Chapter 4

 

                                                            Chapter 4. 

Immune Protection of Embryos and Fetuses in Normal Pregnancy and under Pathological Effects

 

4.1)            Mononuclear phagocytes in embryos and fetuses under normal

                             and pathological conditions of gestation

 

The yolk sac and aorta-gonad-mesonephros region are well recognized as the principal sites of hematopoiesis in developing embryos, and the liver is the principal site of hematopoiesis in fetuses. Moreover, a significant number of committed and multipotent CD34+ progenitors with a capacity for expansion circulates in the fetal blood between weeks 7and 19 of gestation (1). In normal pregnancy, the rate of fetal mononuclear-cell division rate  decreases with gestational age from 1.8% at 18 weeks to 1% at 40 weeks (2). This rate is elevated in early pregnancy and in chromosomally abnormal fetuses, probably as a consequence of the higher number  of circulating haemopoietic precursors. There is a significant association between cell division and the erythroblast count. The rates of both of these parameters increase in chromosomally abnormal fetuses, with an increase in the erythroblast count.

Both the fetal blood and liver provide a rich source of hematopoietic stem and progenitor cells (3), but the fetal liver provides a richer source of more primitive hematopoietic progenitor cells than does the fetal blood. The fetal red blood cells, white blood cells, and platelet counts all increase with gestation, from weeks 8 to 17, reflecting hematopoietic development. The number of normoblasts decreases dramatically with gestation. The number of circulating and hepatic T lymphocytes increases before  week 13 of gestation,  reflecting thymic maturation. The fetal liver contains fewer T lymphocytes than the fetal blood (2.5% vs. 18.6%) and more CD34+ hematopoietic stem and progenitor cells (17.5% vs. 4.3%). Fetal blood at an early (21-22 weeks) gestational age has a higher frequency of primitive hematopoietic progenitor cells (CD34+/CD38- cells) than does the umbilical cord blood at term (39-40 weeks) (4). In fetuses, the cord blood cytokine-receptor network, consisting of IL-1, IL-2, IL-12, IFN-γ and TNF-ą, is biased towards anti-inflammatory activity (5). It should be noted that endothelial progenitor cells  derive  mainly from the monocyte/macrophage-containing CD34- mononuclear cells and only in part from the hematopoietic stem-cell-containing CD34+ mononuclear cells  (6).

The high-affinity protein FcγRI plays a major role in the effector function of circulating monocytes and splenic mononuclear phagocytes, whereas FcγRIII, expressed strongly on the latter effectors, participates in target ingestion (7). Anti-Fcγ RII has no significant effect on the  interaction of fetal spleen mononuclear phagocytes with the red blood cells, whereas anti-FcγRIII causes a significant (43%) inhibition of their phagocytosis. In the absence of any inhibitor, attachment and phagocytotic indices of fetal monocytes are similar to those of their newborn and adult counterparts but markedly lower than those of mononuclear phagocytes from the fetal spleen.

CD95 is the most well-studied receptor mediating a signal for the cell death by apoptosis, and its inducible ligand has been demonstrated to mediate the death of multiple types of CD95-expressing cells. This  molecule  has a crucial role in the homeostasis of haematopoietic cell populations in adults.  Cord blood mononuclear cells enjoy some immune privilege due to their low level of CD95 expression   (relative to adult peripheral blood lymphocytes) and due to expression of the CD95 ligand (8). An increase in IL-2γ receptor expression by the cord-blood mononuclear cells may significantly contribute to the prevention of neonatal infection (9). The blood mononuclear cells in newborns produce less IL-10 than adults, and the primary cells of origin and the regulatory mechanisms may differ from those observed in adults (10). Increased production of IL-6 and decreased production of IFNγ by the cord-blood mononuclear cells appear to be the hallmarks of newborns in the high-risk allergy population (11). The fetal peripheral blood mononuclear cells exhibit a proliferative responses to mitogenic and allergenic stimuli during gestation: fetal exposure in utero to allergens from around 22 weeks of gestation  results in primary sensitization to those allergens, leading to the positive proliferative responses  at birth (12).  

Neonatal monocytes produce a different cytokine-expression profile than adult monocytes (13). ). After lipopolysaccharide exposure, fetal monocytes produce less TNF-ą and more IL-8.  In neonatal sepsis caused by Streptococcus agalactiae, a major cause of severe infection in newborns and pregnant females,  different cytokine expression patterns (IL-6, IL-1 beta, and IL-12p40) have been found in the cord-blood mononuclear cells (14). In contrast to the response to Escherichia coli lipopolysaccharide, where TNFą, IL-1b, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to S. agalactiae results in the production of TNF-ą, but also in the delayed appearance of IL-1b, IL-6, and IL-8 (15). The lymphocyte response to S. agalactiae is manifested by IFNγ and IL-12 secreting, while the E. coli lipopolysaccharide fails to induce production of these critical cytokines. This suggests an important role for TNFą, IFNγ, and IL-12 in S. agalactiae pathogenesis and/or immunity.

Placental malaria in pregnant women is associated with up-regulation of macrophage migration inhibitory factor (MIF)  in the intervillous blood. MIF may play a role in immune responses to malaria during pregnancy by virtue of its ability to activate macrophages and to overcome the immunosuppressive effect of glucocorticoids (16). The level of MIF in the intervillous blood plasma in placental malaria is higher than that in both the peripheral and cord plasma. The intervillous blood mononuclear cells produce  significantly higher levels of MIF, than their peripheral blood counterparts. Placental malaria modulates MIF expression in different placental compartments. A consistent pattern of MIF expression in the syncytiotrophoblasts, extravillous trophoblasts, intervillous blood mononuclear cells, and amniotic epithelial cells, is found, irrespectively, of malaria infection status (17).  Only the amniotic epithelial and intervillous blood mononuclear cells from infected placentas exhibit  significantly higher levels of MIF expression than uninfected placentas.

 

 

 

4.2)                  Immune protective role of the trophoblast

Pregnancy is an immunological balancing act in which the mother's immune system has to remain tolerant of paternal major histocompatibility (MHC) antigens and yet maintain normal immune competence for defense against microorganisms (18). The placenta separates fetal and maternal blood and lymphatic systems, and it is the fetal trophoblast that plays the major role in evading recognition by the maternal immune system.

The trophoblast, the peripheral part of the mammalian conceptus, exerts a crucial role in implantation and placentation, and in the formation of the maternal-fetal interface. Both processes occur as a consequence of an intimate dialogue between fetal and maternal tissues, carried out by membrane ligands and receptors, as well as by the release of hormones and local factors (19).  Chorionic or trophoblastic villi are the main functional units of the placenta within which the fetal blood is separated by only three or four cell layers (placental membrane) from the maternal blood in the surrounding intervillous space (20). After implantation, trophoblast cells proliferate and differentiate along two pathways described as villous and extravillous (21).   Non-migratory, villous cytotrophoblast cells fuse to form the multinucleated syncytiotrophoblast, which forms the outer epithelial layer of the chorionic villi. It is at the terminal branches of the chorionic villi that the most of the fetal/maternal exchange occurs. Extravillous trophoblast cells migrate into the decidua and remodel uterine arteries.  The physiology of pregnancy depends upon the orderly progress of structural and functional changes in villous and extravillous trophoblast, whereas a derangement of such processes can lead to different types of complications, including possible pregnancy loss and life-threatening maternal diseases (19). 

Approximately one week after fertilization, the trophoblast  participates in the contact with maternal cells that enables implantation, a process that quickly sequesters the human embryo within the uterine wall. Through an unusual differentiation process, trophoblastic acquire the properties of leukocytes and endothelial cells that enable many of their specialized functions (22).  Further embryonic development requires the rapid assembly of the basic building blocks of the placenta: the floating and anchoring chorionic villi. The unique structure of the human maternal-fetal interface is established by differentiation of cytotrophoblasts into anchoring villi (23). These fetal cells   form elaborate connections with maternal vessels, thereby diverting uterine blood flow to the placenta. Once the embryo is anchored in the uterine wall, the next major hurdle is rapid formation of extraembryonic lineages, a necessary prelude to assembly of the maternal-fetal interface.  Formation of the placenta, the organ that feeds the fetus, involves a cooperation between maternal NK cells and fetal trophoblast cells that remodels the blood supply. This process and, consequently, human reproductive success, are influenced by polymorphic human leukocyte antigen (HLA)-C ligands and their killer cell immunoglobulin-like receptors (24).

In rhesus monkey, the number of macrophages and CD56+ lymphocytes increases dramatically at implantation and remains high in the early-pregnancy deciduas (25). Macrophages are conspicuously more numerous near the implantation site (decidua basalis) than in sites peripheral to the developing placenta (decidua parietalis), and are found in close association with the cytotrophoblast  adjacent to the decidua, as well as around arteries invaded by the extravillous cytotrophoblast. In contrast to macrophages, CD56+ lymphocytes are more evenly distributed throughout the decidua. A few CD3+ T cells were observed in pregnancy, scattered in the endometrial stroma with occasional aggregate formation.

HLA plays a crucial role in the process of implantation (26). During implantation, the uterine decidua is invaded by extravillous trophoblast   cells whose function is to destroy the walls of the uterine spiral arteries in order to provide adequate blood flow to the fetus. These  cells express an unusual combination of HLA class I molecules, such as HLA-C, HLA-E and HLA-G (27,28). NK cells from the decidua come into close contact with the invading extravillous trophoblasts  and express a variety of receptors which are known to recognize HLA class I molecules. Interaction between these NK cells and extravillous trophoblast cells provides a regulatory influence on implantation. Recognition of HLA-G stimulates uterine NK cells to produce cytokine, via which intrauterine immunosuppression is established (29). Development, growth and differentiation of the placenta have been shown to be regulated by the produced cytokines  (30).

Different  types of HLA are expressed in different parts of the trophoblast and fetal membranes (31). Whereas HLA-A and -B class I genes are down-regulated in human trophoblast cells, nonpolymorphic class-I molecules, e.g., HLA-G class Ib, are expressed in the extravillous cytotrophoblast, in endothelial cells of fetal vessels in the chorionic villi, and in amnion cells and amniotic fluid. HLA-G presents antigens for gamma/delta T cells and at the same time defends the trophoblast from cytotoxic effector mechanisms.

Trophoblast invasion can be seen as a tightly regulated battle between the competing interests of the fetus survival and those of the mother. Successful pregnancy is dependent on the trophoblast invading the mother, attaching the pregnancy to the uterus and securing an adequate supply of oxygen and nutrient for the fetus (32). Trophoblast invasion and migration through the uterine wall is mediated by molecular and cellular interactions, controlled by the trophoblast and the maternal microenvironment (33). The process of migration/invasion of extravillous trophoblast   cells is stringently regulated by many growth factors, their binding proteins, extracellular matrix components, and some adhesion molecules, in an autocrine/paracrine manner at the fetal-maternal interface in human pregnancy (34). For successful invasion to occur, the extravillous trophoblast has to perform a range of functions, such as transform the maternal spiral arteries, tolerate hypoxia, proliferate and die by apoptosis (programmed cell death), differentiate, adhere to and digest the extracellular matrix, and move and interact with the maternal immune system. Each of these functions has multiple overlapping control systems, such that trophoblast invasion is in essence a finely controlled balance of competing mechanisms (35).

Trophoblast invasion of the endometrium shares common features with the inflammatory response. This process is accompanied by the infiltration of uterine NK cells which interact with the nonpolymorphic HLA class-I antigens expressed by the invading extravillous trophoblast (36). In humans, extension of trophoblast invasion beyond the decidual layer into the myometrium presents an additional challenge, which might be relevant in pregnancy complications such as pre-eclampsia.

 

The presence and distribution of different components of the SIS in the human chorion (trophoblast) and decidua  from the first trimester of pregnancy has been described in normal human embryos and early fetuses as well as in those which have been exposed to acute antigenic effects (chorioamnionitis) (37).   The SC, J chain, IgG, IgA,   and macrophages are seen from 3.5-4 to 5 weeks of development and then during the whole first trimester of pregnancy in the syncytio- and cytotrophoblast, and decidual cells (Fig. 4, Tables III and IV).   Macrophages with J chain, IgG and IgA are found in embryonic tissues on week 3.5 to 4, whereas lymphocytes, including those synthesizing  IgA and IgM, appear only at the end of the first trimester of pregnancy. In the decidua, lymphocytes and macrophages are recognized throughout  the entire period studied.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(Color Fig.)

 

         Fig. 4. A 4-week-old normal human embryo.

A. The SC-positive   trophoblast (brown staining) and SC-negative  macrophages. x1000.

B.  J chain-positive macrophages and the trophoblast. x1000.

C.  IgG-positive   monocytes (brown staining) in the trophoblast. x1000.

D. IgA in the syncytio- and cytotrophoblast   and in the monocytes. x400.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table III. The number of lymphocytes and macrophages in the chorionic villi  (in 50,000 µm², mean±SD) 

(After ref. 37)

 

 

^a Significant difference compared to similar parameter in the group I, p < 0.05-0.001.

^b Significant difference compared to similar parameter of the previous age embryos in the same group, p < 0.05-0.001.

 

 

 

 

 

Table IV. The number of lymphocytes and macrophages in the decidua (in 50,000 µm², mean±SD)  (After ref. 37)

 

 

 

                  ^a Significant difference compared to similar parameter in the group I, p < 0.05-0.01.

 

In cases with chorioamnionitis, reactivity of IgG and IgA in the abovementioned fetal cells decreases sharply, while the rate of SC and J chain immunoreactivity    as well as the number of T and B lymphocytes do not change (37). In the decidua, the number of immunoreactive cells increases significantly and the plasma cells appear. Lymphocytes were seen only after week 9 of pregnancy whereas macrophages were observed in high numbers as early as  week 3.5 to 4. In the decidual tissue, the number of all types of immunocompetent cells, including Ig-synthesizing lymphocytes and plasma cells, increases sharply.     

In fetuses of the  second trimester of pregnancy, the Igs, SC and J chain are located in the syncytio- and cytotrophoblast of the chorion (38). The villous stroma contains a small amount of different subsets of lymphocytes. Macrophages account for up to 45% of the stromal cells of the villi and contain IgG and J-chain. In the maternal part of the placenta, the SIS proteins are in the decidual cells. Relatively few lymphocytes and macrophages are observed in the decidual stroma.

A different origin and composition of immunocompetent cells and a different course of immune reactions in fetal and maternal parts of the placenta have been shown (39). Two   types of the SIS have been suggested to be present at the border between maternal and embryonic tissues (37,38).  These systems are already in place at the beginning of the embryonic period, weeks 3.5-4 to 5, function during the entire first trimester of pregnancy and are the main immune mechanism underlying the barrier between these two organisms.  For more details on this topic, see Chapter 1.

 

Trophoblastic villi have several protective mechanisms against the maternal immune system. Trophoblast cells fail to express MHC class I or class II molecules and the extravillous cytotrophoblast cells strongly express the non-classical MHC gene encoding HLA-G, which may downregulate   NK  cell function (18). Extravillous trophoblast cells selectively express the non-classical MHC class I molecules in the form of different types of HLA (HLA-B, HLA-C and possibly also  HLA-G),  which may play an important role in maintaining maternal immune tolerance of the semi-allogenic fetus and  play a protective role during pregnancy (40-43).

 

The trophoblast expresses the complement regulatory proteins CD46, CD55, and CD59, which serve to protect the embryo (18). Moreover, uterine decidual and placental cells produce a huge array of cytokines which, in part, contribute to the deviation of the immune response from Th1 to Th2. This may leave the mother more open to infection whose control is Th1-dependent, but increased production of Th1 cytokines has been linked to spontaneous abortion and small-for-date babies. Th2-type cytokines appear to contribute to the maintenance of pregnancy by controlling the immune and endocrine systems and promoting the function of the trophoblast  at the implantation site  (44).

 

Cytokines released at the feto-maternal interface also play an important role in regulating embryo survival, controlling not only the maternal immune response but also angiogenesis and vascular remodeling (43). The delicate equilibrium established between the mother and her fetus can be compromised in pathological conditions of pregnancy as a result of the mother's humoral and/or cellular response against the trophoblast antigens, leading to spontaneous miscarriage. Cytotoxic cells and antibodies to trophoblast and endothelial cells are frequently found in patients with recurrent spontaneous abortion (for details, see Chapter 5). 

 

In cases with chorioamnionitis, the cellular composition is different in  the embryonic and maternal parts of the gestational sac. In fetal membranes, only the macrophages were found to react to antigenic effects (37,38). The absence of a response by the lymphocytes   showed that in 3.5-4- to 8-week-old embryos even a massive antigenic effect  does not cause an acceleration in their maturation. A weak lymphocyte reaction in the trophoblast  was seen only after 9 to 10 weeks of pregnancy.  In the  decidua, however, the number of lymphocytes, including those synthesizing IgG, IgA and IgM, plasma cells and macrophages increased significantly, even during the earliest embryonic period. 

In patients with pre-eclampsia, a pregnancy complication  with endothelial dysfunction, the sudden onset of maternal hypertension, proteinuria and edema, the cytotrophoblast  fails to differentiate along the invasive pathway  (45).  The functional consequences of this abnormality negatively affect interstitial and endovascular invasion, thereby compromising blood flow to the maternal-fetal interface. In these cases, cytotrophoblast invasion is shallow and vascular transformation incomplete, resulting in abnormal placental production of vasculogenic/angiogenic substances that reach the maternal circulation  (46). Pre-eclampsia has been shown to be  associated with widespread apoptosis of the cytotrophoblast that invade the uterus. Moreover,   the expression of HLA-G by extravillous trophoblast cells appears to be altered, resulting in activation of the maternal immune system (47).    Intrauterine growth retardation in the context of pre-eclampsia is accompanied by reduced trophoblast numbers within smaller and more tortuous arteries and an increase in the proportion of CD56+ uterine NK cells and CD8+ T lymphocytes in the decidua (48). In the case of pre-eclampsia without fetal growth retardation,   no increase in CD56+ uterine NK cells was seen, while CD8+ T lymphocytes were significantly increased compared to normal levels. The development of pathological pregnancies such as pre-eclampsia has been related to the differential expression of epidermal growth factor (EGF) receptor in the syncytiotrophoblast (49). In early-onset pre-eclampsia with intrauterine fetal growth restriction (FGR),  trophoblast invasion into the placental bed is limited by increased apoptosis, resulting in narrower spiral arteries, which is in contrast to findings in anemia (50).

Insulin-dependent diabetes mellitus (Type I) affects the chorionic villi's development causing a significant increase compared to controls in placental volume, and in the volumes of the intervillous space and the trophoblast (51). A significant increase in the volume of the intermediate and terminal villi, the surface area of the villi and fetal capillaries, and the harmonic thickness of the villous membrane was found in the macrosomic subgroup compared to the controls. Morphological changes caused a significant reduction in the villous membrane's specific diffusing capacity in diabetic patients,  and this may contribute to the fetal hypoxia and increased fetal and neonatal morbidity associated with diabetes.

Changes described herein in the placenta, and particularly in the trophoblast, of patients with FGR who died without antigenic effects are similar to those described previously in cases with anti-phospholipid syndrome (APS), especially those  associated with FGR (52,53). APS is characterized by recurrent fetal loss, vascular thrombosis and thrombocytopenia occurring in the presence of antiphospholipid antibodies (54,55). The incidence of APS increases from 5.3% in normal obstetrical patients to  20% in women with recurrent pregnancy loss,  to 37% in women with systemic lupus erythematosus (56), and to 41% in cases of secondary recurrent APS cases  (57). The mean age for APS is 35.6±7.2 years and the mean disease duration is 11.9±8.5 years (57,58).

The antiphospholipid antibodies are acquired antibodies against a phospholipid which has been associated with slow progressive thrombosis and infarction in the placenta (36). The incidence of extensive inflammation and infarction, decidual vasculopathy and vascular thrombosis, and perivillous fibrinoid changes are characteristic lesions of the placenta with APS (59-62).  Although there are no specific histopathological placental abnormalities characteristic of APS patients, primary APS patients may be at increased risk of development of maternal floor infarction or massive perivillus fibrin deposition (63,64). Placental tissue shows large areas with infarctions, intravascular fibrin deposition, syncytial knot formation, and fibrosis. 

APS causes aberrations in early trophoblast differentiation, predisposing the pregnancy to failure. These aberrations manifest themselves as decreased trophoblast area, a constant number of syncytiotrophoblast nuclei (in normal fetuses, this parameter is not constant), a decrease in the number of proliferating trophoblast, and a constant nuclear cytotrophoblast-to-syncytiotrophoblast   ratio (65). These data suggest that abnormal trophoblast differentiation in early gestation may be due to the premature onset of maternal perfusion of the placenta and may be a likely antecedent for conditions associated with failure of placentation, such as recurrent miscarriage. Reduced placental growth and an increase in trophoblastic apoptosis were found in in vitro cultured human placental explants treated with antiphospholipid antibodies  (66). Inflammatory mechanisms in the placental bed may contribute to APS-related pregnancy complications (67). APS biopsies show a high  concentration of inflammatory cells, particularly macrophages, necrosis with hyperplastic vessels, and arterial thromboses.

APS  may affect placental functions through several possible mechanisms. Phosphatidylserine is expressed on the trophoblast surface during differentiation and invasion of the extracellular matrix (68). The antiphospholipid against phosphatidylserine can directly affect trophoblast function by limiting the depth of decidual invasion and by concurrently creating a procoagulant surface on the trophoblast exposed to the maternal circulation. Interaction of antiphospholipid antibodies with cells involved in the coagulation cascade is thought to produce a procoagulant state. Upregulated expression of cell-adhesion molecules  and subsequent stimuation of neutrophil and/or platelet activity within the placental villous tree is unlikely to be a mechanism by which an adverse pregnancy outcome arises in APS pregnancies (69). 

The other mechanism contains the binding of antiphospholipid antibodies to proteins with an affinity for phospholipids, such as beta2-glycoprotein I (b2-GPI) (70). Following the attachment of b2-GPI to anionic phospholipids of the trophoblast, both molecules undergo conformational changes resulting in the exposure of cryptic epitopes within the b2-GPI structure. This may allow the subsequent binding of antibodies, thereby affecting trophoblast functions directly. Moreover, anti-beta2-GPI antibodies induce the activation of endothelial cells, resulting in a proinflammatory state which favors prothrombotic diathesis syndrome.  CD4+ and HLA class II-restricted T cells responsive to b2-GPI are involved in the production of antiphospholipid antibodies in APS patients (71,72). These cells preferentially recognize the antigenic peptide containing the major phospholipid-binding site and have the capacity to stimulate B cells to produce anti-b2-GPI antibodies through IL-6 expression and CD40 ligand engagement (73,74).

There are a series of immune components that are characteristic for APS. B lymphocytes are required for the initiation of antibody-associated disorders, including APS (75). Monoclonal antibodies to B cells, B-cell growth factors, complement proteins and integrin molecules, cell-surface complement regulator proteins or IL-3, all appear to play a role in the antibody-induced disease process (76). Primary APS is accompanied by stimulation of CD4+ and CD8+ T-cell production in patients (77).

 

 

               4.3)  Apoptosis of embryonic cells and its consequences

Programmed cell death, which since Kerr et al. (78)  has been called  apoptosis, is a common and reproducible feature in development of many mammalian tissues and organs. Apoptosis occurs during normal development and it is important in maintaining he correct balance between the loss of old, non-functional cells and the formation of new ones in different organs and tissues. The cell death associated with fusion of the neural folds and the removal of interdigital mesenchymal cells during digit formation  represent  two well-known examples of programmed cell death  (79). Like normal development, abnormal development is also associated with increased cell death in tissues and organs that develop abnormally after exposure to a wide variety of teratogens. Apoptosis  plays an important role in the processes of gamete maturation as well as in embryo development, contributing to the appropriate formation of various organs and structures, organ involution, and ageing, but may arise in pathology when the cell's genetic apparatus  is damaged (80). 

A series of proteins controlling cell death in mammals was identified in the 1990s, i.e., receptors/ligands, caspases, cytochrome c, Apaf-1, bcl-2 family proteins, etc. (79). Apoptosis is triggered by different cell-type-specific signals which involve several pathways, such as te intrinsic mitochondrial and extrinsic receptor-mediated pathways, resulting in caspase-cascade activation (81,82). Morphologically, apoptosis  is   characterized by pronounced cell shrinkage with subsequent fragmentation into apoptotic bodies surrounded by a membrane which are phagocytosed by macrophages without inflammatory reaction (83).

Apoptosis and its associated regulatory mechanisms constitute physiological events that are crucial to the maintenance of placental homeostasis; an imbalance in these processes, however, such as occurs in various pathological conditions, may compromise placental function and, consequently, the pregnancy's success. Increased apoptosis occurs in the placentas of pregnant women with several developmental disabilities, while increased bcl-2 expression is generally associated with pregnancy-associated tumors (84). Bcl-2 protein prolongs cell  survival by blocking apoptosis. In human embryonic differentiation, a low apoptotic index value has been found to be mostly accompanied by the high expression of bcl-2, whereas bax expression was not proportionally related to the  apoptotic index value (85). The apoptotic rate increases during pregnancy with gestational age. Apoptosis is stimulated in maternal peripheral blood during pregnancy, possibly accounting in part for the presence of free fetal DNA in the maternal serum (86).  

Bcl-2 is widely expressed early in embryonic tissues derived from all three germ layers, and this expression becomes restricted as the tissues  mature.  In  human embryos from the 4 to 12 weeks gestation, bcl -2  has been found in many organs of the gastrointestinal tract, in mesenchymal cells surrounding the primitive bronchial epithelium, and in the cells of the metanephronic blasteme and urethral  bud  (87). In  human embryos and fetuses at 7 to 30 weeks  of gestation, bcl -2 is involved in the regulation of apoptosis, and its effect is antiapoptotic. The highest bcl-2 expression has been demonstrated in metanephrogenic blastema cells and the lowest occurrence of bcl-2-positive cells was found in proximal tubules and in branches of the urethral bud (88).. During development of human fetal heart, myocyte undergo apoptosis/mitosis, and CD95 and apoptotic/proliferative processes are present in the early gestation phase, and progressively fading thereafter  (89).  

Apoptosis mediated by the Fas/FasL system may also be associated with maternal immunotolerance to the fetus. The apoptosis, mainly through Fas-FasL or TRAIL-R-TRAIL signalling, may be a defense mechanism against rejection of the fetal allograft by the maternal immune system (81). Presented on trophoblastic cells CD95-L (Fas ligand) plays a part in establishing feto-placental tolerance by inducing apoptosis of immune-defense cells (90).  Expression of FasL  by the human trophoblast is   accepted as a mechanism providing protection against activated decidual immune cells expressing Fas receptor (18,91). Trophoblast apoptosis increases in normal placentas as gestation proceeds, and a greater incidence of trophoblast apoptosis has been observed in pregnancies complicated by pre-eclampsia or FGR. Macrophages presented at the maternal-fetal interface may contribute to trophoblast survival by removing apoptotic cells and producing cytokines and growth factors that influence the progression of the apoptotic cascade (92).

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. Early on, cytotrophoblast cells express most of the important apoptotic proteins, which translocate into the syncytiotrophoblast with the fusion (81). This suggests that apoptosis has a central role in the villous trophoblast turnover. Taking together, the data suggest that regulation of apoptotic events is important in  allowing the correct development, differentiation and functioning of the placenta throughout pregnancy and that an imbalance in this process leads to severe pathologies, such as pre-eclampsia and FGR. 

Apoptosis plays a significant role in trophoblast development and differentiation During the process of implantation, there are a large number of cells at the implantation site undergoing apoptosis, which has been suggested to play an important role in the regulation of endometrial decidulization and trophoblast invasion (93,94). The balance between trophoblast apoptosis, proliferation and expression of cyclins, inhibitors and cyclin-dependent kinases, may represent a mechanism to controlling normal trophoblast invasion (95). Trophoblast apoptosis may be caused by maternal cells such as macrophages but is highly regulated by the trophoblast itself, i.e. trophoblast cells need to be susceptible to be prone to apoptosis. The initial stages of the apoptotic cascade start within the cytotrophoblast, and the execution stages are seen in the syncytiotrophoblast (96).  

During pregnancy, trophoblast cells are shed into the maternal blood from the placenta as they die via apoptosis. Trophoblasts  are fetal cells and they are therefore immunologically foreign to the maternal immune system. The shedding of trophoblasts may not be simply a mechanism used by the fetus to dispose of aged trophoblasts, it  may also provide a chronic source of tolerated paternally derived antigens in order to regulate maternal immune responses to the fetus (97).

Increased apoptosis occurs in the placenta of pregnant women with several developmental disabilities, including hyperglycemia, which may be a key factor evoking apoptosis in the placental trophoblast, and therefore, is relevant to diabetic placenta function (98).  The increased rate of apoptosis seen in the placenta of pregnancies complicated by FGR may have an important compensatory role in transmitting nutrition to, and enabling earlier gas exchange easily with such fetuses (99).

The inflammatory cytokines TNFą and IFNγ  stimulate villous cytotrophoblast apoptosis while EGF  protects these cells. Bcl-2 is reported to be strongly expressed in villous syncytiotrophoblasts, whereas its expression levels are very similar in the first trimester and term cytotrophoblasts (100). Its expression is constitutive, and modulation of its expression levels does not mediate cytokine and growth-factor regulation of apoptosis in these cells. During the first trimester of pregnancy, endogenous expression of  TNFą was detected in villous  as well as in proliferating and invading extravillous trophoblasts,  suggesting this protein's   involvement in trophoblast differentiation (101). The high number of TNFą-positive cells in the first trimester of pregnancy resulted in the appearance of TUNEL-positive cells and an increase in caspase-3 enzyme activity, suggesting that the TNFą-dependent apoptotic cascade is executed in a portion of the early cytotrophoblast.

Apoptosis, which leads to phagocytosis by the mononuclear cells, represents the primary mechanism for removing neutrophils from inflamed tissues and minimizing injury (102).  The activity of caspase 3 and expression of the proapoptotic proteins bax, bad, and bak are lower in neonatal than in adult neutrophils. Prolonged survival of neonatal neutrophils at injured sites is due, in part, to reduced responsiveness to FasL. This may be related to decreased expression of both FasR and bcl-2-family proteins which mediate neutrophil apoptosis. Apoptosis occurs in fetal nucleated erythrocytes that have crossed into the maternal circulation, which might explain the difference between the number of intact fetal cells and the amount of fetal DNA detectable in the maternal plasma (103).   Apoptosis constitutes a mechanism for clearing fetal cells via the maternal circulation. For example, the p53-dependent apoptosis detected in p53+/+ knockout mouse embryos, is considered an immediate reaction detected mostly in the brain, whereas the p53-independent apoptosis is a delayed reaction, with a prominent pattern being observed in the epithelial cells of most organs only in p53-deficient mice (104).

 

Severe hypoxia, which occurs in most preterm infants, also leads to cell death, which may be necrotic or apoptotic. Significant elevation of apoptotic activity may play a role in development of bronchopulmonary dysplasia, ischemic brain lesions, and renal failure in preterm infants who suffered from  infant respiratory distress syndrome, cardiac failure, or periventricular leukomalacia (105).  A high apoptotic ratio is detected in hypoxic injuries of the central nervous system of preterm infants.  In the developing nervous system, sensory organs and orofacial regions of human embryos and fetuses, the  expression of proliferative markers increases with age, whereas  apoptosis is rare in these regions (106).  Enhancement apoptosis was found in the fetal rat's central nervous system, craniofacial tissues and male reproductive organs immediately after the administration of ethylnitrosourea, a well-known DNA alkylating agent, that induces anomalies in different fetal organs (107,108).  In chromosomally abnormal human fetuses, apoptosis was 2.5-fold higher than that found in pregnancies with normal embryos matched for gestational age (86).     

 

 

4.4)  Pathology of the immune organs  in growth-retarded or low-weight fetuses  and newborns under antigen-induced influences

The terms intrauterine or fetal growth retardation (FGR) and low birth weight (LBW) are assigned to newborns born with a birth weight and/or birth length below the tenth percentile for their gestational age. In mammals, size at birth is the outcome of length of gestation and rate of fetal growth (109). In the absence of premature delivery, fetal size within species is determined principally by the fetal growth rate which is dependent on both genetic and epigenetic factors. Failure of either of these mechanisms leads to  FGR/LBW. In mammals, including human infants,  FGR/LBW can occur naturally or pathologically. One major cause for natural FGR/LBW in animals is an increase in litter size. Parental genotype or antigenic differences between the mother and the developing conceptus may be potential causes. Pathological FGR/LBW is due to genetic causes (chromosomal abnormalities or inherited syndromes) or epigenetic causes (intrauterine infections, toxins and chemicals, maternal diseases of pregnancy affecting the placenta).

The underlying pathophysiological processes that occur at the cellular and molecular level in FGR/LBW are still little known. A reduction in the supply of substrates that are necessary for normal cellular function, and an alteration in mediator molecules that regulate cellular growth and differentiation, are important mechanisms. A decrease in growth-promoting factors or an increase in growth-inhibitory factors may lead to growth failure. Growth factors and their receptors are expressed in the developing embryo (at as early as the 1- to 2-cell stage), placenta and maternal uterine tissues, suggesting that these molecules play a role in regulating normal growth and differentiation of the conceptus as well as maternal reproductive tissues. Normal pregnancy is characterized by the transformation of about one half of all spiral arteries within the placental bed. FGR is associated with poor transformation of spiral arteries and is characterized by an increase in uterine NK cells (110). Here we discuss  some morphological and immune aspects of FGR/LBW.

 

Intrauterine mortality and morbidity and premature birth are acute problems in medicine. Infections hold great significance among the various reasons for fetal death and premature birth. The well-known susceptibility of FGR/LBW  fetuses and premature neonates to infections derives from a deficiency in the immunological mechanisms that normally develop during the last tri­mester of gestation. It has been shown that maternal isoantibodies  or other high-molecular-weight substances can enter the fetal blood­stream through the placenta and may act as antigens, causing an immune reaction in the fetuses (111,112). Infections and inflammatory processes resulting in FGR/LBW are considered to be one of the reasons for the associated high rates of pregnancy loss and child death (113,114).  

 

Morphologically, the fetal immune organs are already formed at week 22 of gestation (115). In fetuses at 22 to 23 weeks without antigenic effects, the lymphoid organs are well developed and their differentiation is similar to that of full-term fetuses. In older unaffected fetuses (up to 32 weeks), a significant increase in size of the lymphoid organs and a rise in the rate of lymphoid-cell differentiation are observed. The morphology of the lymphoid organs in unaffected neonates can be regarded as a result of the early death of these newborns, 4 to 8 h  after birth.

 

Different morphological changes are found in the lymphoid organs of fetuses that develop under antigenic influences (115,116).  In 22- to 23-week-old LBW fetuses,   differenti­ation of the lymphoid tissue resembles the normal picture. The only differences are manifested in the smaller weight and size of the lymphoid organs in LBW fetuses and the higher percentage of medium-size lymphocytes.           Development of the lymphoid organs in older fetuses (24-32 weeks) is characterized by changes in some morphometric parameters, by the rate of maturation of the lymphoid cells, and by a marked increase in their number. As a result, the cortex of the thymus, the white pulp of the spleen, and the parenchyma of lymph nodes are all significantly larger in older fetuses. In parallel, maturation of the lymphoid cells is characterized by an increase in the percentage of mature small lymphocytes and a decrease in the number of lymphoblasts and medium-size lymphocytes (112,117).

 

In the thymus,  the first phase of accidental involution can be detected under a light microscope (116). Apoptosis of thymocytes (118) and their concentration around macrophages and phagocytes is rarely identified. In fetuses severely affected by sepsis, with all neonates dying during the first 24 hours, the number and size of the thymic corpuscles showed an increase, with the formation of so-called pearls  (Table V). 

 

The immune reaction of the spleen was clearly recognizable  in mildly affected fetuses suffering from bronchopneumonia and hyaline membrane disease (116). Relative to the unaffected group, the number of lymphoblasts increased sharply and their mitotic rate reached 0.75±0.01/10,000 μm². The number of macrophages rose. The number of lymphocytes remained high, and their ratio to the number of lymphoblasts was 1.6:1. Only about 75% of the follicles were found to react. The number of cells decreased in follicles and the red pulp. As a result of the cells' transformation, the lympho­cytes-to-lymphoblasts ratio decreased to 3.1:1 as compared to 42.9:1 in unaffected neonates (Table VI). This reflects not only the creation of a large number of lymphoblasts but also the high rate of lymphocyte loss.

 

The follicular area in the mildly affected spleen showed a tendency to increase in size. In the severely affected fetuses,  the number of follicles and their areas decreased as a result of the sharp decrease in the number of lymphocytes. The decrease in follicular size was reflected in the appearance of so-called bare central arteries, which were not observed in unaffected or mildly affected fetuses. Where infection was very severe, the follicles disappeared.

 

The immune reaction of lymph nodes was clearly manifested in the mildly affected subgroup. Sinuses were open and contained many macrophages, lymphocytes, and erythrocytes. Phagocytosis was active. The parenchyma contained numerous lymphoblasts and macrophages (Table VI).   In the severely affected subgroup, cell proliferation accompanied the processes of immuno-incompetence: the number of lymphoblasts increased significantly while the number of cells and the size of the parenchymal area decreased as compared to the unaffected group. The parenchymal area was markedly small, and there was a reduction in the number of lymphoid cells per unit of parenchymal area.  In the most severe cases, the lymph nodes appeared to be devastated, containing only stromal cells and a few lymphocytes.

 

The presence of disease-related antigens appears to stimulate an immune reaction in fetuses, with accompanying changes in the morphology of the lymphoid organs (116).   These changes are manifested in an increase in the number of macrophages and their phagocytic activity in the red pulp, in the follicles of the spleen, and in lymph nodes, accompanied by an increase in the number of lymphoblasts and their mitotic activity. The spleen and   lymph nodes of normal fetuses and newborns are characterized by the presence of follicles with the reactive centers in those follicles and mature plasma cells playing a central role in the immune reaction in children and adults. In LBW fetuses affected by antigenic influences, neither the reactive centers nor mature plasma cells are found (115,116). No differences in the immune response are found between the younger (22-23 weeks) and older (up to 32 weeks) LBW fetuses. The immune reaction among such fetuses is generalized in all the lymphoid organs studied, although in regional lymph nodes (in the hilus of the lungs and the mediastinum) this reaction is stronger under pneumonia. We suggest that fetuses exposed to antigen-related diseases undergo morphological changes in the lymphoid organs presumably as a consequence of the primary fetal immune reaction.

 

A characteristic of the immune reaction in LBW fetuses is the rapid development of lymphoid-system decompensation.  This phenomenon is manifested in a progressive decrease in the number of lymphoid cells, especially small lymphocytes (115,116). This was seen  in severely affected fetuses where, as a result of the decrease in the number of lymphoid cells, the number and size of the follicles in the spleen decreased until they disappeared altogether. The number of lymphocytes in lymph nodes also decreased. Macrophages showed higher resistance (their number increased even in severely affected fetuses) and retained their phagocytotic activity for a pro­longed period.

 

 

Table V.

Morphometric parameters of the thymus in different groups of infants (mean ± SE) (After ref. 115,116)

 

^a As a percentage of the whole square of the organ on a slide.

^b Significantly different from unaffected group (p<0.05-0.01)

^c Significantly different from mildly group (p<0.05-0.01)

 

 

 

Table VI.

The lymphocytes-to-lymphoblasts ratio  in the follicles of the spleen and lymph nodes in infants of different ages and groups (After ref. 115,116)

 

^a  Significantly different from unaffected group (p<0.05-0.01)

^b  Significantly different from values of the spleen (p<0.05-0.01)

 

 

 

 

Dystrophic changes in macrophages were seen only in very destructive processes. In such cases, associated with devastation of the lymphoid organs, phagocytosis ceased and the number of macrophages was significantly decreased. In some severely affected cases there was an increase (2-4%) in the number of eosinophils in the lymphoid organs.  The immune reaction of LBW fetuses was also characterized by a weak reaction of the thymus. 

 

The immature lymphoid system in LBW fetuses is characterized partly by the absence of plasmocytes and reactive centers in follicles of the spleen and partly by the weak reaction of the thymus and the absence of follicles in the lymph nodes. These last two features are highly characteristic of LBW fetuses (115,116). Moreover, immuno-incompetence in these fetuses is manifested by the rapid exhaustion of the lymphoid organs. Various morphological and morphometric signs of such devastation were seen among most severely affected fetuses, all of whom exhibited distinct evidence of decompensation in the development of the immune system. We therefore suggest that antigenic effects do not accelerate the normal develop­ment of the immune system in fetuses as proposed elsewhere (119), but interfere with it and may even destroy the processes of normal development, which may be manifested later in childhood. It is suggested that even moderate disease-related antigenic effects can exert a marked influence on the normal development of the immune system. The resulting changes in this system may be regarded as one of the reasons for the LBW of such fetuses.

The immune response to antigenic effects in LBW infants has many features that are typical to fetuses. It is spread throughout the many lymphoid organs  and manifested in the thymus, lymph nodes and mainly in the spleen.   In LBW infants, this type of immune reaction is maintained until up to 10 months or more, while in full-term infants it is retained for no longer than 3 months after birth.(120). It is manifested as an increase in the number of macro­phages and in the transformation of lymphocytes into lymphoblasts. In LBW infants the mature plasmocytes and reactive centers in the follicles of the spleen are absent. This has been explained by the peculiarities of the B-lymphocytes in fetuses and infants (121). Components of the immune system are related to this pathology. For example, the very low levels of IgG serum in LBW infants are connected with a high risk of severe infection and sepsis (122). Septic complications in infants with very low birthweight are accompanied by a significant decreased in the monocyte's phagocytic capacity,  and in HLA-DR expression on the monocytes  (123).   

The morphological changes in the immune organs described herein were seen in all infants studied, who died in the first week and after 4 to 5 months (116). Some peculiarities were found connected with the age of infants and the nature of the effects. In the infants older than 2 weeks, the lymphocytes/lymphoblasts relationship increased under all antigenic effects. Under sepsis, the reaction of neutrophils and eosinophils was found to increase significantly in LBW infants while in similar fetuses it was very weak.

 

The immune reaction in fetuses and infants can be caused not only by infectious (inflammatory) processes within their own bodies, but also by antigenic influences from the mother. The severe morpholog­ical changes in lymphoid organs similar to septical have been seen in fetuses with swelling-type rhesus-HDN  and in infants who were born from mothers with acute appendicitis and periappendicitis or pre-eclampsia and juvenile diabetes (124). 

FGR syndrome has many causes. Severe FGR, for example,  may be associated with placental damage leading to increased feto-maternal cell traffic which results in a significant increase in the proportion of fetal erythroblasts in the maternal blood  (125,126). Artificial FGR   in guinea pig fetuses induced by uterine artery ligation causing the hypoxic stress, leads to an increase in medullary erythropoiesis (127). This was manifested in an increase in total erythrocyte precursors and a decrease in total granulocyte precursors. 

The reasons for insufficiency of the lymphoid system in LBW infants which develop under disease-related antigenic effects are different. This  insufficiency has been found in the weak reaction of the thymus and its reticular epithelium (128), as well as of macrophages (129), of neutrophils (130), of T ­lymphocytes (131) and of B lymphocytes (122). Similar observa­tions was made in a morphological study of the lymphoid organs (115,116). It appears that owing to the close relationship between different parts of the immune system, the underdevelopment of one part causes disturbances in the others and as a result in the entire system. The low mass of the lymphoid tissue, particularly the low number of lymphocytes, can cause rapid devastation of the immune system under antigenic effects.

 

A high rate of relationships between different morpho­logical features was found in unaffected LBW infants which reflected good coordination in all the processes of normal ontogenesis of the lymphoid system. The significant changes in these relationships in mild and especially in severely affected neonates can be considered a result of deep disturbances in the normal develop­ment of lymphoid organs and their insufficiency under the immune reaction.

 

 

 

 

 

 

 

 

 

 

 

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