Danny J. Schust1 and Amanda J. Stephens1
(1)
Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, 500 North Keene Street, Suite 203, Columbia, MO 65203, USA
Danny J. Schust
Email: SchustD@health.missouri.edu
Abbreviations
ADCC
Antibody-dependent cellular cytotoxicity
ANG2
Angiopoietin 2
CMV
Cytomegalovirus
CSA
Chondroitin sulfate A
CTL
Cytotoxic T lymphocyte
DAF
Decay-accelerating factor
DC
Dendritic cell
EVT
Extravillous cytotrophoblast
hCG
Human chorionic gonadotropin
HLA
Human leukocyte antigen
IFN
Interferon
IL
Interleukin
KAR
Killer activation receptor
KIRS
Killer immunoglobulin-like receptors
LIF
Leukemia inhibitory factor
LIRS
Leukocyte immunoglobulin-like receptors
MAC
Membrane attack complex
MBL
Mannose-binding lectin
MCP
Membrane cofactor protein
MHC
Major histocompatibility complex
MS
Multiple sclerosis
NF-κB
Nuclear factor-kappa B
NK
Natural killer
PIBF
Progesterone-induced binding factor
PIGF
Placental growth factor
RA
Rheumatoid arthritis
SLE
Systemic lupus erythematosus
SynT
Syncytiotrophoblast
TCR
T cell receptor
Tfh
Follicular helper T lymphocyte
TGF
Transforming growth factor
Th
T helper
TNF
Tumor necrosis factor
Treg
T regulatory lymphocyte
UL
Unique long
uNK
Uterine natural killer lymphocyte
US
Unique short
VEGFC
Vascular endothelial growth factor C
VZV
Varicella zoster virus
Introduction
Through numerous pathways, the immune system works to protect an individual from exogenous pathogens and from neoplastic cellular changes. During development, immune cells are programmed to discriminate self from non-self and to respond appropriately at initial encounters with self and foreign antigens. When this recognition mechanism fails, the immune system may react inappropriately against self antigens and initiate a series of events that result in autoimmune disorders. During pregnancy, alterations of these recognition processes by the maternal immune system determine the success or failure of continued fetal growth and development until birth. Pregnancy presents a particular immunologic challenge because the tissue antigens presented to the maternal immune system are a combination of self (maternally derived) and non-self (paternally derived) constituents.
The Menstrual and Reproductive Cycle
Throughout the menstrual cycle and pregnancy, changes occur within the lining of the uterine cavity (endometrium) in response to reproductive hormones, particularly the reproductive steroids, estrogen and progesterone [1]. The proliferative phase of the menstrual cycle is characterized by estrogen dominant regeneration of the endometrium [2]. After initial “healing,” regrowth of the ever-changing endometrial “functionalis” layer begins approximately 5 days after the beginning of the menstrual cycle, which is defined clinically as day 1 of bright red vaginal bleeding. This regrowth results from rapid proliferation of the endometrial glands and stroma, which gives this phase of the menstrual cycle its common name—the proliferative phase. Important to this regrowth is a revascularization of the endometrium, which was poorly vascularized during the relatively hypoxic “sloughing” phase of menstruation. Alterations in the length of the proliferative phase are largely responsible for variations from the classical 28 day menstrual cycle. Near the end of the proliferative phase, endocrine, autocrine, and paracrine events within the hypothalamic–pituitary–ovarian axis cause a rapid increase or surge in luteinizing hormone (LH) secretion and ovulation occurs soon thereafter. During this time, local and systemic progesterone levels begin to increase while estrogen levels decrease somewhat. If implantation follows, progesterone levels continue to rise. This progesterone dominant part of the menstrual cycle is called the luteal phase and its length is fairly consistent from cycle to cycle. The endometrium of the luteal phase responds to this new hormonal milieu by undergoing a transformation in preparation for implantation that is called decidualization. The endometrium is now renamed the decidua. Between cycle days 20–24, specific morphologic changes in the decidua characterize the “window of implantation,” including decreased microvilli and the development of cilia with luminal protrusions on the apical glandular surface called pinopodes [3]. The maternal uterine spiral arteries develop and continue to grow. The dominant follicle that released the oocyte at the time of ovulation develops into the corpus luteum which produces progesterone to maintain an early pregnancy until the placenta is capable of sufficient progesterone production, approximately 7–9 weeks of gestation. If implantation does not occur, the corpus luteum regresses in a predictable fashion. In response to falling levels of estrogen and progesterone, a series of cytokine-, chemokine-, and prostaglandin-mediated events lead to endometrial hypoxia, endometrial shedding, and menstruation. If implantation occurs and the pregnancy progresses normally, estrogen, progesterone, human chorionic gonadotropin (hCG), and a variety of other hormones continue to increase to support the developing embryo.
Implantation is one of the most complex and important events of pregnancy and continues to be targeted in many investigations of pregnancy immunology. At least 50 % of all pregnancies fail to synchronize the necessary events of implantation and only 25 % of all fertilized ova will generate a live birth. The majority of early pregnancy losses are of chromosomally abnormal human embryos [4–6]. Major histocompatibility antigens that have the potential to induce an alloimmune response in the maternal host are expressed on the surfaces of human preimplantation embryos but the role of these antigens in pregnancy has not been fully elucidated (described in detail below) [7]. While it is generally accepted that the mother recognizes and responds to these alloantigens, it is possible that aberrant maternal recognition of these antigens in certain pregnancies may play a role in implantation failure [8].
Approximately 6 days after fertilization in the fallopian tube, the developing embryo becomes a blastocyst that has an inner cell mass that will develop into the fetus and an outer trophectoderm layer, which will subsequently differentiate to become the multilayered placenta. Once the blastocyst attaches to the decidua, the trophectoderm differentiates into the syncytiotrophoblast and cytotrophoblast. Initially, the syncytiotrophoblast invades into the decidua and allows the blastocyst to be enveloped with maternal tissue. The trophoblast quickly perforates the maternal capillaries and the spaces within the syncytium are filled with maternal blood. These areas enlarge and fuse to become the intervillous space of the human placenta, the site at which nutrient and gas exchange occurs between the mother and her developing embryo. Two weeks after implantation, the cytotrophoblast cell subpopulation in the placenta proliferates into buds that grow through the syncytium. The trophoblast cells of the post-implantation placenta are generally divided into two populations: (1) villous trophoblast that covers the chorionic villi and interacts with maternal blood in the intervillous space and (2) extravillous trophoblast (EVT) that migrate into the decidua and surround the maternal spiral arteries, destroying the muscular walls and leading to endothelial cell swelling. Like syncytiotrophoblast, EVT come into direct contact with maternal peripheral blood (Fig. 1.1) [9]. Remodeling of the maternal spiral arteries by endovascular trophoblast creates low resistance vascular channels that are largely unable to respond to maternal vasoactive stimuli; this prevents compromise of the utero-placental blood flow during maternal stressors. The remaining extravillous cytotrophoblast cells will be in direct contact with the immune cells of the maternal decidua. EVT typically invade through the decidua and invasion can extend as far as the inner third of the myometrium in healthy pregnancies. Alterations in the depth of such invasion have been seen in pregnancy pathologies such as preeclampsia and intrauterine growth restriction (poor invasion) and placenta percreta (overly robust invasion).
Fig. 1.1
The human maternal–fetal interface in early pregnancy. The fetal aspect of the maternal–fetal interface is comprised of a very large number of branching placental villi that are bathed by the maternal blood filling the intervillous space (IVS). Placental villi contain fetal vessels (FV) embedded in stroma and covered by trophoblast. Floating villi (FV) and anchoring villi (AV) are covered by a mostly continuous (in early pregnancy) layer of syncytiotrophoblast, the multinucleated syncytium of cells that coats the IVS and comes into direct contact with maternal blood. Syncytiotrophoblast is the product of fusion of the underlying cytotrophoblast progenitor cells. Unlike floating villi, which float freely in the IVS, anchoring villi cross the IVS and attach to the maternal decidua (MD). At the tips of the anchoring villi, some cytotrophoblast cells cease proliferating and transform into invasive extravillous cytotrophoblast (EVT) cells. These cells leave the anchoring villi to invade through the decidua, often reaching as far as the inner third of the uterine myometrium. A subset of extravillous cytotrophoblast cells, called endovascular trophoblast (EnT) remodels the maternal uterine spiral arteries (SpA), replacing cells of the maternal vascular wall and creating a vaso-inert conduit for the maternal blood that dumps into the IVS after about 11–12 weeks of gestation. From soon after initial implantation until about 10–11 weeks of gestation, extravillous trophoblast plugs the ends of the SpA and the IVS is filled with nutrient rich exudates
Basic Principles of Immune Response
The immune system is divided into two general methods of response, the innate immune response and the acquired immune response. Cooperation between these two systems is often needed to provide effective responses to a foreign pathogen as these responses differ in intensity, onset, and specificity.
Innate Immunity
When a foreign pathogen enters the body, the innate immune mediators are the first to encounter the pathogen. The innate immune response is comprised of a variety of cells and tissues that provide initial host defense. Epithelial tissues containing protective tight intercellular junctions, such as those in the skin and mucosal membranes, are often the first location of pathogen exposure. Other components of the innate immune response include phagocytic and cytotoxic cells and a range of effector molecules, including inflammatory response molecules, antimicrobial peptides, and cytokines. The innate immune response is a rapid generalized response that is not specific to the pathogen or other foreign antigen. It is unable to establish memory toward a pathogen or other foreign antigen and therefore cannot develop adaptations to the antigen that promote more rapid or robust immunologic responses upon future exposure.
Acquired Immunity
The first exposure and resulting primary immune response to a foreign pathogen induce other cells and pathways in the acquired immune system to form an adaptive response to a subsequent exposure. When that same antigen is encountered a second time, it will be confronted by a quantitatively and qualitatively different immune response called a secondary response. Antigen-specific cells and molecules of the acquired immune system often interact and cooperate with components of the innate immune defense systems. For instance, antibodies of the acquired immune system may bind to bacterial surface antigens leading to phagocytosis by macrophages of the innate immune system. Macrophages can also process and present antigens to specific T cells during a primary response.
Antigen-specific lymphocyte responses are characterized by their proliferative capacity as well as by functional differentiation into cells with effector capacities (i.e., the production of antibodies and cytokines) and the capacity for antigen-specific memory.
Primary and Secondary Immune Responses
When naïve B cells recognize peptide antigens, they interact with CD4+ T cells to produce both primary and secondary humoral immune responses. The primary response to an antigen requires a large amount of antigenic stimulus and peaks 5–10 days after exposure. Primary responses typically secrete more IgM than IgG and the scale of response is lower than the second exposure. Primary exposure promotes the generation of memory B cells which then contributes to the secondary immune response.
After subsequent exposures, the secondary humoral response occurs. This response is faster acting, taking only 2–5 days to reach peak intensity and much more robust than a primary response. A larger amount of IgG is secreted compared to IgM. The IgG has multiple effects and can cross the placenta from mother to fetus in increasing amounts beginning by about 15 weeks of gestation. IgG can bind antigen in its variable region, causing recognition and internalization by phagocytic cells. IgG also promotes antibody-dependent cellular cytotoxicity (ADCC) by signaling lysis by cytotoxic T cells, Natural Killer (NK) cells, or NKT cells.
After antigen-induced proliferation, some of the newly developed lymphocytes may commit to become long-term memory cells that survive and maintain their antigen specificity for many years. Activated B cells will also undergo isotype switching and change their antigen-specific immunoglobulin secretory product from the IgM subtype to IgG, IgE, or IgA, each of which has distinct physiologic and biologic functions. Through these processes, a maturing immune response maintains antigen specificity while simultaneously establishing memory and functional diversity.
Cellular Effectors of an Immune Response
Leukocytes, including lymphocytes, monocytes, macrophages, dendritic cells, neutrophils, basophils, and eosinophils, are the cellular effectors of the immune system (Table 1.1). Lymphocytes are further divided based on their function and on cell surface markers, called “cluster of differentiation” or CD markers. Lymphocytes include T cells, B cells, and natural killer cells. B and T lymphocytes originate in the bone marrow and play a role in antigen-specific immune responses.
Table 1.1
Cells involved in the innate and adaptive immune response
|
Innate immune system |
|
|
Macrophages |
Phagocytosis Antigen presentation Produce IL-1, IL-8, TNF |
|
Neutrophils |
Kill opsonized pathogens |
|
NK cells |
Recognize cells lacking MHC class I products, viral infected cells, and oncogenically transformed cells Cytotoxic |
|
γδ T cells |
Produce IL-10 and TGFβ |
|
Adaptive immune system |
|
|
Cytotoxic T-cell |
Lyse infected target cells |
|
T-helper (Th) cells |
Cytokine-producing lymphocytes |
|
Th1 |
Produces IL-2 and IFNγ Activates cytotoxic T cells Initiates delayed hypersensitivity |
|
Th2 |
Produces IL-4, IL-5, and IL-13 Promotes antibody responses |
|
Th17 |
Promotes inflammation Produces IL-17 Recruits neutrophils |
|
Treg |
Suppresses inflammation Produces IL-10 and TGFβ |
|
B-cell |
Produces antibodies Presents antigen to Th cells |
|
Dendritic cell |
Antigen presentation |
IL interleukin, TNF tumor necrosis factor, NK natural killer, MHC major histocompatibility complex, IFN interferon, TGF transforming growth factor
T (Thymus-Derived) Cells
Prior to their entrance into the thymus, T cell precursors lack antigen receptors as well as CD3, CD4, and CD8 surface proteins. All mature T cells have CD3 proteins on their surface. As CD4 and CD8 negative T cells pass through the thymus, they mature into T cells that initially express both CD4 and CD8 cell surface receptors. Maturation of the T cell continues as the cell migrates from the cortex of the thymus to the medulla where the T cells will now express either CD4 or CD8. These cells then migrate out of the thymus into the peripheral blood and tissues. CD4 positive T cells develop into helper T cells upon reaching peripheral lymphoid tissues. Despite its certain oversimplification, helper T cells are often subdivided into several groups, with categorization based largely on effector function and the identity of the helper cells’ dominant cytokine secretory products (for further detail, see section on cytokines). For example, T helper cell type 1 (Th-1) cells activate cytotoxic T cells by producing interleukin (IL)-2; they aid in the initiation of delayed-type hypersensitivity through their effects on macrophages and they produce cytokines that help to orchestrate B cells isotype switching. Th-2 cells produce IL-4 and IL-5, which promote B cell differentiation into antibody-producing plasma cells. In contrast to CD4 positive helper T cells, CD8 positive T cells become cytolytic T cells (CTLs) or suppressor T cells (currently called regulatory T (Treg) cells) upon leaving the thymus [10]. CTLs lyse infected or otherwise altered target cells.
B (Bone Marrow-Derived) Cells
B cells mature within the bone marrow prior to their migration into the peripheral immune system. B cells differentiate into plasma cells that produce the antibodies of the humoral immune response. They may also present antigen to helper T cells [11].
NK (Natural Killer) Cells
Natural killer cells are circulating lymphocytes that are classically ascribed non-antigen-specific responses, although this concept is still being fully clarified. NK cells recognize cells lacking major histocompatibility complex (MHC) class I products (see below) and attack virally infected or oncogenically transformed target cells. They also display cell surface receptors (specifically CD16) that enable recognition of antibody-coated target cells and allow NK cells to function as a major effector of ADCC. The NK cell can kill target cells through the release of cytotoxic granules containing perforin and granzymes [12]. Activated NK cells can also secrete cytokines, including interferon (IFN)-γ and tumor necrosis factor-alpha (TNF-α) [12].
Monocytes, Macrophages, and Dendritic Cells
Monocytes are derived from bone marrow stem cells and circulate in the peripheral blood and tissues. Within specific tissues, monocytes mature into macrophages. Macrophages are capable of phagocytosis, antigen presentation, and cytokine production [13, 14]. When tissue macrophages encounter bacteria, viruses, and other foreign antigens, they may phagocytize some of the encountered antigens. The macrophage phagosome can then fuse with lysosomes, resulting in the production and/or release of reactive oxygen and nitrogen compounds and lysosomal enzymes that can destroy the pathogen. After ingestion, class II MHC proteins on the cell surface of macrophages can also present fragments of the antigen to CD4 positive helper T cells. Macrophages can produce IL-1 and tumor necrosis factor which are important for inflammation. They can also secrete IL-8 and attract neutrophils and T cells to the site of infection. Dendritic cells, like macrophages, are antigen-presenting cells that express class II MHC proteins and react with CD4 positive T cells [15]. They are also important in the primary antibody response of B cells.
Other Effector Cells
Neutrophils, eosinophils, and basophils are effector cells of the innate immune system whose reactivities are most specific for certain pathogens. Each has also been associated with specific immune-mediated diseases. Eosinophils have classically been described as central in defense against parasites and in asthma. Basophils are important in immediate hypersensitivity reactions.
Soluble Components of Immune Responses
Immunoglobulins and Humoral Immunity
Immunoglobulins (Ig) are composed of dimerized heavy and light chains [13]. The N-terminal portions of each chain are highly polymorphic and are therefore termed the variable regions. The variable region of one heavy change combines with the variable region of a light chain to confer the antigen specificity of the immunoglobulin. The C-terminal segments of each immunoglobulin chain have minimal polymorphism and are called the constant regions. Constant segments are responsible for the specific biologic functions of the immunoglobulin molecule, including complement activation and binding to cell surface receptors. Ig isotypes include IgA, IgD, IgE, IgG, and IgM. IgG, IgE, and IgD typically present as single Ig monomers; IgA circulates as dimers; and IgM as pentamers.
Each Ig isotype has characteristic functions. IgA dimers are associated with mucosal surfaces. IgE molecules are involved in immediate hypersensitivity reactions by releasing mediators from mast cells and basophils upon exposure to antigens. Membrane-bound IgD and IgM interact with antigen-recognizing B cell receptors on naïve B cells. Pentavalent IgM can fix and activate the complement cascade. Due to its small size, IgG is the only immunoglobulin that can pass through the placenta in significant amounts.
Complement
The complement components represent an important effector arm of immune protection. The system is composed of an array of circulating proteins in the maternal blood stream that can be activated by classical, alternative, and lectin pathways (Fig. 1.2). Activation of these pathways causes a cascade of downstream effects involving proteolysis and assembly in a manner analogous to the coagulation cascade. Through the classical pathway, complement component C1q binds to antigen–antibody complexes (IgG or IgM) or directly to the surface of a microorganism. In the alternative pathway, spontaneously activated C3b binds directly to the pathogen. The lectin pathway is similar to the classical pathway; however, mannose-binding lectin (MBL) binds to the pathogen surface. After initial binding, a series of enzymatic cleavages ensues, creating C4b and C2a components that bind and produce C3-convertase, which ultimately drives the production of C5a, the most potent of the anaphylatoxins. The outcomes of activation of the complement cascade include opsonization or phagocytosis of antigens, chemotaxis or recruitment of macrophages and neutrophils, and direct cell lysis. The latter can occur through the formation of a membrane attack complex (MAC) that creates an ion permeable transmembrane pore in the target cell and promotes osmotic lysis of this target.
Fig. 1.2
The complement cascade. The complement cascade can be activated via several pathways. The classical pathway is activated by the pathogen surface itself or by the binding of complement component C1 (C1q) to free antibody complexes or antibody bound to pathogen. The lectin pathway is activated by the binding of mannose-binding lectin (MBL) to components of the pathogen surface. In the alternative pathway, active complement component C3b binds directly to the pathogen. All pathways set in motion a series of enzymatic cleavage steps involving several complement components (C5–C9) that result in direct or indirect attack on the inciting pathogen. Here, direct cell lysis occurs through formation of a membrane attack complex (MAC)
Cytokines
In conjunction with immunoglobulins and the components of the complement cascade, cytokines (e.g., interleukins, IFNs, tumor necrosis factors, transforming growth factors) and chemokines complete the list of soluble mediators of immune responses. Cytokines, secreted proteins made by immune cells, are pleiotropic and have a variety of effects on a number of different cell surface receptors. Cytokines can activate or inhibit other cells of the immune system. Cytokines often have complementary and/or redundant effects at cell surface receptors. Once produced, they are secreted rapidly and produce autocrine, paracrine, or endocrine responses.
Cytokines are particularly integral to T helper cell differentiation. CD4+ T helper cells travel from the thymus through the peripheral tissues as naïve Th0 cells. When first presented with an antigen, the direction of CD4+ T helper cell differentiation is based upon the cytokines released from other antigen-presenting cells, such as dendritic cells and macrophages as well as the cell surface co-receptors expressed on the Th0 cell [16]. The differentiation of the CD4+ T helper cell into T helper cell subtypes is termed polarization and specific cytokines appear to direct this process (Fig. 1.3). Interleukin 12 (IL12), IL 18, and interferon gamma (IFNγ) stimulate the Th0 cell to differentiate into a Th1 cell capable of secreting inflammatory cytokines including IFNγ, IL1, and TNF-α. If the Th0 cell is exposed to IL4, the Th0 cell then develops into a Th2 cell that secretes IL4, IL5, IL9, and IL13 [17]. Th2 cells participate in allergic-type responses, including mast cell and eosinophil activation and antibody production. Although the Th1:Th2 paradigm was the first to be described in detail, other types of T helper cells, their cytokine secretory profiles, and their characteristic transcription factors have been and continue to be reported. Th17 cells are produced from exposure to transforming growth factor beta (TGFβ) and IL-6. These cells secrete IL17 and IFNγ [17]. T regulatory (Treg) cells are produced in the presence of TGFβand the transcription factor forkhead box P3 (Foxp3) and secrete IL10, IL35 and TGFβ [18]. Tregs have several activities, including: (1) reducing the cytolytic activity of NK cells [19], (2) inhibiting the development of dendritic cells [20], and (3) decreasing CD3+ T cell proliferation and cytokine release [21]. Peripheral and endometrial Tregs may be particularly important in pregnancy health and maintenance [22, 23]. Specific T helper cell subtype secretory products tend to further amplify the production of identical cells through positive and negative feedback loops. Although the development of T helper cell subtypes was once thought to be unidirectional, recent descriptions of T helper cell plasticity show that regulation of this development is quite complex [16].
Fig. 1.3
T helper cell differentiation. Differentiation of T helper cells from naïve Th0 cells to T helper cell subpopulations characterized by distinct cytokine secretory profiles is largely dependent on the cytokine microenvironment present at the site of antigen presentation to the naïve cell. The number of described T helper cell subtypes continues to grow and one depiction of these subtypes is shown here. The transcription factors related to a particular differentiation pathway are included below the subtype (e.g., FoxP3 for T regulatory cell differentiation) as are several of the characteristic secretory products of a given subtype. While T helper cell differentiation was once thought to be unidirectional, this is now being questioned. Activity and re-differentiation of particular T helper cell subpopulations can be modulated by the secretory products of other T helper cell subpopulations (depicted as dotted and solid curved lines). ILinterleukin, IFN interferon, TGF transforming growth factor
Basis of Immune Specificity and Immune Cell Education
Unlike the innate immune response, effector cells of the acquired immune response typically cannot recognize free antigen. Rather, they must recognize antigen in the context of an antigen-presenting cell. These antigens are typically processed and presented on the cell surface of antigen-presenting cells in the innate immune system to the effector cells of the acquired immune system. These specific effector cells distinguish the cell presenting the antigen as “self” and the antigen as “foreign,” which the effector cell can do because it has been previously “educated” to recognize these distinctions (see below). Antigen presentation is essential for development of cellular and humoral immune responses [24]; the specificity of this presentation is exquisitely sensitive at the level of a particular effector cell but remarkably broad when all “educated” effector cells are considered.
The MHC
The MHC is a large cluster of highly polymorphic genes that are found on the short arm of human chromosome 6 (Fig. 1.4). The protein products of the MHC aid in the distinction between self and non-self or altered-self (e.g., pathogens, foreign tissues, and oncogenically transformed cells/proteins). MHC-encoded proteins include two major types of antigen-presenting molecules called MHC class I and MHC class II products. MHC class I molecules are further subdivided into classical, class Ia (human leukocyte antigen (HLA)-A, -B, and -C), and nonclassical, class Ib (HLA-E, -F, and -G) constituents.
Fig. 1.4
The MHC region of human chromosome 6. Many of the genes encoding proteins integral to antigen presentation are located within a fairly well-characterized region of the short (p) arm of human chromosome 6. Called the MHC, the region is further subdivided into a three general groups of loci. The class I region contains genes encoding MHC class I molecules, such as human leukocyte antigens A, B, C, E, F, and G (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G). The class II region contains genes encoding MHC class II molecules such as HLA-DR and HLA-DQ. The class III region contains a varied group of genes encoding molecules important to immune recognition and response. These include MHC-like molecules involved in innate immune recognition (MICA and MICB), several of the complement components (C2, C4A, C4B) and many others
Class Ia molecules are expressed on the surface of nearly every nucleated cell in the human body. Class Ia molecules provide defense against intracellular pathogens such as viruses by presenting pathogen-derived antigens to cytotoxic T cells. They also aid in the detection of oncogenically transformed cells by presenting altered intracellular protein antigens to immune effector cells. MHC class Ia molecules are central to the distinction between self and non-self, which is best exemplified in transplant rejection. MHC class Ia molecules act as ligands for the T cell receptor (TCR) on CD8+ cytotoxic/suppressor T cells. MHC class Ia antigens can also participate in the inhibition or activation of NK cells through inhibitory NK-cell receptors and activating NK-cell receptors (killer activation receptor, KAR). Nonclassical MHC class Ib molecules are less polymorphic and likely have less importance in antigen presentation; however, continued research is investigating their role in immunologic disease. These MHC class I subtypes appear to be particularly relevant in the immunology of the maternal–fetal interface (see below)
MHC class II molecules (HLA-DR, -DP, and -DQ) are present on the surface of a smaller fraction of cells than that expressing MHC class I. MHC class II expression is generally limited to antigen-presenting cells, including dendritic cells, macrophages, monocytes, B cells, and a variety of tissue-specific antigen presenters (e.g., Langerhan’s cells in the skin). MHC class II molecules are important in the removal of extracellular pathogens such as bacteria. MHC class II molecules interact with CD4+ T helper cells leading to humoral immune responses.
Lymphocyte Education
The ability to determine self from non-self is an imperative function of lymphocytes. Immature B cells within the bone marrow that recognize self antigens with high avidity undergo negative section and die by apoptosis. The remaining B cells within the bone marrow are tolerant to self antigens and this tolerance is independent of MHC molecules [25].
T lymphocytes, however, undergo a more complex education during which MHC molecules play a central role. Upon entering the thymus, immature T cells that lack the TCR (the CD3 antigen is part of the TCR) and the co-receptors CD4 and CD8 come to express all three cell surface molecules and are considered “double (CD4 and CD8) positive” cells. T cells that recognize self MHC with low avidity undergo positive selection. Among positively selected cells, those with receptors that recognize self MHC class I molecules become CD8+ cytotoxic/suppressor T cells. Those positively selected T cells with receptors that recognize self MHC class II molecules become CD4+ helper T cells. Negative selection occurs when double positive cells recognize self MHC with high avidity and these cells are selected for deletion. If this process is imperfect, there is potential for autoimmunity.
The Fetal Allograft
The maintenance of a successful pregnancy requires an intricate coordination and balance between the mother and the developing embryo and fetus. In 1953, Medawar proposed the idea of the fetal allograft [26]. He theorized that there was anatomic separation between the mother and the fetus, that the fetus itself must be antigenically immature, and that the mother was immunologically inert during the pregnant state. Over the last six decades these theories have helped guide the field of reproductive immunology and have been advanced upon. It is now known that there is no anatomic separation between the mother and the fetus. The fetus, a semi-allograft carrying maternal and paternal antigens, possesses fetal trophoblast cells that are antigenic and carry nonclassical MHC class I antigens. Finally, although altered, the maternal immune system is fully functional during pregnancy.
Cellular Immune Effectors in Pregnancy
Peripheral Immune Cells During Pregnancy
One of the first changes noted within the maternal immune system during pregnancy is an increase in overall peripheral leukocyte number (Fig. 1.5) [27]. The distribution of leukocyte types, quantities, locations, and functions is modified during pregnancy and results in altered immune responses. Many of these alterations likely contribute to the relapsing and remitting nature of some autoimmune disorders during pregnancy.
Fig. 1.5
Interfaces between maternal immune cells and trophoblast in the human placenta. Fetally derived trophoblast comes into direct contact with maternal blood at three distinct sites within the maternal–fetal interface. After about 10 weeks of gestation syncytiotrophoblast contacts the maternal peripheral blood filling the intervillous space. Endovascular trophoblast contacts maternal peripheral blood flowing through the remodeled maternal spiral arteries. The immune cells populating the peripheral blood during pregnancy are composed of: 50–70 % neutrophils, 25–30 % T cells, 5–10 % B cells, 5–10 % NK cells, and 5 % monocytes and immature dendritic cells. Extravillous cytotrophoblast contacts the maternal immune cells populating the decidua. The immune cell subtypes in the decidua differ dramatically from those in the peripheral blood. Decidual immune cells are comprised of: 50–70 % NK cells, 15–20 % macrophages, 10–15 % T cells, and 10–15 % monocytes and immature dendritic cells, but almost no B cells or neutrophils
Peripheral T Lymphocytes
Peripheral T cells exhibit decreased mitogen responses and diminished proliferative responses during pregnancy [28]. Women with recurrent pregnancy loss do not dampen their proliferative responses to recall antigens to the same extent as their counterparts with uncomplicated pregnancy histories [29]. Memory CD4+ T cell numbers decrease, naïve CD4+ T cell numbers increase, and there is an overall decrease in the surface expression of activation markers HLA-DR and CD25 [30]. The quantity of circulating maternal Treg cells has also been shown to increase during the first and second trimesters of human pregnancy [31], likely secondary to increasing levels of systemic estradiol. Failure of Treg expansion in the periphery (and in the decidua) has been linked to adverse pregnancy outcomes, including early pregnancy loss [32, 33], preeclampsia, and preterm delivery [34].
Maternal progesterone levels are elevated throughout pregnancy and peak in the third trimester. Progesterone may stimulate the synthesis of an incompletely characterized progesterone-induced binding factor (PIBF) by lymphocytes [35]. Like progesterone, circulating levels of this substance appear to increase as gestation progresses, then drop after delivery. Investigators have demonstrated that in pregnancies resulting in preterm labor, miscarriage, or pregnancy-induced hypertension, PIBF levels are abnormally low [36, 37]. Further, high concentrations of PIBF can promote differentiation of CD4+ T cells into Th2 cells that favor anti-inflammatory regulators [36], as further discussed below. Whether directly related to PIBF or induced via alternative mechanisms, the overall decrease in Th1 responses and shift toward Th2 responses that occurs during human pregnancy can be documented at the maternal–fetal interface and systemically [38].
Peripheral NK Cells
During pregnancy, NK cells undergo dramatic and important alterations in surface receptor expression. The majority (90 %) of human peripheral NK cells express small amounts of CD56 (CD56dim) and large amounts of CD16 on their cell surfaces (CD56dimCD16+). The remaining (10 %) of peripheral NK cells exhibit high levels of CD56 (CD56bright) and low levels of CD16 [39]; this latter expression pattern is similar to that of the majority of decidual NK cells (CD56brightCD16−) [40]. The overall number of peripheral NK cells is decreased in pregnant women when compared to their nonpregnant counterparts [27, 38, 41]. NK cells produce less IFNγ during pregnancy [38] which decreases their cytotoxic capacities. These changes also are responsible for a shift from cellular to humoral immune responses. When these changes do not occur and NK cells remain highly cytotoxic and in nonpregnant proportions, the rate of spontaneous pregnancy loss increases and the success of in vitro fertilization (measured by per cycle live birth rates) decreases [42].
Peripheral Neutrophils
The majority of the increase in total circulating leukocyte cell numbers characteristic of pregnancy is accounted for by an increase in neutrophils [43]. These neutrophils, however, have reduced anti-microbicidal effects, reduced chemotaxis, and reduced phagocytic activities [44].
Decidual Immune Cells
The number, subclasses, and functional phenotypes of the immune cells populating the uterus are transformed throughout the menstrual cycle and, even more dramatically, during pregnancy. These changes are largely in response to alterations in local and circulating levels of the reproductive steroids (Fig. 1.5) [45, 46]. During the proliferative phase of the menstrual cycle, less than 10 % of the cells in the endometrium are leukocytes. This number increases to 20 % in the decidua of the late secretory phase of the menstrual cycle and to over 40 % in early pregnancy [47]. Within the pregnancy decidua, a remarkable 70 % of CD45+ leukocytes are uterine NK (uNK) cells (CD56bright). Other cells within the decidua include macrophages, monocytes, and a small number of T cells (Fig. 1.5). Neutrophils and B cells are rare in the human endometrium and decidua at any time during the menstrual cycle or pregnancy. Changes in the number and/or relative frequency of any of these immune cell populations can lead to dramatic alterations in the immune response to the developing pregnancy and may be related to increased rates of pregnancy loss.
Special Decidual Immune Cell Subpopulations
NK Cells
The phenotypes of the NK cells that populate the decidua during the luteal phase of the menstrual cycle and early pregnancy are very different from those of the peripheral blood, with an apparent dramatic reduction in the subpopulation of CD56dimCD16+ NK cells that dominate the periphery and a marked increase in the CD56brightCD16− cells that are a small minority in the periphery. It is becoming increasingly clear that this change in NK cell phenotype, while the result of a combination of recruitment from the peripheral compartment and proliferation in situ, is mainly dependent on the latter [48].
The predominant NK cell subtype in the decidua has been called by numerous names: uNK cells, decidual NK cells, decidual granular lymphocytes, and large granular lymphocytes. These cells make up approximately 70 % of the total endometrial lymphocyte population in early pregnancy [40, 49]. This number decreases after 20 weeks of gestation and uNK cells are nearly absent in the endometrium at term. uNK cells are particularly prevalent at the implantation site, suggesting they may specifically recognize extravillous cytotrophoblast cells as fetal [40]. While their exact function within the decidua is still under investigation, it is hypothesized that uNK cells influence maternal endometrial mucosal and arterial function and/or placental trophoblast invasion [50]. Unlike the majority of their peripheral counterparts, uNK cells display fairly limited cytotoxic capabilities [51, 52]. Instead, they are efficient and potent cytokine producers [49]. Human uNK cells produce a variety of cytokines including: macrophage inflammatory protein-1α, granulocyte-macrophage colony-stimulating factor, interferon-γ, TGFβ, vascular endothelial growth factor C (VEGFC), placental growth factor (PIGF), and angiopoietin 2 (ANG2) that play a role in angiogenesis and vascular stability. Through these cytokines, uNK cells may help to mediate trophoblast invasion and modify maternal spiral arteries to lead to the increase in blood flow necessary for normal pregnancy [53]. Note that the minority of decidual NK cells that have the peripheral phenotype will maintain nonpregnant expression of activation markers in anembryonic and ectopic pregnancies and may play a role in early pregnancy loss [54, 55].
Macrophages, T-Cell Receptor γδ+ T Lymphocytes, and Dendritic Cells
Macrophages increase early in pregnancy, then stabilize throughout the remainder of gestation, and make up approximately 10 % of decidual cells at the implantation site [56]. This increase is believed to play a role in the proinflammatory environment seen very soon after conception that appears necessary to prepare the endometrium for implantation [1]. However, for much of normal human pregnancy, the predominant activities of decidual macrophages are immunosuppressive [56]. Like T helper cells, macrophages have been subcategorized by their secretory products into several phenotypes. After early pregnancy, most decidual macrophages are of the M2 phenotype, secreting IL-10, prostaglandin E2 (PGE2), and indoleamine 2, 3-dioxygenase (IDO). IL-10 inhibits proinflammatory cytokine production from T cells and decreases the ability of macrophages and dendritic cells to present antigens by inhibiting their surface expression of MHC class II and costimulatory molecules [56]. These actions may also help with the development of T-cell anergy to fetal cells and may contribute to the Th2 shift typical of uncomplicated pregnancies (discussed below).
While the majority of circulating T cells in humans carry the αβ T cell receptor, a small proportion have a different TCR subtype called the γδ TCR. γδ+ T cells are more prevalent within the tissues of the mucosal immune system and within the decidua in pregnant and nonpregnant women. Their numbers in the decidua are elevated during the luteal phase of the menstrual cycle when compared to the follicular phase. Their frequency is even higher during the first trimester of pregnancy and, in fact, both circulating and decidual γδ + T cells comprise a larger proportion of T cells in pregnant women when compared to nonpregnant women [51]. Decidual γδ+ T cells secrete IL-10 and TGF-β; they promote trophoblast invasion and inhibit trophoblast apoptosis [51]. The true physiologic function of these cells has not yet been determined and their role in pregnancy maintenance is still under investigation.
Dendritic cells (DC) within the decidua express MHC class II molecules and are CD14−. It is thought that DCs are more specialized than macrophages at presenting antigen to T cells [56]. Uterine DCs may play a very early and important role in the maintenance of pregnancy since depletion of uterine DCs inhibits endometrial decidualization and angiogenesis [57].
Soluble Immune Effectors and Pregnancy
Complement
In pregnancy, maternal complement levels are equal to or greater than in the nonpregnant state [55]. Activation of complement against paternal antigens could potentially be harmful to the developing fetus and result in adverse outcomes that include those disorders that result from poor placentation (e.g., intrauterine growth retardation, preeclampsia) or pregnancy loss. Several complement regulatory proteins are expressed by the trophoblast that may be central to inhibiting such activation. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), and CD59 [54]. DAF inhibits the alternative complement pathway by preventing the formation and accelerating decay of C3/C5 convertase [58]. MCP inhibits the classical complement pathway by binding C3b on the cell surface and by functioning as an inactivating cofactor for C3b and C4b. CD59 inhibits the assembly of the MAC.
Cytokines and the Cytokine-Shift Hypothesis
Few individual cytokines appear to be absolutely necessary for pregnancy maintenance. Of the cytokines that are, leukemia inhibitory factor (LIF) and IL-11 have been shown to be imperative for blastocyst implantation but not for continued fetal development. LIF functions at the blastocyst attachment/adhesion phase and IL-11 functions in the controlled response to the implanted blastocyst [59].
The actions of T helper cells have been shown to greatly affect the outcome of a pregnancy. As discussed previously, Th1 cells produce IL-2 and interact with cytotoxic T cells; the majority of their actions are proinflammatory. Th-2 cells, on the other hand, produce IL-4 and IL-5 which promote B cell development into antibody-producing plasma cells. The overall effect is largely anti-inflammatory. In 1993, the cytokine-shift hypothesis was proposed, which posited that pregnancy was an anti-inflammatory condition [60]. It was thought that the intrauterine environment of normal pregnancies was Th2 dominant, whereas pregnancies resulting in fetal losses were associated with increased amounts of interferon-γ, IL-2, and tumor necrosis factor-α which are harmful to the developing fetus [47]. Currently, it is debated whether pregnancy is an inflammatory or anti-inflammatory condition and to call it purely one or the other may be an oversimplification.
It may be more useful to divide human pregnancy into three distinct immunologic phases: implantation/placentation, fetal growth and development, and parturition [61]. From blastocyst attachment to the early second trimester, a proinflammatory environment is noted within the woman’s body. Decidual NK cells secrete IL-8 and interferon-inducible protein-10 as well as other angiogenic factors that aid in successful decidualization and in trophoblast invasion [62]. The maternal experience of this overwhelming proinflammatory response and dramatic hormonal changes may partly explain the symptoms of morning sickness [61]. The second trimester to near birth is a period of rapid fetal growth and is thought to be anti-inflammatory in nature. Although anti-inflammatory IL-10 is thought to be essential for maintenance of a pregnancy, IL-10 levels are markedly reduced in term placentas that are not yet in labor, when compared with first- and second-trimester placentas [63]. The downregulation of IL-10 near term may, in fact, contribute to the onset of labor. It has been demonstrated that parturition is characterized by a migration of inflammatory cells into the cervices, myometria, chorioamniotic membranes, and amniotic cavities of women with spontaneous labor at term [63, 64]. Local accumulation of these cells dramatically increases the local levels of inflammatory cytokines such as IL-1β, IL-6, TNF-α and IL-8 that promote the onset of contractions and progression through delivery of the infant and separation of the placenta.
The Implantation Site and Transplantation Antigens
The implanting human blastocyst is characterized by an inner cell mass representing the developing fetus and an outer layer of trophectoderm that will become the placenta [65]. After several weeks of initial development and differentiation, the human placenta is characterized by a complex collection of branching villi. Each of these tree-like structures contains a core of fetal vessels surrounded by a loose stroma. Some villi float freely in the intervillous space (floating villi), while others reach across this space to attach to the maternal decidua (anchoring villi). Both types of villi are covered by two layers of trophoblast along surfaces that face the intervillous space (Fig. 1.1). The inner layer is comprised of cytotrophoblast cells. These cells are covered by a multinucleated syncytium of the cytotrophoblast cells called the syncytiotrophoblast. The syncytiotrophoblast layer is generally continuous during early pregnancy and this layer is the source of human placental lactogen and hCG [66]. Later in pregnancy the syncytiotrophoblast is characterized by scattered “knots” of syncytialized cells.
At sites where anchoring villi meet the maternal decidua, a subset of cytotrophoblast cells further differentiates into invasive extravillous cytotrophoblast (EVT) cells. These cells move through and populate the maternal decidua and can even be found in the inner third of the uterine myometrium in normal pregnancies. EVT also surround and remodel the maternal spiral arteries, replacing vasoactive vascular cells with vaso-inert endovascular trophoblast cells. This ensures continuous and robust blood supply to the developing fetus even in the face of fairly significant maternal stressors. The human placenta is unique in its level of trophoblast invasion into the maternal tissues and is one of the few placentas in nature that permit direct access of fetally derived trophoblast cells to maternal immune cells [67]. Three subsets of human trophoblast display this level of intimate association with maternal tissues: (1) the syncytiotrophoblast (SynT) layer that coats the placental villi and is directly exposed to the maternal peripheral blood in the intervillous space, (2) extravillous cytotrophoblast (EVT) cells that directly interact with maternal decidual immune cells, and (3) endovascular cytotrophoblast cells of the remodeled uterine spiral arteries carrying maternal peripheral blood to the fetus (Fig. 1.1). All of these trophoblast subtypes are initially derived from villous cytotrophoblast cells. Consistent with Medawar’s allotransplantation paradigm, syncytiotrophoblast lacks both MHC class I and class II molecules [68], making it antigenically “invisible” to maternal T cells. This is not true for the other trophoblast subtypes making contact with the maternal immune system and the lack of MHC class I on syncytiotrophoblast is problematic from the standpoint of NK cell recognition anyway (see below). EVT [69] and endovascular trophoblast cells [70] display at least three potentially allogenic MHC class I subtypes on their cell surfaces: the classical MHC class Ia molecule, HLA-C, and the nonclassical class Ib molecules, HLA-E and HLA-G [45, 46]. The MHC class Ib molecules have a limited degree of polymorphism and are therefore less effective in antigen presentation. This alteration in antigen presentation may actually be protective for the EVT and endovascular trophoblast cells as it would be unlikely that the paternal HLA-G would be recognized as foreign [71]. Further, the presence of an MHC class I molecule of any type on their cell surface protects these trophoblast cell subsets from NK cell-mediated cytolysis, since such recognition is based not on self vs. non-self, but rather on the absence of any MHC class I or class I-like products [72]. While often described as fairly non-polymorphic, the trophoblast expressed MHC class Ia molecule, HLA-C, has actually been shown to display a fairly high degree of polymorphism [73, 74]. Still, for uncertain reasons, HLA-C molecules do not appear to stimulate robust antipaternal adaptive immune responses [75].
HLA-C’s major role at the maternal–fetal interface may be to modulate the activities of the voluminous NK cells in the decidua. Since natural killer cells recognize and destroy cells that lack MHC class I antigens, the expression of HLA-G, -E, and -C on EVT and endovascular trophoblast cells may protect these cells from uNK-mediated attack [69, 75]. For example, although all NK cells express a variety of activating and inhibitory receptors on their cell surfaces, HLA-E molecules bind to an inhibitory receptor that is expressed on all uNK cells (but not all peripheral NK cells) [76]. HLA-C molecules are the preferential ligands for NK killer inhibitory receptor subtype, KIR2D, and interactions between particular genotypes of this polymorphic MHC molecule and of the polymorphic KIR2Ds have been associated with adverse pregnancy outcomes [67]. Overall, interactions between trophoblast MHC molecules and activating and inhibitory receptors on decidual NK cells and macrophages are poor inducers of cytotoxicity [52], but important to maintenance of pregnancy [77, 78]. These interactions have also been shown to be necessary for decidual and vascular trophoblast invasion, spiral artery remodeling, and angiogenesis [79, 80]. Trophoblast MHC class I molecules have also been associated with the cytokine shifts that occur in normal pregnancy. For example, EVT-expressed HLA-G suppresses Th1 cytokine secretion and induces anti-inflammatory Th2 cytokine production by decidual cells [71].
Interestingly, despite studies showing support for an important role for HLA-G in the immune modulation that occurs during pregnancy, homozygosity for a null allele of the HLA-G gene does not appear to affect human fertility and pregnancy outcomes [81]. When approached from an evolutionary viewpoint, this should not be overly surprising. The success of human pregnancy may be too central to the survival of the species to expect anything other than redundancy and overlap in protective mechanisms. Similar findings were also mentioned in the section on cytokines and the cytokine shift. Here too, these molecules are deemed to be important to pregnancy maintenance, yet few play absolutely essential roles.
Pregnancy as a State of Immune Modulation
It is now widely accepted that during pregnancy, the implanting fetus is recognized by the maternal immune system and robust immune responses at the maternal–fetal interface, including proinflammatory and proangiogenic responses, have been demonstrated in developmentally normal pregnancies [80, 82]. These immune responses, however, occur in an environment consisting of dramatic hormonal and metabolic changes. As a result of the dynamic background upon which the immunologic changes of pregnancy occur, it is often unclear which particular alterations are necessary for pregnancy success.
Hormonal Regulation
As previously discussed, circulating levels of estrogen and progesterone rapidly increase after ovulation, and if conception occurs, continue to rise until after delivery of the placenta. Among the many immunomodulatory roles ascribed to it, progesterone inhibits mitogen-induced proliferation of CD8+ T cells and cytokine secretion by these cells; it also promotes Th2 responses and increased secretion of LIF [83–85]. All of these progesterone-induced changes aid pregnancy maintenance. Progesterone also inhibits TNF-α, a cytokine that can have deleterious effects on a developing fetus. The role of estrogen in immune modulations of pregnancy is less well accepted than that of progesterone. Estrogen favors pregnancy maintenance by down regulating delayed-type hypersensitivity reactions, promoting Th2 type immune responses, and protecting against chronic allograft rejection [85]. Estrogen is largely responsible for the increases in the circulating and decidual Treg populations characteristic of normal pregnancy [86, 87]. Other, nonsteroidal hormones also increase dramatically during pregnancy and several have been linked to the immune modulation seen in the pregnant female. For example, hCG has been shown to increase IL-27 and IL-10 (anti-inflammatory), to decrease IL-17 (pro-inflammatory) expression, and to increase the number of circulating Tregs when administered exogenously to women [88]. hCG can decrease HLA-DR expression on dendritic cells in culture [89] and will attract Treg cells toward trophoblast cells in models of the human maternal–fetal interface [90]. Interestingly, prolactin, which reaches fairly high levels in the circulation of pregnant women, exerts fairly pro-inflammatory effects on the adaptive and innate arms of the maternal immune system [91]. The fact that many of the maternal immune changes vary throughout pregnancy despite continually increasing levels of anti-inflammatory (progesterone, hCG, estrogen) and pro-inflammatory (prolactin, estrogen) reproductive hormones speaks to the complexity of this system.
Autoimmune Diseases and Pregnancy
Many observed alterations in immune function and autoimmune disorders have been demonstrated in pregnancy. Th1-mediated autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (MS), have been noted to be improved with fewer flares during pregnancy. In contrast, the severity and number of flares in Th2-mediated disorders, such as systemic lupus erythematosus (SLE), characteristically increased during pregnancy [92]. Overall, pregnant women with underlying autoimmune disorders may experience varying, albeit somewhat predictable, changes in their disease status and symptomology. Although certainly oversimplified, rheumatologic diseases whose pathophysiology involves inflammatory changes may improve during pregnancy while those with more allergic or antibody-mediated underpinnings may worsen. Autoimmune diseases and their alteration and management during the pregnant state will be discussed in much greater detail throughout this textbook.
Conclusion
Maternal immune system changes begin prior to conception as the cellular milieu within the endometrium and developing decidua varies with the menstrual cycle. These changes, including increasing numbers of uNK cells and a shift toward Th2 immune responses, are imperative for the proper implantation and continuance of a successful pregnancy, but may place the mother at risk for exacerbation of select autoimmune disorders. Inadequate development of these altered immune responses has been linked to increased risk of pregnancy wastage and pregnancy complications, including preeclampsia and intrauterine growth restriction.
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