Immunobiology of the human placenta. I. IgGFc receptors in trophoblastic villi.

Qualitative and quantitative studies were performed on IgGFc receptors in the trophoblastic villi of human placentae ranging in gestational age from less than 4 weeks to full term. IgGFc receptors were detected on cells of the syncytiotrophoblast (ST) using two different assay systems; EA rosette formation and direct immunofluorescence with deaggregated human IgG. The ST IgGFc receptors had high affinity for native IgG molecules (deaggregated IgG) as well as affinity for antigen-antibody complexes (EA). The receptors for deaggregated IgG were present on a majority of ST cells in first and second trimester trophoblast, but were significantly less frequent on ST cells in older placentae. Similar receptors also were detected on cells lining some fetal vessels in the trophoblastic villi. Not only were the IgGFc receptors expressed on ST cells, but in vivo bound IgG was detected in association with the ST and the two patterns of IgG binding were essentially identical. In contrast to the qualitative nature of the receptors on ST cells, cells in the stromal (central) region of the trophoblastic villi expressed IgGFc receptors that had high affinity for EA but failed to bind the deaggregated IgG except at a high concentration. The results are discussed with respect to the possible role of the IgGFc receptors in the specific transfer of IgG from maternal to fetal circulations.     

Methodological approaches to immunohistochemical demonstration of beta-hexosaminidase in human placental and renal tissue with monoclonal antibodies

The lysosomal enzyme, beta-hexosaminidase (Hex), was studied in full-term human placentas and in renal tissue using monoclonal antibodies raised against Hex purified from human placentas. The immunohistochemical reaction for Hex was pronounced in trophoblastic cells and macrophages of the basal plate and the smooth chorion, but was faint or negative in the amnion as well as in the syncytiotrophoblast and Hofbauer cells of the chorionic villi. The maternal decidual cells of the basal plate were negative. Biochemical enzyme analysis showed the highest activity in basal plate cells (containing trophoblast, decidual cells, macrophages and neutrophils) and a low activity in the chorionic villi. Placental tissue was less positive with monoclonal antibodies specific for Hex A, compared with antibodies reacting with both Hex A and Hex B. Epithelial cells of the renal proximal tubules were positive to the same degree with antibodies recognizing both Hex A and Hex B as well as those recognizing only Hex A.     

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

Summary In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the intial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Proteolytic activity of first trimester human placenta: localization of interstitial collagenase in villous and extravillous trophoblast

In human placentation, events of implantation and early blastocyst development are mediated by fetal trophoblastic cells which penetrate into the maternal endometrium and myometrium. Although highly regulated in its biological behavior, trophoblast simulates a malignant neoplasm by virtue of invading the uterine wall and uterine spiral arteries and by embolizing throughout the systemic circulation. This process is at least in part dependant on the regulated production of proteolytic enzymes to degrade extracellular matrix. The most abundant extracellular protein is connective tissue type (interstitial) collagen. The uterine remodeling during the establishment of the embryo requires collagenase which catalyzes the initial step in the breakdown of collagen. This study demonstrates the presence of interstitial collagenase in villous and extravillous trophoblast of first trimester placenta using immunocytochemical methods on light microscopic and ultrastructural levels. Intracytoplasmic staining for interstitial collagenase was present in cyto- and syncytiotrophoblast covering the chorionic villi as well as in extravillous intermediate trophoblast invading spiral arteries in the placental bed. Furthermore, outgrowth cultures of chorionic villi were studied with the immunogold method. Gold labelling was associated with the cell surface of trophoblastic cells as well as with fibrillary collagen like proteins of newly synthesized extracellular matrix. We speculate that interstitial collagenase plays a role in the degradation of uterine collagen within the developing human placenta.     

Monoclonal antibodies to cytotrophoblast and syncytiotrophoblast of human placenta

Mouse monoclonal antibodies were raised against plasma membranes-enriched fractions of 8-12 week placental villi. Several antibodies were obtained and three of these are described in detail: ED822 reacts with the outer membrane of the syncytiotrophoblast; ED235 reacts with the cytotrophoblast layer; ED341 reacts with the whole trophoblastic layer. Antibodies ED235 and ED341 were useful markers for identification of trophoblast cells in culture.     

Lectin histochemistry of human placenta

Abstract. The human placenta was studied histochemically using 23 fluorescein-isothiocyanate-labeled lectins Distinct patterns of staining, as well as some differences between first-trimester and term placenta, were discerned. Eleven lectins (HPA, VVA, BPA, HAA, SBA, PNA, GSA-I, MPA, RCA-I, RCA-II, and UEA-I) did not react with the trophoblast. Two lectins (LCA and PEA) reacted with the trophoblast of first-trimester placenta but not with the trophoblast of third-trimester placenta. The remaining ten lectins (ConA, Suc.ConA, WGA, GSA-II, LAA, STA, DBA, LBA, PHA-E, and PHA-L) reacted with the trophoblast of both first- and third-trimester placenta, and two of these lectins (ConA and Suc.ConA) reacted preferentially with the syncytiotrophoblast. Five lectins (LAA, STA, DBA, GSA-II, and LBA) reacted with nuclei of the cytotrophoblast. The nuclei of some stromal and syncytiotrophoblastic cells were also reactive. Eighteen lectins reacted with the trophoblastic basement membrane, and all reacted with Hofbauer cells and the stroma of the villi. Latin binding was influenced by the mode of fixation and tissue processing. These data show that some lectins can be used to identify components of the placental villi (e.g., basement, membrane) to characterize differences between the first- and third-trimester trophoblast, and to distinguish the cytotrophoblast from the syncytiotrophoblast.     

Fc receptors on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

Immunohistochemistry in human placental tissue--pitfalls of antigen detection.

Because incongruous controversial staining results are a common phenomenon in the placenta, methodical investigations are important to prevent researchers from obtaining misleading results. While investigating dendritic cells (DC) at the human fetomaternal interface, we observed staining of endothelial cells (EC) in chorionic villi for CD83. Given the high specificity of this antigen for DC, this did not seem credible. Previous studies had revealed the same surprising staining pattern with human leukocyte antigen (HLA)-G antibodies. We therefore analyzed human placental EC staining more closely. Both CD83 and HLA-G antibodies were of the same mouse IgG2b isotype. We also observed EC staining with a panel of control antibodies of the IgG2b isotype. This suggests a high affinity of human placental capillaries for mouse IgG2b. Several commonly used techniques for blocking nonspecific binding of antibodies could not prevent this nonspecific EC staining. A new preincubation step with purified human IgG was introduced. This abolished any placental EC staining with CD83, HLA-G, and IgG2b isotype control antibodies, presumably by blocking Fc receptors, whereas specific staining patterns remained unchanged. Mouse antibody of the IgG2b isotype are bound nonspecifically by vascular endothelial cells in human placenta and this can be overcome by blocking with purified human IgG. This blocking procedure could also be appropriate for frozen tissues other than placenta in which Fc receptors are expressed.     

The different roles of two distinct Fc gamma receptors on guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes

The functional roles of two distinct types of Fc gamma receptors (Fc gamma 1/gamma 2R specific for both IgG1 and IgG2, and Fc gamma 2R specific for IgG2 alone) on the surface of guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes (EA) were investigated by the use of two Fab's of monoclonal anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibodies. The binding and subsequent ingestion of IgG1 antibody-sensitized erythrocytes (EA gamma 1) by macrophages were completely inhibited by anti-Fc gamma 1/gamma 2R Fab', indicating that the reactions are mediated only by Fc gamma 1/gamma 2R. On the other hand, the binding and subsequent ingestion of IgG2 antibody-sensitized erythrocytes (EA gamma 2) were substantially inhibited by anti-Fc gamma 2R Fab', but not by anti-Fc gamma 1/gamma 2R Fab'. The inhibitory activities of anti-Fc gamma 2R Fab' were dependent upon the amount of IgG2 antibody bound on erythrocytes; increasing the amount of bound IgG2 antibody from 0.15 to 0.91 micrograms/2 X 10(8) erythrocytes resulted in a decrease in the inhibition of binding of EA gamma 2 by anti-Fc gamma 2R Fab' from 50 to 0%, and also a decrease in the inhibition of ingestion of EA gamma 2 from 100 to 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat

Summary Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells, T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.     

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.     

Immunohistochemical aspects of immunological cross-reaction and masking of epitopes for localization studies on pregnancy-associated plasma protein A

Summary The influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined.Apparently monospecific IgG fraction of the anti-plasma protein applied directly on fixed tissue resulted in staining of connective tissue and a thin apical rim of the syncytiotrophoblast. Further absorption of the antibody with foetal connective tissue abolished this staining reaction. Pre-treatment of the fixed placental tissue with trypsin prior to application of the antibody, which had been absorbed with connective tissue, resulted in staining within the cytoplasm of the syncytiotrophoblast exclusively. Identical staining was seen when this IgG preparation was used directly on frozen placental tissue.The results point to the importance of the specificity of the antibody preparations and of proteolytic unmasking of epitopes when fixed tissues are used for localization studies of pregnancy-associated plasma protein A by immunoperoxidase technique.     

Expression of Bcl-2 and p53 at the fetal-maternal interface of rhesus monkey

To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis.     

The immune response in the hamster. VII. Studies on cytophilic immunoglobulin.

Hamster 7S IgG1 and IgG2 antibodies to hen-egg albumin (HEA) were tested for their capacity to bind to macrophage cytophilic Ig receptors. Both IgG1 and IgG2 were cytophilic for hamster macrophages though the membrane receptor had a predominant specificity for IgG1. Hamster IgG1 bound primarily to homologous macrophages whereas IgG2 bound to macrophages from other rodent species as well. The binding of hamster Ig to hamster macrophages was inhibited by a wide range of heterologous rodent sera. The only exception was guinea pig serum since guinea pig IgG2 was found to bind only to homologous macrophages. Sheep red blood cells (SRBC) coated with hamster IgG2 were ingested by macrophages more readily than those coated with hamster IgG1. Thus, there appeared to be a paradoxical relationship between the apparently strong affinity of IgG1 for the hamster macrophage Ig receptor and its reactivity weak ingestion promoting activity. Implications of this phenomenon are discussed.     

Isotype Specificity and Heterogeneity of Fcγ-Receptors on Guinea Pig Splenic B and T Lymphocytes

The isotype specificity of Fc-receptors for IgG (Fcγ-Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti-sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B-cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette-forming B cells bore the Fcγ-R monospecific for IgG2, whereas 80% of the cells had two distinct Fcγ-Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fcγ-R monospecific for IgG1 was not definitively demonstrated in the B-cell fraction. In the T cell-enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1)rosette-forming cells, however, comprised only 6% of the total cells, indicating that most of the EA(IgG2) rosette-forming T cells bear essentially the Fcγ-R monspecific for IgG2 alone. The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fcγ-Rs, which are not equally distributed on the B- and T-cell populations and also on their respective subsets.     

Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation

The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DH1 in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DH1 monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.     

Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation

Summary The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DHl in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DHl monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.