Danny J. Schust, Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, 500 N. Keene St., Suite 203 Columbia, MO 65201, USA. E-mail: firstname.lastname@example.org
Citation Nagamatsu T, Schust DJ. The contribution of macrophages to normal and pathological pregnancies. Am J Reprod Immunol 2010
Macrophages represent one of the major leukocyte subsets in the uterine decidua. Owing to their remarkable phenotypic plasticity, decidual macrophages can participate in diverse activities during pregnancy. At baseline, decidual macrophages are characterized by an immunosuppressive phenotype and M2 polarization, supporting feto-maternal immune tolerance. In early pregnancy, macrophage-derived pro-angiogenic factors prompt vascular remodeling within the uterine wall to ensure appropriate utero-placental circulation. Upon invasion by pathogens, pattern recognition receptors on decidual macrophages help to alter the characteristics of these malleable cells toward an M1, inflammatory phenotype. Similar inflammatory characteristics are seen in those macrophages that accumulate in the lower segment of the uterus to drive cervical ripening. Disturbances in the tight control that balances macrophage function during pregnancy can trigger the development of pregnancy complications. Here, we discuss the physiologic role of uterine macrophages at different stages of pregnancy and describe their relevance in selected pregnancy disorders.
Macrophages exhibit a high level of phenotypic plasticity and participate in very diverse physiologic processes, including host defense against external pathogens, the removal of apoptotic cells and angiogenesis.1 Macrophages acquire specialized roles in response to the demands of their microenvironment and, in some organs, are given specific names: Kupffer cells in the liver,2 osteoclasts in the bone,3 etc. Throughout pregnancy, a large number of macrophages reside within the specialized uterine endometrium of pregnancy, the uterine decidua. Although their physiological significance at this site has not been fully elucidated, accumulating evidence suggests the involvement of uterine macrophages in a wide range of gestational process including implantation, placental development and cervical ripening.4–9 Soluble factors and direct cell-to-cell interactions exquisitely and distinctively regulate the trafficking and functional maturation of macrophages at the fetomaternal interface throughout pregnancy. These roles may be very different at different stages of gestation.
Macrophages are included among the several types of professional antigen presenting cells (APCs). In their role as an APC, they bridge innate immune reactions resulting from initial contact with external antigens and antigen-specific adaptive immune responses. The interactions of macrophages with T cells are modulated by co-stimulatory pathway signaling that is critical to the delicate balance between immunity and tolerance.10 During pregnancy, decidual immune cells, including macrophages, are required to negotiate seemingly opposing demands: immune tolerance toward allogenic fetal antigens in the face of constant risk for infection by ascending and blood-borne pathogens. In this light, tight control of macrophage immune function appears to be essential to successful pregnancy.
This review will address the important contributions of decidual macrophages to pregnancy and describe the role of these diverse and changeable immune cells in the pathology of complicated pregnancies.
The phenotype of decidual macrophages
Macrophages are the predominant subset of human leukocyte antigen (HLA)-DR+ APCs in the human decidua and comprise about 20–25% of the total decidual leukocytes.11,12 Immunodetection of decidual macrophages is characterized by combined expression of CD14 and CD68. Dendritic cells (DCs) are CD14−, but HLA-DR+, and make up less than 2% of human decidual immune cells.13 B cells are extremely scarce in the human decidua. In mice, DCs appear to account for a higher percentage (5–10%) of decidual leukocytes, although the sum of macrophages and DCs in the murine decidua is similar to that in humans.14 This discrepancy is mainly attributable to the molecular markers used to discriminate macrophages and DCs in each species. Although CD11c is generally used as a marker for murine DCs,15 the expression of this molecule is shared between macrophages and DCs in humans.16 In both species, macrophages and DCs originate from common precursors in the bone marrow and circulating blood monocytes and they both belong to the mononuclear phagocyte system.17 Therefore, in terms of cellular function, the CD14+ HLA-DR+ APC subset in the human decidua may be a heterogeneous ensemble of CD11c+ DCs and classical macrophages (F4/80+ cells) in the murine decidua. Supporting this concept, human macrophages in the decidua of early pregnancy, but not in non-pregnant endometrium, express dendritic cell–specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, a C type lectin receptor), the expression of which was originally reported on immature DCs.18,19 Additionally, these early, pregnancy-derived macrophages will differentiate into mature CD83+ DCs under in vitro stimulation with inflammatory cytokines.18 The CD14+ immune cells that have been categorized as macrophages in the human decidua may function as a reserve cellular pool to provide functionally matured DCs that can quickly respond to the local cytokine milieu.
There are a variety of reasons to characterize decidual macrophages as immunosuppressive. First, T cell responses in mixed lymphocyte reactions are inhibited by the presence of decidual macrophages in co-culture when compared to those in co-culture with peripheral monocytes.8 Prostaglandin E2, produced by decidual macrophages, putatively accounts for these suppressive effects.20,21 Second, the anti-inflammatory cytokine, IL-10, is produced in greater amounts by decidual macrophages when compared to peripheral monocytes and in vitro-differentiated macrophages.5,7 Finally, tryptophan metabolites produced by indoleamine dioxygenase (IDO) activity function as potent suppressors of T cell proliferation.22 The significance of the IDO pathway in pregnancy maintenance has been demonstrated in a murine model in which rapid T cell-induced rejection of allogeneic concepti was induced by pharmacologic inhibition of IDO.23 IDO transcripts and protein expression have been confirmed in decidual macrophages isolated from term decidua, 5,24 although the actual enzymatic activity has not been studied. Taken together, these immunosuppressive capabilities have been hypothesized to mechanistically link decidual macrophages with the establishment of immune tolerance toward the allogenic fetus (Fig. 1).
The functional maturation of macrophages has recently been re-envisioned in a manner analogous to the well-described concept of Th1/Th2 polarization of effector T cells. This has resulted in subcategorization of macrophage effector phenotypes as either M1 or M2.25–27 Macrophages activated under the influence of pro-inflammatory cytokines and lipopolysaccharide (LPS) are termed as M1-type, secrete tumor necrosis factor (TNF) and IL-12 and participate in the progression of inflammation. In contrast, M2 polarization is induced by exposure to Th2 cytokines (IL-4, IL-10 and IL-13) and glucocorticoids27. M2 polarization is characterized by enhanced expression of innate immunity receptors, including scavenger receptors and the macrophage mannose receptor, as well as an upregulation in arginase activity, which counteracts nitric oxide (NO) synthesis (see below and Fig. 1).28 Additionally, M2 macrophages exhibit increased secretion of IL-1 receptor antagonist29 and a reduction in IL-12 production. These properties of M2 macrophages are involved in tissue repair and the inhibition of inflammation.27 Congruent with the well-accepted Th2 dominance in pregnancy,30 a gene expression study using microarray techniques revealed that CD14+ macrophages isolated from first trimester decidua are polarized toward an M2 phenotype.31 These decidual macrophages demonstrated an up-regulation of M2 marker genes closely related to immunomodulation and tissue remodeling, a finding that supports the important role of macrophages in the maintenance of fetomaternal immunological homeostasis.
Decidual macrophages and the development of uterine vasculature
Dramatic remodeling of the uterine vasculature occurs during the first and second trimesters of human gestation, thereby securing appropriate circulation of maternal blood in the placental intervillous space to support healthy fetal growth. This process is accompanied a marked loss in vascular smooth muscle and endothelial content in uterine spiral arteries along with replacement of the vascular wall by extravillous trophoblast cells.32 These extravillous trophoblast cells decrease baseline spiral artery resistance as well as vascular responsivity. The role of decidual macrophages in this process is becoming increasingly clear. Upon tumor progression and wound healing, macrophages display angiogenic functions mediated by the production of a variety of pro-angiogenic factors in response to hypoxia and other micro-environmental signals.33 Several lines of evidence have demonstrated the involvement of decidual immune cells, including macrophages, in uterine vascular remodeling. Immunohistochemical studies have shown that the earliest stages of vascular remodeling are characterized by an abundant infiltration of CD68+ macrophages and CD56+ uterine NK cells prior to vascular cell apoptosis.34 Interestingly, the production of matrix metalloproteinase (MMP)-7 and -9 was detected in these leukocytes, supporting a role for these cells in degrading the extra-cellular matrix of local tissues to prepare a microenvironment that promotes endovascular invasion by trophoblast cells. The conditional ablation of CD11c+ DCs at the time of implantation in a transgenic mouse model resulted in a striking impairment in decidual vascular development.35 Considering the aforementioned classification problems among human and murine decidual macrophages, the results in this murine model may also support the importance of decidual macrophages in the establishment of utero-placental circulation during human pregnancy.
The role of vascular endothelial growth factor (VEGF) family molecules in the development of placental vasculature has been extensively studied. 36–38 VEGF potently promotes angiogenic processes through its binding to two receptors, fms-like tyrosine kinase-1 (Flt-1) and kinase inert domain receptor (KDR). Placental growth factor (PlGF), another VEGF family ligand, is exclusively produced in the placenta under physiologic conditions and shares some biological functions with VEGF by interacting with Flt-1, but not KDR.39,40 VEGF expression in the uterine endometrium is enhanced by the hormonal stimulation of early pregnancy36 and extra-villous trophoblast cells produce abundant amounts of VEGF and PlGF.38,41 It has been reported that the binding of VEGF or PlGF to monocyte-expressed Flt-1 increases the production and expression of integrins and cytokines, prompts monocyte transmigration through the vascular endothelial layer and enhances the participation of tissue macrophages in the angiogenic process.42–44 The expression of VEGF and Flt-1 has been described in macrophages in early pregnancy.41,45 In short, the combined literature would suggest that the VEGF signaling system may be intimately involved in the angiogenic activities of decidual macrophages by controlling their migration and their level of activation.
Soluble (s)Flt-1 is a secretory isoform of Flt-1 that is generated as the result of alternative splicing of a shared gene transcript.46 Through competitive ligation to VEGF and PlGF, sFlt-1 antagonizes angiogenic signaling via Flt-1 and KDR on cell membranes.47 A significant elevation in placentally derived peripheral sFlt-1 levels is seen in pre-eclamptic women prior to the appearance of clinical symptoms.48–50 The excessive placental production of sFlt-1 in these patients supports a placental origin for pre-eclampsia. Disturbances in uterine vascular remodeling at the site of implantation are a key characteristic of pre-eclamptic placentas. As macrophages have the ability to produce both of VEGF and sFlt-1,51 it can be hypothesized that an imbalance in the production of these molecules by macrophages may be central in the pathogenesis of this disease. Interestingly, sFlt-1 expression in macrophages is induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and placental GM-CSF is elevated in pre-eclamptic placentas when compared to normal placentas.52 Using the abortion-prone mouse model (CBA/J × DBA/2), a recent study found that activation of the complement system stimulates sFlt-1 secretion from monocytes/macrophages and leads to fetal demise.53 Abnormalities in the pro-angiogenic functions of decidual macrophages appear to be closely linked to several pregnancy pathologies, including pre-eclampsia and pregnancy loss.
Pattern recognition receptors and decidual macrophages
The innate immune responses of macrophages are controlled by signaling mediated via pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) and C-type lectin receptors (CLRs). As is the case with the other tissues, the recognition of microbes by TLRs functions as a primary host defense mechanism in the decidua.54 In murine models, however, excessive PRR stimulation during pregnancy by administration of specific ligands for different TLRs induced fetal resorption and pre-term labor, depending on gestational age at the time of exposure.55–59 In each of these studies, elevated production of macrophage-derived inflammatory cytokines, such as IL-1, IL-6, and TNF-α, were confirmed. Notably, the depletion of macrophages or the neutralization of TNF-α rescued pregnancies in a mouse model of deoxy-cytidylate-phosphate-deoxy-guanylate (CpG)-induced pre-term delivery (CpG is a ligand for TLR-9).59 This same study also demonstrated that IL-10(−/−) mice are more susceptible to CpG administration when compared with wild-type mice, suggesting a protective role for IL-10 in PRR-mediated pregnancy loss. Whereas IL-10 is abundantly produced by M2 macrophages, bacterial components such as LPS (a ligand for TLR-4) induce M1 polarization. Therefore, TLR signaling triggered by external pathogen exposure may alter decidual macrophage polarity, shifting the immunosuppressive M2 dominancy in the unexposed state to an inflammatory M1 phenotype.
Progesterone inhibits the innate immune responsivity of macrophages by modulating TLR signaling.60–62 The inhibitory effect of progesterone on the TLR-4 pathway is mediated by the glucocorticoid receptor as well as the progesterone receptor60 and is associated with the suppression of NF-κB and the enhancement of suppressor of cytokine signaling 1 (SOCS1) expression. Together, these alterations in signaling result in a decrease in NO, IL-6 and IL-12 production.61,63 Although this progesterone-mediated immune modulation can be protective by preventing excessive TLR signaling, the same mechanisms may correlate with the increased susceptibility of pregnant women to certain macrophage-tropic organisms, including Toxoplasma gondii and Leishmania species.64,65
Through the recognition of specific carbohydrate patterns on glycosylated proteins, CLRs are implicated in broad range of physiologic cellular functions, such as cell-to-cell contact, leukocyte trafficking and macrophage phagocytosis.66–68 TLRs mainly promote pro-inflammatory processes following binding to pathogen-specific substances. In contrast, CLRs recognize both foreign ligands derived from microbes and endogenous antigens, giving them the potential to aid in maintaining balance in tissue homeostasis. The expression of several types of CLRs has been described in decidual macrophages. Macrophage mannose receptor (MMR, also called CD206 and mannose receptor, C type 1) participates not only in host defense against microbes but also in the clearance of harmful substances produced during inflammatory processes, thereby contributing to the resolution of inflammation and immunosuppression.68,69 MMR is expressed on decidual macrophages throughout the gestational period19,70, and gene expression analysis of early decidual macrophages showed an up-regulation of MMR as a marker of M2 polarization.31 MMR-mediated signaling is promoted within the Th2-dominant local and systemic immune environment71 seen in pregnant women. Although its discrete function at the fetomaternal interface has not been elucidated, it is conceivable that MMR expression on decidual macrophages is a sign of their anti-inflammatory role in human pregnancy.
DC-SIGN, a C-type lectin receptor belonging to the type II receptor family, was originally discovered as a dendritic cell–specific receptor involved in interactions with intracellular adhesion molecule-3 (ICAM-3) expressed on T-cells.72 However, later studies demonstrated that DC-SIGN is also expressed on tissue macrophages residing in the decidua13,18,19 and in pulmonary alveoli.73 A detailed study using pregnant rhesus monkeys revealed that the appearance of CD68+ DC-SIGN+ decidual macrophages starts soon after implantation and that these macrophages are located in close proximity to extravillous trophoblast cells. This localization is in distinct contrast to the diffuse distribution of CD68+ DC-SIGN− decidual macrophages.74 Soluble factors, including cytokines and chemokines derived from trophoblast cells, may control the differentiation and migration of these unique, DC-like decidual macrophages.
DC-SIGN recognizes Lewis X- and Lewis Y-type carbohydrate patterns.75,76 Carcinoembryonic antigen (CEA), carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) and ICAM-3 are known to display Lewis X antigens77–80 and ICAM-2 displays Lewis Y antigens.75 In human colorectal cancers, the condensation of Lewis X/Y sequences on tumor-associated CEA following carcinogenesis enhances the binding of the neoplastic cell to DC-expressed DC-SIGN.77 These DC-SIGN-mediated interactions are proposed as potential immune evasion strategies for cancer cells. As ICAM-3 is expressed on the cell surface of CD56bright uterine NK cells18 and CEA is expressed on trophoblast cells,81 the glycosylation patterns of these molecules, and the consequence of their ligation to decidual macrophage-expressed DC-SIGN, is an interesting subject for future investigation.78
There is evidence to suggest that alterations in CLR expression at the fetomaternal interface are associated with pregnancy complications. In one immunohistochemical study, the basal plates in pre-eclamptic placentas were devoid of CD14+ MMR+ macrophages, while those in normal placentas had abundant CD14+ MMR+ macrophages.82 Increased expression of DC-SIGN and DEC-205 (a CLR mainly expressed on mature DCs) was observed in the placentas women with HELLP syndrome (the most severe pre-eclampsia phenotype), while the placentas from patients with mild pre-eclampsia and intrauterine growth retardation lacked these changes.83 These findings indicate that abnormal regulation of the CLR system in decidual macrophages may be closely linked to the pathophysiology of certain pregnancy abnormalities.
Decidual macrophage interactions via co-stimulatory molecules
In addition to primary antigen presentation via major histocompatibility complex/T cell receptor (MHC-TCR) ligation, co-stimulatory interactions are necessary to induce efficient T cell responses. Co-stimulatory ligands on APCs positively or negatively modulate the level of T cell activation by binding to their corresponding receptors on these T cells, thereby regulating the extent of T cell clonal expansion and downstream effector functions.84 The B7 family of ligands is a major co-stimulatory molecular group and decidual macrophages express several members of this family.85,86 It has been suggested that inhibitory co-stimulatory signaling from decidual APCs (including macrophages) may aid in pregnancy-specific immune tolerance by preventing harmful T cell responses to allogeneic fetal antigens.87,88
Co-stimulatory signaling involving programmed death-1 (PD-1), an inhibitory receptor for B7-H1 and B7-DC, is involved in peripheral tolerance by controlling the development and function of regulatory T cells.89–91 Blockade of B7-H1 signaling increases the rate of rejection of allogenic, but not syngeneic, fetuses in murine models.88 This effect was not observed in RAG-1 deficient mice, suggesting the prevention of allo-destructive fetal reactions via blockade of PD-1 signaling is T cell–mediated. Our own recent work demonstrated that T cell/decidual stromal cell interactions involving PD-1 and its ligands function as a negative feedback system to regulate T cell cytokine production.92 The importance of this pathway at the human fetomaternal interface is also supported by observations of B7H1 expression on trophoblast cells and decidual macrophages87 and extensive PD-1 expression on decidual T cells.93
B7-H4 is another B7 family member that delivers inhibitory signals upon binding to an as-yet-unknown receptor on T cells.94 In human ovarian carcinomas, B7-H4+ macrophages residing within the tumor are able to suppress tumor-associated, antigen-specific T cell responses.95 IL-10 secreted from regulatory T cells induces B7-H4 expression on monocyte-derived APCs, promoting the development of an immunosuppressive phenotype.96 B7H4-positive macrophages have recently been reported to reside in term decidua97, although their functional role remains unclear.
The expression level of B7-1 and B7-2, ligands for an activating receptor, CD28,84 is relatively low on decidual macrophages isolated from term compared to early- and mid-gestation pregnancies.5,19 This suggests that decidual macrophages are activated at an early stage of gestation, probably in response to fetal antigens. Cytotoxic T-lymphocyte antigen 4 (CTLA-4), a second receptor for B7-1 and B7-2, delivers suppressive signals and is constitutively expressed on CD4+ CD25+ regulatory T cells. In vitro investigations have shown that that signaling via B7-1/-2 ligation up-regulates IDO expression in decidual macrophages stimulated with a chimeric CTLA-Ig protein.24 These actions may partially account for the tolerogenic activity of the regulatory T cells that accumulate in the decidua during normal pregnancies.98,99
Relevance of decidual macrophages to the progress of parturition
In contrast to the overall immunosuppressive phenotype of decidual macrophages during pregnancy, several aspects of parturition, including cervical ripening, the onset of labor and post-partum repair of the uterine cervix, are associated with pro-inflammatory macrophage activities. Alterations in the uterine cervix during the peripartum period are characterized by a significant infiltration of leukocytes with inflammatory characteristics.100–102 During human pregnancy, the number of macrophages and granulocytes, but not T cells, selectively increases in the cervix of patients who deliver vaginally or by cesarean section, but only when the latter operation is performed after the onset of spontaneous labor.101,103 In murine cervices, macrophage numbers peak on day 18 of pregnancy (a day prior to birth) and return to non-pregnant levels on the day after delivery.100 Macrophage trafficking to the pre-partum cervix is thought to result from the combined effects of progesterone withdrawal and enhanced local chemokine production.104 One murine model for pre-term labor uses exposure to a P4 receptor antagonist. In these mice, the blockade of progesterone effects combines with mechanical stretch to induce myometrial production of monocyte chemoattractant protein-1 (MCP-1), a potent chemoattractant for monocytes/macrophages.105
The ripening of the uterine cervix is associated with the production of a variety of inflammatory mediators.106 IL-1β, IL-6, IL-8 and TNF-α are generated in large amounts in the peri-partum cervix103,107 and are most likely the product of the leukocytes and macrophages that accumulate rapidly at this site during this time.103 Cervical macrophages acquire a pro-inflammatory phenotype in this cytokine milieu and increase their production of NO, cyclooxygenase (COX)-2 and MMPs.108,109 A critical role for NO in cervical ripening has been established. In rat pregnancies, a marked increase in cervical NO synthesis can be detected during term labor, and NO synthase (NOS) inhibitors decrease cervical distensibility and prolong the duration of labor and delivery.110 In both humans and animals, cervical ripening is stimulated by the administration of NO donors.109,111 Inducible NO synthase (iNOS) activity and NO production are enhanced in CD45+ leukocytes (mostly macrophages) in the third trimester of pregnancy.112 The enhancement of NO production following iNOS activation is associated with the M1, classically activated macrophage phenotype. Alternatively activated macrophages (M2) are characterized by reduced NO synthesis resulting from enhanced arginase function, as arginase and iNOS competitively share the substrate, arginine.113 Active NO synthesis and inflammatory cytokine production suggest that cervical macrophages are polarized toward an M1 phenotype in the peri-partum period. This is in distinct contrast to the immunosuppressive, M2-polarized phenotype of decidual macrophages during the prolonged period of uterine quiescence characterizing earlier pregnancy.
>Abnormal alterations in the activity of cervical macrophages may result in pre-term delivery. Local intrauterine infection, a major initiator of pre-term labor, is thought to trigger a series of inflammatory cascades that lead to cervical change and uterine contractions.114 Murine models for pre-term delivery include some in which labor is induced upon exposure to specific TLR ligands.55–59 During infection-induced pre-term labor, bacterial and viral components may activate innate immune cells, including macrophages, through similar TLR pathways and promote the production of inflammatory cytokines at the fetomaternal interface. An increase in the production of chemokines such as MCP-1 and macrophage migration inhibitory factor (MIF) is described in the amniotic fluid and the uterine myometrium in the presence of infection.115–117 In addition, trophoblast cells can produce regulated on activation, normal T cell-expressed and -secreted (RANTES) in response to TLR-3 signaling.118,119 All of these chemokines may promote de novo recruitment of monocytes and macrophages to the cervix during pregnancy. This, in turn, may cause feed-forward progression of the inflammatory process and infection-associated pre-term labor.
Decidual macrophages participate in a wide range of gestational processes by adapting their cell phenotype to the local microenvironment. The appearance of DC-SIGN+ macrophages in the vicinity of invading trophoblast cells suggests that this DC-like subset of cells might support initial immune tolerance of the fetus by maternal cells. As the placenta develops, macrophages gather around spiral arteries and support vascular remodeling by removing apoptotic cells and producing pro-angiogenic factors such as VEGF family molecules and MMPs. The M2 polarization of decidual macrophages isolated from normal pregnancies indicates that their immunosuppressive activities are required for the maintenance of immunological homeostasis during pregnancy. At the same time, the recognition of dangerous microbes via TLRs and CLRs on macrophages is a central mechanism for host defense in the decidua. The remarkable phenotypic plasticity of uterine macrophages allows balance of these seemingly discrepant activities. At the end of pregnancy, macrophages with an inflammatory phenotype participate in the progression of cervical maturation leading to the onset of labor. In summary, the trafficking and phenotypic differentiation of macrophages are exquisitely controlled based upon the distinct requirements of specific stages of pregnancy. Impairments in uterine macrophage function are closely linked to the pathophysiology of abnormal gestations, including those complicated by pre-eclampsia and pre-term delivery.