several changes of PBS for 5 min, incubated for 30 min with PE-conjugated secondary mouse anti-rat IgG (50 µg/ml), and rinsed in PBS. For 2-color staining, tissue sections were then incubated with 10% normal rat serum in PBS, pH 7.1, for 15 min and exposed for 30 min to FITC-conjugated rat anti-mouse cytokeratin, B220, Gr-1, Mac-1, or Thy1.2 mAbs. Either rat anti-human CD44 mAb Hermes-1 or mouse IgG were substituted for primary antibodies as controls for nonspecific staining. The staining patterns of antibodies were tested on at least 10 different tissue samples. Sections were analyzed on the day of staining. Samples were examined with a phase/epifluorescence microscope UFX-II (Nikon, Garden City, NY) or with a DMLM microscope (Leica, Bensheim, Germany) equipped with the appropriate filters. A permanent record of immunofluorescence staining was obtained by a sensitive silicon-intensified target video camera allowing rapid capture of immunofluorescence images. In some instances, the same field was captured under transillumination to allow visualization of lukonyl blue as a marker of maternal vessels. The images were then processed and overlaid (Photoshop; Adobe, Mountain View, CA), as appropriate for presentation in the figures.
To confirm luminal expression of E-selectin in some experiments, we examined staining of trophoblast cells by circulating mAbs. Rat mAb against E-selectin (0.5 mg) was injected i.v. and allowed to circulate for 1–2 h. Mice were then killed and perfused to remove free antibody. Frozen sections were stained with PE-conjugated mouse anti-rat IgG. This approach has been used previously to distinguish luminal from abluminal and cytoplasmic reactivity of HEV for vascular addressins [24].
Immunoperoxidase Staining
For imunoperoxidase staining, acetone-fixed frozen sections were exposed for 60 min to individual mAbs diluted in PBS. Samples were washed with several changes of PBS for 5 min and then incubated for 10 min with 3% H2O2 in methanol to quench endogenous peroxidase activity. After washing for 5 min in PBS, the sections were incubated for 60 min with the biotinylated secondary donkey anti-rat IgG (50 µg/ml) and rinsed in PBS. To increase sensitivity the sections were then incubated with preformed avidin-biotinylated horseradish peroxidase macromolecular complex (ABC reagent; Vector Laboratories, Burlingame, CA) for 30 min, washed with PBS for 5 min, and treated with diaminobenzidine/H2O2 solution (Vector) for 2–10 min. Sections were lightly counterstained with methyleneblue.
Antibody Blocking of TK1 or L1-2 Homing to the Pregnant Uterus In Vivo
TK1 or L1-2 cells were fluorescently labeled with tetramethyl rhodamine isothiocyanate (TRITC, Sigma, Taufkirchen, Germany) as previously described [32]. TK1 or L1-2 cells (5 x 107) were injected i.v. into Day 9 pregnant mice and allowed to circulate for 1 h (2 animals for each cell line) or 2 h (2 animals for each cell line). To investigate whether cell accumulation could be inhibited by specific mAbs, in some experiments Day 9 pregnant mice were injected with 500 µg blocking anti-MAdCAM-1 mAb MECA-367 30 min before injection of TK1 cells (2 animals for each time period) or with 500 µg blocking anti-VCAM-1 mAb MK2.7 30 min before injection of L1-2 cells (2 animals for each time period). Anti-human CD44 mAb Hermes-1 was used as a rat IgG2a negative control (2 animals for each cell line and time period). Uteri were sectioned to identify vascular sites of cell accumulation. Samples were examined with a DMLM microscope (Leica) equipped with the appropriate filters.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We investigated the homing mechanisms at the maternal/fetal interface at several stages of mouse pregnancy from the critical period of initial placenta development (Day 9) over midgestation (Day 12) and the second half of pregnancy (Day 14) to the development of the mature placenta (Day 20). The specialized leukocyte-vascular interactions at the time of initial placenta development were described recently for Day 9 of pregnancy (corresponding to Day 10 according to the terminology in the present study) [6]. Although no striking differences were detected between Day 9 and Day 10 of pregnancy, the results obtained here for Day 9 are summarized for a better comparison of variations in patterns of vascular homing receptors, microenvironmental relationships, and infiltration of specialized leukocyte subsets during gestation. The organization of the pregnant uterus from gestational Days 9, 12, 14, and 20 and a schematic summary of the infiltrating leukocyte subsets and the expression of vascular adhesion molecules are shown in Figures 1 and 2, respectively. The distribution patterns of adhesion molecules and leukocyte subsets in the pregnant uterus are described seperately for each day investigated in the presentation of the immunohistology results.
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Microenvironments of Leukocyte Recruitment in the Day 9 Allopregnant Uterus
During the first half of pregnancy, the uterus is composed of 3 histologically defined zones: the central decidua basalis, the vascular zone (a region of sinusoidal vessels within the decidua basalis), and the decidua capsularis. At Day 9 of pregnancy, the decidua at the maternal/fetal interface displays an unparalleled compartmentalization of microenvironmental domains associated with highly differentiated vessels expressing vascular adhesion molecules in nonoverlapping patterns and with recruitment of specialized leukocyte subsets.
Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood spaces displays the neutrophil ligand E-selectin, which is only weakly expressed at this time of gestation (Figs. 3a and 4a). Double staining with FITC-conjugated anti-cytokeratin mAb confirmed that these maternal blood spaces are lined by trophoblast, suggesting that these trophoblastic cells are themselves E-selectin+ (Fig. 3, a and b). To further evaluate the localized expression of E-selectin, we injected mice with 0.5 mg anti-E-selectin mAb. After 60 min, recipients were killed and perfused under low pressure to remove free mAbs in serum. Sections of uterus were stained with PE-conjugated anti-rat IgG. Double staining with FITC-conjugated anti-cytokeratin mAb confirmed that the trophoblastic cells lining the blood spaces are E-selectin+ (Fig. 3, c and d). These E-selectin+ blood spaces contain mainly neutrophils (Gr-1high, Mac-1low, 4-, ß7- cells) in their lumen and occasional monocytes (not shown). The trophoblast at Day 9 is remarkably devoid of other adhesion molecules, including ICAM-1, ICAM-2, VCAM-1, MAdCAM-1, P-selectin, and peripheral lymph node addressin (PNAd), and 4, ß2, and ß7 integrins (not shown). Cells with the phenotype of monocytes (Mac-1high, LFA-1high, Monts-1+, L-selectin+) but expressing 4ß7 integrin (60%–70% of total cells in the lumen) are localized in the blood vessels of the specialized vascular zone (Figs. 4b and 5d), which display the unusual combination of P-selectin (partially associated with platelets) (Fig. 5a) and the 4ß7 ligand MAdCAM-1. These vessels failed to stain with the mAb against VCAM-1 (Fig. 5c) or E-selectin (not shown). Staining with anti-human CD44 mAb Hermes-1, which was used as a rat IgG2a isotype-control mAb, confirmed that the reactivities are specific (Fig. 5b). As illustrated in Figure 5e, Thy1+ granulated metrial gland (GMG) cells constitute a well-defined cluster positioned in the central decidua basalis (Fig. 4c) around and in venules prominently expressing the 4ß1 ligand VCAM-1 (but not MAdCAM-1) (Fig. 5, e and f). The luminal cells are phenotypically similar to the GMG cells in the tissue: Thy1+, LFA-1high, and 4high (Fig. 5g) but negative for ß7 (Fig. 5h), Mac-1, CD4, and CD8. The expression of ICAM-1 on maternal vessels in the central decidua basalis and the vascular zone is low in contrast to expression of ICAM-2, which is uniformly highly expressed on maternal vessels in the pregnant uterus (not shown). Only rare scattered lymphocytes and small numbers of granulocytes were detected in the decidua basalis. Lymphocytes are predominantly localized in the blood vessels of the vascular zone, representing only 2% of luminal leukocytes (not shown).
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Expression of Vascular Adhesion Receptors and Leukocyte Infiltration at Day 12 of Gestation
At Day 12, the basic decidual zones defined at Day 9 remain almost intact (Fig. 6, a and b). Differences from the Day 9 pregnant uterus include quantitatively more intense staining for most of the vascular adhesion receptors and a dramatic increase of all leukocyte subsets infiltrating the decidua basalis. The degree of separation of microenvironments is not so dramatic as that observed at Day 9, and the maternal vessels of the decidua basalis now start to express vascular adhesion molecules in overlapping patterns. E-selectin expression increases dramatically at Day 12 of pregnancy but remains restricted to the trophoblast-lined maternal blood spaces of the outer spongy zone (Fig. 3, e and f), which now also express ICAM-2 (Fig. 6, c and d). The increase of E-selectin expression at this time is associated with a high accumulation of granulocytes in these vessels and an increase of monocytes. Rare scattered T cells and B cells are seen in the E-selectin+ maternal blood spaces of the outer spongy zone (not shown).
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One of the most striking observations is that maternal blood vessels of the vascular zone (Fig. 6b), which display a combined expression of MAdCAM-1 and P-selectin, now additionally stain weakly but significantly with anti-VCAM-1 mAb (not shown). The vessels of the vascular zone, which remain a site of predominant monocyte recruitment at this stage of pregnancy, now also contain increased numbers of CD4+ (Fig. 6h) and CD8+ T cells, B cells, granulocytes (not shown), and single Thy1+ GMG cells (Fig. 6g) in their lumina, apparently migrating through vessel walls. The luminal GMG cells are phenotypically similar to the GMG cells in the tissues of the vascular zone and decidua basalis: 4highß7-, Thy1+, and LFA-1high but Mac-1-, CD8-, and CD4-. GMG cells reach their maximal accumulation at this time, infiltrating the whole decidua basalis, and are in close contact with the invading trophoblast. Some of the LFA-1high GMG cells can also be found in the maternal blood spaces associated with the giant cell trophoblast, spongiotrophoblast, and labyrinthine zone of the placenta, and these cells apparently interact with trophoblast cells lining the maternal blood spaces (Fig. 6, e and f). In contrast to Day 9 of pregnancy, trophoblast cells in intimate contact with the decidua basalis and trophoblast cells lining the maternal blood sinusoides in the spongy zone are ICAM-2high, which represents the ligand for LFA-1 expressed by GMG cells (Fig. 6, c–f). The central decidua basalis (not shown) retains natural killer (NK)-like GMG cell and macrophage regions but now also contains increased numbers of recruited CD4+ T cells and rare scattered granulocytes. Venules in the central decidua basalis express high levels of VCAM-1 but remain ICAM-1low, ICAM-2high, E-selectin-, and MAdCAM-1-.
Leukocyte-Vascular Homing Interactions at the Maternal/Fetal Interface at Day 14 of Gestation
At Day 14, the major infiltrating leukocyte types are no longer comparmentalized into discrete, well-defined domains within the decidua basalis as seen at Day 9 and to a lesser degree at Day 12 of gestation. E-selectin reaches maximal expression at this time of gestation and predominates on maternal blood spaces lined by trophoblast of the outer spongy zone, which also express ICAM-2 (not shown). These maternal blood spaces contain predominantly neutrophils (40%–50%) and monocytes (20%) but also increased numbers of B cells (10%–20%), T cells (10%–20%), and single GMG cells (not shown). Within the decidua basalis, the predominant remaining populations represent GMG cells (which are fewer in number compared with the Day 9 and Day 12 decidua), monocytes, and neutrophils, which infiltrate the maternal blood vessels and the tissue of the decidua basalis. At Day 14, dilated maternal blood vessels are characterized by MAdCAM-1 expression (reduced compared with Days 9 and 12) (Fig. 7a) and significantly higher expression of P-selectin (Fig. 7c) and VCAM-1 (Fig. 7d). These MAdCAM-1+, P-selectin+, VCAM-1+ vessels contain large numbers of luminally adherent leukocytes presumed to be in the process of adherence and extravasation (Fig. 7, b and e). The great majority of EC-associated leukocytes in these vessels now appear to be granulocytes (representing 40%–60% of luminally leukocytes) (Fig. 7e) followed by 4ß7+ monocytes (25%–30%) (Fig. 7b). However, increased numbers of recruited T cells (15%–20%) and B cells (5%–8%) were also seen within the lumina of these vessels (not shown). At sites of extremely high P-selectin expression, 80%–90% of the adherent mononuclear cells are monocytes (not shown). A bright-field photomicrograph showing the maternal/fetal interface of the Day 14 pregnant uterus is presented in Figure 7f.
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Maternal blood vessels in the central part of the decidua basalis (not shown) express high levels of VCAM-1 but lack immunohistologically detectable E-selectin and PNAd. P-selectin expression is low to negative, and MAdCAM-1 is near background levels on EC lining these vessels. These VCAM-1+ vessels are surrounded by Thy 1+ GMG cells, which infiltrate the tissue of the decidua basalis in large numbers. A significant number of these GMG cells are also seen within the lumina of VCAM-1+ vessels. Only small numbers of T cells, B cells, and granulocytes and almost no 4ß7+ monocytes could be detected. Occasionally vessels are seen lacking immunohistologically detectable MAdCAM-1, P-selectin, VCAM-1, or E-selectin but containing scattered monocytes and lymphocytes.
Strongly Decreased Leukocyte Recruitment in the Mature Prenatal Decidua Basalis (Day 20)
Our studies revealed only minimal infiltration of CD45+ leukocytes in the mature prenatal decidua at Day 20 (not shown). In addition, fewer leukocytes are present within vessels. The expression of vascular addressins on ECs lining maternal blood spaces is dramatically decreased, ICAM-2 expression is weak (Fig. 8, c and d), and E-selectin (Fig. 3, g and h) is near background levels on trophoblast cells lining maternal vessels of the outer spongy zone (Fig. 8, a and b). These vessels contain only small numbers of granulocytes and monocytes, rare scattered T and B cells, and single GMG cells. MAdCAM-1 expression is low on dilated maternal blood vessels, and only P-selectin+ maternal vascular segments remain, defining sites of extremely low residual traffic of strongly reduced numbers of Mac-1+ 4ß7+ monocytes (not shown). VCAM-1 reactivity has also declined, and only a few maternal blood vessels in the decidua basalis still stain brightly with anti-VCAM-1 mAb (Fig. 8e). The number of GMG cells is very much reduced. The 4 integrin reactivity on these cells has declined and is now low to negative (Fig. 8f) in contrast to the strong expression at Days 9 and 12. CD4, CD8, and B cells are rare (not shown).
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Involvement of Adhesion Receptor Pairs in Leukocyte Trafficking to the Day 9 Pregnant Uterus
To assess directly the importance of 4ß7/MAdCAM-1 and 4ß1/VCAM-1 ligand pairs in leukocyte traffic to the decidua basalis, TRITC-labeled 4ß7high, LFA-1+ cell line TK1 or 4ß1+ß7-, LFA-1- cell line L1-2 were injected i.v. into Day 9 pregnant mice and allowed to circulate for 1 h or 2 h (Fig. 9, c–h), respectively. The uterus was sectioned to identify vascular sites of cell accumulation. Day 9 of pregnancy was chosen because at this stage the maternal/fetal interface displays an unparalled compartmentalization of microenvironmental domains associated with highly differentiated maternal vessels expressing vascular addressins in nonoverlapping patterns. TK1 cells expressing the 4ß7 receptor for MAdCAM-1 at very high levels were localized predominantely in the MAdCAM-1+ (Fig. 9a), P-selectin+ (Fig. 5a) dilated maternal blood vessels of the vascular zone (Fig. 9c), which represents the site of predominant monocyte recruitment in Day 9 allogeneic pregnancies (Fig. 9b). Occasionally, single TK1 cells could also be observed in the vessels of the central decidua basalis (not shown). Injection of the specific blocking anti-MAdCAM-1 mAb MECA-367 30 min prior injection of TK1 cells inhibited accumulation of TK1 cells in vessels of the vascular zone (Fig. 9d), whereas the control anti-human CD44 mAb Hermes-1 did not influence the accumulation of TK1 cells (Fig. 9e). Conversely, 4ß1+ L1-2 cells interacted with VCAM-1+ vessels in the central decidua basalis (Fig. 9g) and with the VCAM-1+ venous channels at the base of the mesentery (not shown) but not with the vessels of the vascular zone (Fig. 9f). Injection of the blocking anti-VCAM-1 mAb MK2.7 inhibited accumulation of L1-2 cells in the vessels of the central decidua basalis (Fig. 9h), although some L1-2 cells could still be seen in the tissue of the mesentery (not shown). Control mAb Hermes-1 did not influence the localization of L1-2 cells (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the mouse, the most prominent component of the maternal/fetal interface is the specialized uterine NK cell. These unique cells, termed GMG cells in the mouse [34], are seen in the endometrium during receptive phases for implantation but infiltrate the decidua in large numbers during the first half of pregnancy. Their numbers decline in the second half of pregnancy, and few GMG cells remain by Day 18 of gestation [35, 36]. GMG cells have received considerable attention because of their abundance, unusual phenotype, and still not fully understood function at the maternal/fetal interface [5, 37]. Published speculation about possible roles of GMG cells include regulation of trophoblast development or invasion, but GMG cells may also participate in regulating many other aspects of the local immune environment [38]. These uterine NK cells are a major source of interferon-
, which modifies the expression of genes in the uterine vasculature and stroma, initiating vessel instability and facilitating pregnancy-induced remodeling of decidual arteries [39]. Macrophages and granulocytes are also present. B and T cells are rare, especially early on. However, the proportion of T cells and monocytes/macrophages increases with gestational age, followed by a decline in the term pregnant uterus [40, 41]. These observations indicate that changes in the uterine architecture and alterations in the expression of trophoblast antigens and especially in the fetal/maternal relationship during development may be associated with variations in the patterns of vascular differentiation, microenvironmental relationships, and infiltration of specialized leukocyte subpopulations, suggesting that these factors may influence the nature of the local maternal immune response at different stages of gestation. Thus, our key goal was to characterize the homing mechanisms at the maternal/fetal interface from initial trophoblast invasion to development of the mature placenta.
At Day 9 of pregnancy, which represents the critical period of initial placenta development, the major infiltrating leukocyte types are compartmentalized into discrete, well-defined domains within the decidua basalis. At this time of gestation, the degree of separation of microenvironments is dramatic and reminiscent of the organized architecture thought to play a significant role in regulating immune responses at the tissue level in lymphoid tissues. One of the most striking features of these specialized decidual microenvironments is the distinctive local vascular differentiation associated with unique patterns of vascular adhesion receptor/addressin expression.
Neutrophils are almost exclusively limited to the region of necrosis associated with the enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood spaces display the neutrophil ligand E-selectin. At this stage of gestation, trophoblast cells are remarkably devoid of other immunohistologically detectable adhesion molecules, including VCAM-1, MAdCAM-1, ICAM-1, ICAM-2, P-selectin, and PNAd. GMG cells (4high, ß7-, and probably 4ß1+) constitute a well-defined cluster positioned in the central decidua basalis around venules prominently expressing the 4ß1 ligand VCAM-1. Consistent with this, Burrows [42] observed that human decidual NK-like large granulated lymphocytes located near VCAM-1+ vessels at the implantation site also express the 4ß1 integrin. However, it is not clear whether endothelial VCAM-1, selectively and highly expressed by venules in the GMG cell-rich zone, may help mediate the recruitment of GMG cells to the decidua basalis or whether GMG cells rather differentiate in the uterus from previously recruited precursors.
Monocytes expressing 4ß7 integrin are localized in dilated maternal blood vessels of the vascular zone, which display the unusual combination of P-selectin (partially associated with platelets) and the 4ß7 ligand MAdCAM-1. The expression of 4ß7 integrin by the intraluminal monocytes is of interest because 4ß7 is the receptor for MAdCAM-1 displayed by vascular zone EC but also because most circulating monocytes are 4ß7- [43]. Rare scattered B cells and CD4+ T cells and almost no CD8+ T cells were present. The unusual coexpression of P-selectin and MAdCAM-1 in this site, as discussed recently [6], may provide a mechanism for selecting specialized subsets of leukocytes displaying a unique combination of P-selectin and MAdCAM-1 binding activies (e.g., the 4ß7+ monocytes observed as the dominant luminally adherent cell type in the vascular zone). The same mechanisms perhaps unique to the decidua may effectively bar entry to potentially harmful immunocytes, including most immunocompetent T and B cells. In other studies, it also has proven difficult to demonstrate infiltration of paternally alloreactive T cells, except in the setting of fetal resorption where TH-1 cytokines are discussed to be involved in pregnancy loss [2, 37].
The above results point to the involvement of 4ß7/MAdCAM-1 and 4ß1/VCAM-1 ligand pairs in leukocyte trafficking to the decidua basalis. Short-term homing studies using defined cell lines expressing known homing receptors may be valuable by permitting evaluation of the ability of particular adhesion pathways to participate in leukocyte interaction and arrest in decidual vessels. For this purpose, TRITC-labeled 4ß7high, LFA-1+ TK1 cells or 4ß1+, LFA-1- L1-2 cells (4ß1 binds to VCAM-1 but not to MAdCAM-1) [21] were injected i.v. and allowed to circulate for 1 or 2 h. The uterus was sectioned to identify vascular sites of cell accumulation. The observed localization of TK1 cells in the dilated maternal blood vessels of the vascular zone is particularly interesting because these cells expess the 4ß7 receptor for MAdCAM-1 at very high levels and are therefore predicted to interact well with the MAdCAM-1+ vessels at Day 9 of pregnancy. Conversely, L1-2 4ß1+ß7- cells were not observed in the vascular zone vessels, instead interacting with vessels in the central decidua basalis expressing VCAM-1. Inhibition of TK1 or L1-2 accumulation by specific mAbs against MAdCAM-1 or VCAM-1, respectively, [19–21] supports the importance of predicted adhesion pathways in the localization observed.
At Day 12, the decidua basalis is characterized by a striking influx of all leukocyte subpopulations. The increased recruitment of leukocytes at this time has also been described by Kearns and Lala [40] and may result from a recognition of paternal major histocompatibility complex (MHC) class I alloantigens, whose transcripts can initially be detected at Day 9.5 postcoitus (corresponding to Day 10 in the present study) in the primary and secondary trophoblast giant cell populations [44]. One of the most striking observations at this period is the beginning of loss of microenvironmental specialization, a trend that continues to term. At Day 12 of gestation, the GMG cells reach their maximal accumulation, infiltrating the whole decidua basalis, and are seen in close contact with the invading trophoblast. Some of the GMG cells can also be found in the maternal blood spaces associated with the giant cell trophoblast, spongiotrophoblast, and labyrinthine zone of the placenta, and these cells apparently interact with trophoblast cells lining the maternal blood spaces, as also described by Bulmer et al. [35]. It has been suggested that to control placentation GMG cells must recognize the semiallogeneic fetal trophoblast cells. Evidence that MHC class I molecules are target ligands for NK cells is widely accepted (reviewed in [38]). In humans, decidua-associated NK cells express 1 or more inhibitory receptors specific for human leukocyte antigen G, thus preventing attack of fetal tissues [3]. However, other molecules might also be involved in interactions between the trophoblast and GMG cells. At this stage of pregnancy, trophoblast cells of the outer spongy zone in intimate contact with the decidua basalis and with the GMG cells and trophoblast cells lining the maternal blood sinuses of the deeper spongy zone or labyrinthine zone express ICAM-2 (in contrast to Day 9), which represents the ligand for LFA-1. Thus, GMG cells that show intense reactivity to the anti-LFA-1 mAb, as also described by Stewart [45], also might bind to trophoblast cells via LFA-1/ICAM-2 interaction.
The dilated maternal vessels of the vascular zone have an entirely unusual phenotype at Day 12 of pregnancy, displaying high levels of P-selectin in combination with MAdCAM-1 and VCAM-1. This combination of vascular adhesion receptor expression has not been described previously and may be unique to this setting. However, at Day 12 VCAM-1 expression is only weak. The MAdCAM-1, P-selectin, VCAM-1+ vessels of the vascular zone are surrounded by GMG cells. Single GMG cells, however, are also seen within the lumina of these vessels and extend through the vascular EC lining as if in the process of diapedesis. The GMG cells within the lumina of the dilated vessels are phenotypically indistinguishable from the GMG cells in the tissue and from the GMG cells at Day 9 of pregnancy. They are 4high but ß7-, suggesting that they display the VCAM-1 receptor 4ß1. Their lack of ß7 expression suggests that it is unlikely that they use MAdCAM-1 as a vascular ligand. Expression of endothelial VCAM-1 by vascular zone vessels at this stage of pregnancy and the appearence of GMG cells in the lumina of these vessels and the surrounding tissue suggest an involvement of VCAM-1 in the recruitment or spreading of these cells. However, it has been suggested that during decidualization, activation of small agranular lymphocytes, believed to be precursors of uterine NK cells, occurs and leads to the appearance of GMG cells that rapidly proliferate within the uterine tissue [46]. Thus, the possibility of intravasation of single GMG cells and their seeding from the uterus must be considered seriously; Stewart [47] has seen GMG cells in lungs of pregnant mice.
Within the vessels of the vascular zone, adherent leukocytes are predominantly neutrophils and monocytes, with increased numbers of CD4+ T cells and B cells and occasional CD8+ T cells. The mechanisms involved in lymphocyte recruitment to these vessels are not clear. The exclusion of lymphocytes in the P-selectin+ MAdCAM-1+ vascular zone vessels in the Day 9 pregnant uterus suggests that it is unlikely that the recruited lymphocytes use MAdCAM-1 as a vascular ligand. VCAM-1, however, is induced on vessels involved in mononuclear cell recruitment in nonintestinal sites of inflammation. On most circulating lymphocytes (including 4ß7high T cells, which tend to be 4ß1low but not negative), binding to VCAM-1 is dominated by 4ß1, and 4ß7/VCAM-1 interaction is difficult to demonstrate except under artificial experimental conditions [9]. Thus, lymphocytes and especially T cells expressing 4ß1 or even 4ß7 might be allowed access to the decidua at this time of gestation by binding to endothelial VCAM-1.
At Day 14 of pregnancy, the distinct specialized microdomains defined at Day 9 and to a lesser extent at Day 12 are no longer readily identified, and the maternal vessels of the decidua basalis express vascular adhesion receptors in overlapping patterns. Dilated maternal blood vessels show a strongly increased expression of VCAM-1 and a slightly increased expression of P-selectin. MAdCAM-1 reactivity has declined in comparison to that at Day 12 and at Day 9. This change in vascular adhesion receptor expression (decline of MAdCAM-1 with upregulation of VCAM-1) parallels a dramatic alteration in recruited leukocyte subsets. The great majority of EC-associated leukocytes in these vessels now appear to be granulocytes followed by 4ß7+ monocytes, which are concentrated at sites of extremely high P-selectin expression. Most monocytes are in the tissue and are the predominant cell population next to the GMG cells. Decreased numbers of luminal monocytes might be associated with decreased MAdCAM-1 expression in this site. Increased numbers of recruited T and B cells were also seen within the lumina of these vessels, supporting the involvement of VCAM-1 in their recruitment. An increased recruitment of T cells during the second half of pregnancy was also described by Kearns and Lala [40]. Normal pregnancy is characterized by a lack of strong maternal cell-mediated anti-fetal immunity and a dominant humoral immune response, although paternal antigens are present at the fetal/maternal interface. This finding prompted Wegmann [48] to suggest that the maternal immune response in the pregnant uterus is biased to the less damaging antibody-mediated TH2 type, as indicated by the fact that mouse fetoplacental tissues spontaneously secrete the TH2-type cytokines interleukin (IL) 1, IL-5, and IL-10. The predominance of monocyte/macrophages and GMG cells, however, suggests a critical role for these populations in the pregnant uterus. Thus, suppression of the specific arm of the maternal response might be accompanied, and perhaps compensated for, by activation of the nonspecific innate immune system, as also proposed by Sacks et al. [7]. A unique dysregulation between the innate and specific arms of the maternal immune system, in which monocytes and GMG cells rather than lymphocytes assume a central role in maternal immunological adaptation, must be considered seriously.
The major change in the term pregnant uterus is that the decidua basalis contains remarkably few maternal leukocytes in the lumina of the maternal vessels and in the tissue, suggesting decreased recruitment at this stage. The expression of vascular addressins on EC lining maternal blood spaces also decreases dramatically. Impaired adhesion molecule switching has also been described in the human placenta [49], suggesting that trophoblast cells and maternal EC lose their selective antigenic characteristics when the process of placentation is complete. The number of GMG cells, which represent the predominant cell population during pregnancy, is dramatically reduced and few remain in the term pregnant decidua. The fate of the GMG cells remains uncertain. It has been suggested that some of the depletion of GMG cells in about the last week of pregnancy might be due to migration into the maternal blood vessels [35]; however, there is evidence that beginning at Day 12 of gestation uterine NK cells undergo progressive nuclear fragmentation [36].
The results of this study reveal an elegantly orchestrated progression of selective leukocyte events from the critical peroid of beginning placenta development through development of the mature placenta. One of the most striking features is the partial loss of microenvironmental specialization and switches in vascular specifity during the course of pregnancy. These changes parallel dramatic changes in the populations of leukocytes recruited to the maternal/fetal interface, transforming the leukocyte-rich midterm decidua basalis into the relatively leukocyte-poor phenotype at term. However, our understanding of how decidual leukocytes interact with each other or with the fetal trophoblast during the course of pregnancy is rudimentary. What is now clear is that the decidua in pregnancy is a highly complex tissue containing unique, highly specialized leukocyte subpopulations for each stage of gestation. All of these cells have the potential to secrete a variety of cytokines, thus influencing trophoblast differentiation and modulating other maternal immune cells or regulating the TH1-TH2 cell bias. The highly regulated expression of vascular addressins clearly indicates that adhesion receptor expression and, more generally, mechanisms of vascular differentiation and specialization are fundamental to this process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH grants GM37734 and AI37832, by the FACS Core and the Molecular Biology and Cell Imaging Core Facilities of the Stanford Digestive Disease Center under grant DK38707, and by an award from the Department of Veterans Affairs. A.K. was a recipient of a fellowship from the Deutsche Forschungsgemeinschaft. 
2 Correspondence: Andrea Kruse, Institute of Immunology and Transfusion Medicine, School of Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. FAX: 49 451 5003069; kruse{at}immu.mu-luebeck.de
Accepted: September 13, 2001.
Received: May 4, 2001.
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75% CD4+, 25% CD8+, Thy1+, 
