Characterization of tubular basement membrane antigens in human kidney

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.     

Expression of epithelial membrane antigen on malignant mesothelioma cells. An immunocytochemical and immunoelectron microscopic study

The presence of epithelial membrane antigen (EMA) on malignant mesothelial cells found in pleural and ascitic fluids was demonstrated immunocytochemically using a monoclonal anti-EMA antibody. Serous fluids of 25 patients with malignant mesotheliomas were investigated. In 23 cases, varying numbers of EMA-positive tumor cells were present; in 2 cases, no such cells were found. Immunoelectron microscopy was performed both on Lowicryl-embedded sediments of serous fluids and by application of preembedding techniques using the immunogold method. Expression of EMA by the immunogold method was found selectively on the villi of the malignant mesothelioma cells whereas the nonvillous, flat surfaces were largely EMA-negative. The results indicate that immunoelectron microscopy may offer a useful adjunct in the diagnosis of malignant mesothelioma in serous fluids.     

The basement membrane of the atrophic kidney tubule. An electron microscopic study of changes in rats.

In a light and electron microscopic study of the rat's kidney after obstruction of the renal vein or after subtotal nephrectomy the tubular basement membrane changes occurring during the ensuing atrophy of the tubules have been examined. Characteristically, they consist of diffuse widening, focal thickening with vesicular and granular inclusions, circumscribed dissolution, or reduplication of this structure. These observations have led to the conclusion that the tubular basement membrane is formed by both interstitial and tubular cells, although the interstitial cells contribute the greater share to its formation, maintenance and renewal. The thickening of the basement membrane taking place in tubular atrophy must, however, be attributed entirely to the interstitial cells.     

Alpha-actinin and actin in the outer retina: a double immunoelectron microscopic study

Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.     

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.     

Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis.     

Nuclear origin of progesterone receptor of the chick oviduct cytosol. An immunoelectron microscopic study

Progesterone receptor (PR) was studied immunoelectron microscopically from fixed vibratome sections of the chick oviduct and biochemically from the fractionated oviduct homogenate. Immunoelectron microscopically both unoccupied and occupied PR were localized inside the nuclei. Only a few cells showed PR immunoreactivity in the endoplasmic reticulum which probably represents newly synthetized PR. Biochemically unoccupied PR was in the cytosol fraction. The cytosol and nuclear PR as well as the non-transformed 8S-form and the transformed 4S-forms of cytosol PR were recognized by the anti-PR antibody (IgG-RB). The lack of PR immunostaining in the cytoplasm is therefore not due to lack of recognition by IgG-RB. We propose that in the chick oviduct progesterone receptor is a nuclear protein but synthetized in the endoplasmic reticulum.     

Synthesis of biotinylated glyfoline for immunoelectron microscopic localization

Antineoplastic glyfoline (1) has potent antitumor efficacy against various murine and human solid tumors. To elucidate the actual mechanism of action, we synthesized biotinylated glyfoline (B-Gly) and used it for the visualization of glyfoline-binding sites in nasopharyngeal carcinoma (NPC) cells. Under the electron microscope (EM), after cells were incubated for 6-36 h, the reaction products of anti-B-Gly were seen on some areas of the external cell surface and on the outer and inner membranes of the mitochondria. Pure EM morphology of NPC cells after glyfoline treatment revealed the similar morphological change of mitochondria. These findings indicate that the binding site of glyfoline in NPC is the inner membrane of the mitochondria, suggesting that B-Gly can be used as a marker for glyfoline localization.     

Immunoglobulin A (IgA) polymerization sites in human immunocytes: immunoelectron microscopic study

The cytoplasmic affinity of polymeric IgA for secretory component (SC) and the expression of joining (J) chain were examined in pokeweek mitogen (PWM)-stimulated human peripheral blood lymphocytes (PBL) to determine, on the ultrastructural level, the polymerization sites of human IgA. SC-binding was found in 5.7% of transformed PBL on day 7 of culture; SC-binding was observed in a high proportion of IgA-producing cells. A low proportion of IgM-producing cells also bound to SC, while there was virtually no SC-binding by IgG-producing cells. A high proportion of IgA- and IgM-producing cells expressed intracellular J chain, while approximately half of the IgG-producing cells were positive for J chain. The number of J chain-positive cells exceeded the number of SC-binding cells among transformed PBL on day 7 of culture. Immunoelectron microscopic study of the sites of SC-binding, and of IgA and J chain expression, revealed that polymerization of human IgA and the addition of J chain occur in the perinuclear space and endoplasmic reticulum, prior to immunoglobulin secretion.     

Evaluation of basement membrane degradation during TNF-alpha-induced increase in epithelial permeability

We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 +/- 0.04 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 1.47 +/- 0.05 x 10(-6) cm/s, P < 0.05). The permeability increase in the TNF-alpha-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 +/- 0.07 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 2.82 +/- 0.02.10(-6) x cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 microg/ml TIMP-1 or 10(-6) M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha-induced increase in alveolar epithelial monolayer permeability.     

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.     

Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.     

alpha 2-macroglobulin traps a proteinase in the midregion of its arms. An immunoelectron microscopic study

alpha 2-Macroglobulin, one of the major plasma proteinase inhibitors with Mr = 720,000, is known to inhibit proteinases of all four classes through the "trap mechanism" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724), but the proteinase binding site of alpha 2-macroglobulin has not been identified precisely. We localized bound proteinase molecules on the electron microscopic images of alpha 2-macroglobulin, using anti-proteinase IgG. Serratial Mr = 56,000 proteinase produced by Serratia marcescens was chosen as the antigenic probe in this study because its affinity to specific antibodies was retained in its bound state to alpha 2-macroglobulin. Dimers of alpha 2-macroglobulin/Mr = 56,000 proteinase complexes cross-linked with anti-Mr = 56,000 proteinase IgG were prepared and subjected to electron microscopic observations. The electron microscopic image of alpha 2-macroglobulin complexed with Mr = 56,000 proteinase had four straight arms with an overall shape looking like the character "H." From the way anti-Mr = 56,000 proteinase IgG linked two alpha 2-macroglobulins, it was concluded that the proteinase existed in the midregion of one of the arms. This result helps us to form a more concrete view of the trap mechanism in that one of the arms of alpha 2-macroglobulin wraps the trapped proteinase and holds it isolated from high molecular weight substrates in the surrounding medium.     

抗体形成细胞发育的免疫电镜研究

The pre-embedding immunoelectron microscopic method was used to study the development of antibody-producing cells in the guinea pig popliteal lymph nodes of 2, 3, 5, 8 and 10 days after a second challenge with horseradish peroxidase. The results indicated that the antibody activity was located in the perinuclear space, the endoplasmic reticulum and Golgi complex. According to the cellular developmental stages and the characteristics of distribution of the antibody activity, the antibody-producing cells (APC) were divided into four types: (1) Type I cells (lymphocytes) exhibited many positive granules throughout the cytoplasm; (2) Type II cells (proplasmacytes) contained many positive granules and positive short bars, some of them were parallel; (3) Type III cells (proplasmacytes) contained numerous parallel positive lamellae in cytoplasm; (4) the parallel lamellae in cytoplasm of type IV cells (plasmacytes) were arranged into a network-endoplasmic reticulum. According to the kinetic change from granules, short bars to parallel lamellae and the network, the results indicated the developmental course of AFC from lymphocytes, proplasmacytes to plasmacytes.     

Purification of Drosophila hsp 83 and immunoelectron microscopic localization

Heat-shock protein (hsp) 83 was purified from Drosophila culture cells. Analysis by gel filtration revealed that this hsp exists in a dimeric form under nondenaturing conditions. Monoclonal and polyclonal antibodies produced against this hsp have been used to determine its intracellular localization by indirect immunofluorescence and immunogold electron microscopy in normal cells, after heat shock, during recovery and after a second heat shock. Under normal conditions, hsp 83 is predominantly cytoplasmic. Immunogold labeling reveals that this hsp is associated with vacuole-like structures containing numerous dense bodies. In addition, hsp 83 is detected, albeit at a lower level, in the nucleus where it is found within the network of perichromatin ribonucleoprotein (RNP) fibrils. This distribution changes during heat shock: hsp 83 is then found in increased concentrations at the cell periphery close to the plasma membrane. After a recovery period, hsp 83 appears associated with the nuclear membrane and/or with the neighboring endoplasmic reticulum. Following a second heat shock at 37 degrees C after recovery, a renewed deposition of hsp 83 is observed at the cell periphery. A small population of cells also shows an increased concentration of this protein in the nucleus. This intracellular distribution of hsp 83 is consistent with its reported association with various cellular proteins and suggest that this hsp may be involved in their intracellular transport and/or in the modulation of their activity.     

Immunoelectron microscopic study of the insoluble antigens of bone marrow cells in rats

An immunoperoxidase study of the localization of insoluble antigens was carried out on the rat bone marrow cells. The effect of different fixatives and inhibitors of endogenous peroxidase on the cell ultrastructure and the preservation of immunoreactivity of the cell antigens. The best results were obtained while fixing with 1% paraformaldehyde and 0.05% glutaraldehyde mixture added with 0.5 saponin and using 10% acetic acid as an inhibitor of endogenous peroxidase. Differences were found in the localization of antigens and the intensity of immunoperoxidase staining in cells of different lines of differentiation and degree of maturity.     

Effects of light deprivation on prolactin cells in golden hamsters: an immunoelectron microscopic study.

In the golden hamster light deprivation has been shown to induce gonadal regression and reduction of pituitary and plasma levels of prolactin (PRL). In the present study we examined changes in morphology and population ratios of three types of PRL cells 8 weeks after light deprivation, by means of blinding or exposure of hamsters to continuous darkness. In the pituitary of intact hamsters of either sex, which were entrained to a 14-h light: 10-h dark cycle, Type C cells with large secretory granules were the most numerous and Type A with smaller granules the least. After light deprivation the pituitary was found to contain remarkably atrophic PRL cells and showed a profound change in population ratio of PRL cell types, i.e., Type A cells prevailed over the other two types. Pituitary glands from light-deprived and concurrently pinealectomized hamsters exhibited structures and a population ratio of three types of PRL cells similar to those from intact animals. It is suggested that small-granule-containing PRL cells represent an inactive stage of PRL cells, whereas medium- and large-granule-containing cells are functionally active cells. The atrophy of PRL cells can account for the decreased pituitary level of PRL in light-deprived hamsters reported previously.     

Heterogeneity of rat macrophages recognized by monoclonal antibodies: an immunohistochemical and immunoelectron microscopic study

Three monoclonal antibodies, designated RM-1, TRPM-1, and TRPM-2, were raised against rat peritoneal macrophages. By the immunoperoxidase method, antigens recognized by these antibodies were distributed throughout most tissue and free macrophages examined, including those of splenic red pulp, lymphatic sinus, connective tissue, and peritoneal cavity, as well as Kupffer cells of liver and alveolar macrophages. The numbers of positive cells were different for each antibody. RM-1 and TRPM-1 were also reactive with interdigitating cells (IDCs) in the thymus-dependent area and with Langerhans cells in the skin, whereas TRPM-2 failed to demonstrate IDCs in thymic medulla and Langerhans cells. The reactions of each antibody were observed by immunoelectron microscopy in the different ultrastructural compartments of the cells. RM-1 recognized a cell surface antigen; reaction products for TRPM-1 were found on a part of the cell membrane and in the cytoplasmic vacuoles; and those of TRPM-2 were present along the nuclear envelope and intracytoplasmic vacuoles. These antibodies seem to be useful not only for the detection of macrophages in tissue sections but also for investigation of macrophage heterogeneity in different tissues.     

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.