Antigens of human trophoblast. Effects of heterologous anti-trophoblast sera on lymphocyte responses in vitro.

This report describes the inhibition of human mixed lymphocyte culture (MLC) reactions by rabbit antisera to intact and detergent solubilized, fractionated, human trophoblast membranes. Heat-inactivated antisera were passed through solid-phase immunoabsorption columns of normal human serum and extensively absorbed with human erythrocytes, lymphocytes and liver powder. Immunohistological experiments with these absorbed antisera showed that they reacted brilliantly with syncytiotrophoblast in cryostat sections of human but not baboon or monkey placentae, and not with other normal adult tissues including peripheral blood lymphocytes (PBL). Addition of these antisera to MLC reactions produced significant and reproducible suppression of responses without affecting cell viability. Absorption studies demonstrated complete removal of MLC inhibition and trophoblast membranes but not with PBL or suspensions of HEp-2 cells. Timed experiments showed that optimal inhibition occurred when the antisera were added between 2 and 6 h after culture initiation, and that little suppression was achieved after 18 h. Lymphocytes harvested from MLC reactions after 2 h showed that 3--5% of the cells reacted with PBL/liver-absorbed anti-trophoblast sera, and that unstimulated PBL were negative. Cultures of subhuman primate lymphocytes in the presence of heterologous antisera to human trophoblast membranes showed total inhibition of rhesus:human and human:rhesus MLC, and no suppression of baboon:human or human:baboon reactions, whereas human lymphocytes responded in an exagerated manner when stimulated by baboon cells. Modulated MLC responses to human, rhesus, or baboon lymphocytes, in the presence of anti-trophoblast sera indicate that the antisera recognize trophoblast cross-reactive lymphocytes antigens. We propose that these antigens are reaction products of cell-cell interactions, and that the nature of the antigens is determined by the specificity of the recognition signals which initiate the reaction.

Immunohistological and elution studies of the human placenta.

An immunohistological survey of 28 full-term human placentas has demonstrated deposits of IgG, beta1C, beta1E, and fibrinogen/fibrin in areas of fibrinoid necrosis and on the trophoblast basement membrane in approximately 35% of placental villi. Traces of IgM were detected at similar sites in 18 of 28 full-term placentas. In 11 specimens of immature placentas (10-18 wk gestation) traces of IgG and beta1C and deposits of fibrinogen/fibrin were also present, but IgM was not detected in this material. IgG was recovered in acidic eluates from an homogenized placenta which behaved as an antibody reactive with unidentified material present in fibrinoid deposits and on the thickened trophoblast basement membrane of some villi. It could not be determined whether this IgG was derived from the maternal or fetal circulation.

Peyer's patches: immunologic studies.

The immune capabilities of the Peyer's patches have been investigated by the use of an in vitro system. Despite our failure to stimulate Peyer's patch lymphocytes in vivo it appears that Peyer's patches behave immunologically as peripheral lymphoid tissues. Cultures prepared from the dissociated Peyer's patches of normal rabbits respond to sheep erythrocytes. The response is comparable to that obtained with spleen cultures from the same animals and is not dependent on the presence of the epithelial cells which line the lumen. Similar thymic cultures do not respond. Our experiments with cultures prepared from rabbits which have received one or two injections of SRC show that the Peyer's patches contain both IgM and IgG "memory" cells which have migrated from the spleen. The concentration of these cells in the spleen remains several hundredfold higher.

Human trophoblast-lymphocyte cross-reactive (TLX) antigens define a new alloantigen system.

Antisera to human syncytiotrophoblast microvillous cell surface membranes from different placentas are cytotoxic for lymphocytes from some people but not others, demonstrating the presence of allotypic trophoblast-lymphocyte cross-reactive (TLX) antigens. Exploratory principal components factor analysis, performed on limited data consisting of 300 cytotoxic reactions produced by ten separate trophoblast antisera on a panel of lymphocytes from 30 random donors, suggested the presence of three distinct TLX antigen groupings. It is proposed that such TLX alloantigens are central in establishing maternal recognition and protection of the blastocyst, and that lack of recognition results in implantation failure and spontaneous abortion. These findings are compatible with contemporary results of immunotherapy to prevent recurrent spontaneous abortions, and their implications extend to other conditions of allogeneic coexistence, such as organ transplantation and the tumor-host relationship.

Increased frequency of the MZ phenotype of alpha-1-protease inhibitor in juvenile chronic polyarthritis.

Pi phenotypes of alpha-1-protease inhibitor were determined by isoelectric focusing and print immunofixation in 96 English children suffering from juvenile chronic polyarthritis. A significantly increased frequency of the MZ phenotype (10.41%) was found in comparison with a geographically matched control population (1.6%). The results of this study suggest that the possession of a Z-deficient allele of alpha-1-protease inhibitor could be a predisposing, aggravating, or perpetuating factor in the articular damage occurring in juvenile chronic polyarthritis.

Immunological studies of the human placenta. Characterization of immunoglobulins on trophoblastic basement membranes.

Immunohistological and elution studies of the human placenta revealed the presence of IgG on the trophoblastic basement membrane (TBM) which demonstrated specificity for placental but not lung, thyroid, or kidney basement membranes, suggesting the presence of a placenta-specific antigen in TBM. IgG comprised the bulk of immunoglobulin in eluates, and small amounts of IgA, trace amounts of IgM, but no IgE or IgD were identified in eluates. The distribution of IgG subclasses in eluate was not unusual as compared to maternal and neonatal sera, and Gm and Inv typing of eluates indicated that it was of maternal origin. Small amounts of eluate-IgG effectively inhibited the blastogenic response of unrelated lymphocytes to old tuberculin, phytohemagglutinin, and in one- or two-way mixed lymphocyte culture reactions. The inhibition was distinct from nonspecific inhibitors, and dose-response analysis indicated that eluate was very much more potent as an inhibitor than were the nonspecific inhibitors. Inhibition was shown to not be due to anti-HL-A activity, and was probably not due to aggregated IgG or immune complexes. Binding of eluate to lymphocytes was very loose as shown by washing experiments, and no binding could be shown by immunofluorescence. The capacity of eluate IgG to inhibit MLC was retained after pepsin digestion to F(ab')(2), suggesting that the inhibition reactions were immunological. It is suggested that eluate-IgG is maternal blocking antibody to a hitherto uncharacterized trophoblast antigen, and it is speculated that either abnormal antigen or aberrant responses to antigen could result in fetal wastage.

Diferric transferrin reduction by K562 cells. A critical study.

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.

Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates.

Transplasma membrane electron transport from HeLa cells, measured by reduction of ferricyanide or diferric transferrin in the presence of bathophenanthroline disulfonate, is inhibited by low concentrations of adriamycin and adriamycin conjugated to diferric transferrin. Inhibition with the conjugate is observed at one-tenth the concentration required for adriamycin inhibition. The inhibitory action of the conjugate appears to be at the plasma membrane since (a) the conjugate does not transfer adriamycin to the nucleus, (b) the inhibition is observed within three minutes of addition to cells, and (c) the inhibition is observed with NADH dehydrogenase and oxidase activities of isolated plasma membranes. Cytostatic effects of the compounds on HeLa cells show the same concentration dependence as for enzyme inhibition. The adriamycin-ferric transferrin conjugate provides a more effective tool for inhibition of the plasma membrane electron transport than is given by the free drug.

A passive agglutination method using collagen-coated tanned sheep erythrocytes to demonstrate collagen-glycosaminoglycan interaction.

A haemagglutination method originally designed to detect and measure antibody has been shown to be capable of monitoring interactions of glycosaminoglycans and collagen in vitro under physiological conditions of pH and ionic strength. In this system, dermatan sulphate and chondroitin 4-sulphate interact at higher dilutions ('higher titres') with collagen than do chondroitin 6-sulphate or keratan sulphate. The results are relevant to studies of anti-collagen antibodies in connective tissue fluids and extracts, as well as to connective tissue physiology.

Propidium iodide as a nuclear marker in immunofluorescence. II. Use with cellular identification and viability studies.

The use of a fluorescence method employing propidium iodide to determine nuclear morphology of different cell types by epi-illumination in membrane immunofluorescence is described. Its usefulness is illustrated by differentiating nucleated and anuclear transferrin receptor-bearing cells as well as by simultaneously determining the viability of cell suspension without requiring a change from fluorescence microscopy, this causing no loss of visual accommodation.

Propidium iodide as a nuclear marker in immunofluorescence. I. Use with tissue and cytoskeleton studies.

Some examples are given of immunofluorescence with tissue sections and microtubular cytoskeletons of cultured cells where the fluorescent dye propidium iodide (PI) has been used as marker of nuclei. The emission wave length of IP is longer than that of fluorescein, making it possible to use several different and commonly available filter combinations. The use of nuclei as positional indicators is often a more suitable method than phase microscopy combined with immunofluorescence because of low background illumination against which morphology is viewed, circumventing the need for often expensive phase optics.

Myocardial fibrin deposits in the first month after transplantation predict subsequent coronary artery disease and graft failure in cardiac allograft recipients.

PURPOSE: To determine whether fibrin deposition during the first month following cardiac transplantation predicts development of coronary artery disease and graft failure in cardiac allograft recipients. PATIENTS AND METHODS: We prospectively studied 121 consecutive adult patients who received cardiac transplants between 1988 and 1995. Serial endomyocardial biopsies obtained during the first month posttransplant (2.3 + 0.6 biopsies/patient) were studied immunohistochemically for fibrin deposits. Patients were followed up with annual angiograms (3.2 + 1.7/patient) evaluated with side-by-side comparisons for the presence and progression of coronary artery disease. RESULTS: All pretransplant biopsies were fibrin-negative; 60 allografts (50%) remained without fibrin, and 61 (50%) contained fibrin during the first posttransplant month. Of allografts with fibrin, 72% developed coronary artery disease, while 27% of allografts without fibrin developed the disease (P <0.001). Coronary artery disease was progressive in 61% of allografts with fibrin, and in 25% of allografts without fibrin (P <0.001). Graft failure was more frequent and time-to-graft-failure occurred earlier in patients whose allografts had fibrin during the first month after transplantation (P <0.001). CONCLUSIONS: Fibrin in biopsies during the first month after transplantation identifies patients at high risk for developing coronary artery disease or graft failure, thereby allowing the opportunity to initiate preventive procedures.

MHC class II reactivity of human villous trophoblast in chronic inflammation of unestablished etiology.

Absence of class II MHC antigens from human syncytiotrophoblast is a common finding in normal-term placentae. Since chronic villitis of unestablished etiology is a placental lesion frequently found in normal and abnormal term placentae, and fetal stem vessels are MHC class II-positive in these lesions, we asked if syncytiotrophoblast in villitis is reactive for MHC class II antigens. We found segments of syncytiotrophoblast that were reactive for the MHC class II HLA-DR, DP, and DQ antigens in villitis areas of normal-term placentae and in placentae from women with a history of recurrent spontaneous abortions. This reactivity was not due to trophoblast replacement by activated macrophages, though the possibility of crossreactive antigens and binding of soluble MHC class II antigens by receptors developed in areas of villitis could not be excluded. MHC class II antigen expression on syncytiotrophoblast could be due to cytokine release from activated macrophages and helper T lymphocytes which we have previously described in areas of villitis of unestablished etiology.

Natural anticoagulant and fibrinolytic pathways in renal allograft failure.

This is an immunocytochemical study of the relationship between depletion of natural anticoagulant and fibrinolytic pathways and allograft survival following renal transplantation. Patients (n = 44) were classified in three groups according to the length of time between transplantation and allograft failure: group 1 (n = 14) failed within a month of transplantation; group 2 (n = 14) failed between one month and one year after transplantation; and group 3 (n = 16) failed after one year of transplantation. Control biopsies were from donor kidneys (n = 16) prior to transplantation. There were no statistically significant differences in recipient age, gender, donor kidney type (living-related versus cadaver), histocompatibility, and plasma cholesterol, triglycerides, or creatinine concentrations between groups. However, group 1 allografts had a greater depletion of the vascular heparan sulfate proteoglycan-antithrombin III natural anticoagulant pathway than allografts in group 2 or 3 (P < or = 0.05), and this depletion was associated with significantly greater fibrin deposition in group 1 than in either group 2 or 3 (P < or = 0.05). All three groups demonstrated severe depletion of tissue plasminogen activator from arteriolar smooth muscle cells and depressed fibrinolysis as evidenced by increased fibrin/plasmin ratios. However, no significant differences were found for either endothelial thrombomodulin or T cell, neutrophil, or macrophage infiltration between the groups. These data indicate that differences in graft outcome may be determined more by compromised vascular function than by the presence of cellular infiltrates.

Tissue plasminogen activator in human cardiac allografts.

The activation of hemostasis and fibrinolysis frequently is observed in allografted organs. Plasminogen is activated by urokinase and tissue plasminogen activator (tPA). We have studied human hearts before and after transplantation to determine if fibrin deposition within the microcirculation is associated with a depletion of myocardial tPA, and if such depletion of tPA is associated with decreased fibrinolysis. We found that tPA in pretransplanted hearts and in biopsies from hearts of most patients with a stable clinical course is confined to arterial and arteriolar smooth muscle cells. The depletion of smooth muscle cell reactivity was associated with microvascular fibrin deposition in unstable allografts, and the appearance of endothelial cell tPA reactivity heralded a bad prognosis. Successful medical management was signaled by a loss of endothelial tPA reactivity and a return of tPA reactivity in arterial and arteriolar smooth muscle cells. These findings indicate a central role for tPA in maintaining the integrity of the microcirculation in transplanted human hearts.

The endothelial heparan sulfate-antithrombin III natural anticoagulant pathway in normal and transplanted human kidneys.

This is the first study of the antithrombin III-heparan sulfate natural anticoagulant pathway in human kidneys. Immunocytochemical experiments were done to demonstrate the pathway on normal renal endothelial cells. Enzymatic studies were done to show that the antithrombin III was anchored to endothelium by molecules of heparan sulfate. Displacement studies were done with glycosaminoglycans to show that the antithrombin III was bound to its glycosaminoglycan anchor via a heparinlike binding site, and replacement studies showed that antithrombin III could be returned to the same endothelial cells from which it was displaced. Immunocytochemical studies of biopsies showed that normally functioning renal allografts manifested the endothelial antithrombin III-heparan sulfate anticoagulant pathway. The pathway was compromised or absent from the microcirculation of biopsies from rejecting or rejected renal allografts, and the diminishment of endothelial ATIII was associated with the presence of fibrin deposition. It is concluded that compromise of the antithrombin III-heparan sulfate natural anticoagulant pathway results in compromised renal function in transplanted kidneys.

Tissue factor in normal and transplanted human kidneys.

Tissue factor (TF) plays a central role in the initiation of blood coagulation that frequently is enhanced in renal allografts. The identification and localization of TF was studied immunocytochemically in biopsies from normal and transplanted human kidneys and classified according to its distribution. The clinical status of each allograft was then correlated with the TF classifications. From these correlations, four distributional types of TF were identified. In normal kidneys, TF was localized to glomerular epithelium and basement membranes. Glomerular TF expression did not colocalize with mesangial or endothelial HLA-DR reactivity as determined by double antibody techniques. Tissue factor in donor kidneys also was identified in the renal capsule and in the adventitia of large arteries. These structures were not reactive in long-term transplanted grafts. Some cadaver kidneys prepared for transplantation had depleted glomerular TF, and exhibited TF reactivity within stromal tissues. Long-term allografts with progressive loss of renal function and kidneys with advanced rejection exhibited diminished TF reactivity of glomerular epithelium and basement membranes. This was frequently associated with fibrin deposition within the glomeruli and in the intertubular microcirculation. These findings indicate that the evaluation of TF in transplanted kidneys is related to the prognosis of graft survival.

Novel immunohistochemical markers of human renal allograft dysfunction--antithrombin III, Thy-1, urokinase, and alpha-smooth muscle actin.

We have studied the expression of alpha-smooth muscle actin (alpha sm-1) by mesangial cells, and the expression of Thy-1 glycoprotein, antithrombin III (ATIII), and urokinase by tubular epithelial cells in normal kidneys and dysfunctional renal allografts. Kidney biopsies were studied immunocytochemically for changes in each of these markers and the findings were classified into two groups and compared with creatinine plasma levels at the time the biopsies were taken. In dysfunctional grafts, mesangial alpha sm-1 and tubular epithelial Thy-1 reactivities were greatly diminished, and urokinase and ATIII were missing from proximal renal tubular epithelial cells. Urokinase, which was absent from normal renal glomeruli, appeared in glomeruli of some dysfunctional allografts. The possible usefulness of these markers in patient evaluations was supported by our finding that the distribution of vinculin, fibronectin, myosin, actin B4, desmin, glomerular HLA-DR, and the tubular expression of CD15 remained unchanged. These data prompt us to suggest that the immunocytochemical localization and evaluation of alpha sm-1, Thy-1, ATIII, and urokinase in kidney allografts may be useful adjuncts in the assessment of function in renal allografts.

Vascular endothelial growth factor expression in transplanted human hearts.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen thought to play an important role in coronary collateral vessel formation. We used immunocytochemistry to determine VEGF expression in biopsies (n = 283) of transplanted human hearts (n = 109) with and without microvascular fibrin. Measures of vascular fibrin, alpha 2 plasmin-inhibitor (a2Pl), macrophages, neutrophils, and serum cardiac troponin T titers were used to evaluate myocardial damage. Antibody to T lymphocytes was used to evaluate cellular rejection, and HLA-DR, ICAM-1, and PAL-E antibodies were used to assess endothelial cell activation and phenotypic changes in the microcirculation. No VEGF immunoreactivity was detected in control donor hearts without fibrin, but the proportion of biopsies demonstrating VEGF immunoreactivity increased significantly in allografts with increasing fibrin and a2PI reactivity (P = 0.0001). VEGF immunoreactivity was confined to areas of fibrin deposition and was associated with infiltrates of macrophages and neutrophils (P < 0.0001), but not with T cells (P = 0.10). Biopsies with fibrin/VEGF reactivity were associated with increased capillary endothelial cell HLA-DR, ICAM-1, and PAL-E reactivity. In a subset of patients, serum cardiac troponin-T values were greater in patients with VEGF-positive (n = 21) than VEGF-negative (n = 19) biopsies (P = 0.05). Nested RT-PCR demonstrated that biopsies with and without fibrin/VEGF immunoreactivities expressed VEGF121, VEGF165, and VEGF189 variants, with VEGF165 being the dominate variant. These results indicate that endogenous VEGF is expressed locally following vascular thrombosis and myocardial cell damage, and that VEGF expression may be related to endothelial cell activation and phenotypic changes found in the microcirculation of cardiac allografts.

Tubular antithrombin at transplantation determines subsequent renal allograft function.

BACKGROUND: Antithrombin is found in the microvasculature and tubules of normal and transplanted human kidneys. Although depletion of vascular antithrombin is associated with renal allograft dysfunction, neither the distribution nor clinical significance of tubular antithrombin is known. METHODS: Changes in tubular antithrombin in biopsy specimens (n=41) obtained from donor kidneys at transplantation were studied immunohistochemically. The relationship between these changes and subsequent graft function was analyzed. RESULTS: Granular intracellular antithrombin was found only within the proximal tubular epithelium. Allografts with depleted tubular antithrombin at transplantation (n=20) had significantly greater plasma creatinine concentrations at posttransplant days 3 (P < 0.001) and 5 (P < 0.03) than allografts with normal tubular antithrombin (n=21). Indeed, depletion of tubular antithrombin at transplantation correlated with the degree of graft dysfunction at 3 days after transplantation. CONCLUSIONS: Depleted tubular antithrombin at transplantation is associated with reduced early graft function, and this may identify patients at risk of a complicated follow-up.