Exocytosis of cytolytic T-lymphocytes studied by cryofracture and stereophotogrammetry

Cryofracture, stereophotogrammetry and three-dimensional reconstruction were used to study the membranes of cytolytic T lymphocytes (CTL) and target cells (TC) conjugates. Circular bulges free of intramembrane granules and ring-shaped structures, analogous to the sites of vacuole release in secretory cells, appear on the surface of the lymphocyte plasmalemma after 30 to 60 minutes of interaction with TC. Polymorphic vacuoles 70 to 140 nm in diameter are observable in the contact space between lymphocytes and TC. Part of the vacuoles are found on the surface or near the CTL plasmalemma. The data confirm an assumption about the secretory mechanism of the cytolytic effect of T killers.     

Scanning electron microscopy of the oviduct of Salamandra salamandra (L.) (Amphibia, Urodela)

The oviduct of non-pregnant females of the ovoviviparous salamander, Salamandra salamandra, was examined using SEM-techniques. In the luminal epithelium polygonal ciliated cells were found along the entire surface of the oviduct, except the uterus, and non-ciliated cells with a varying number of short or long microvilli. The ciliated cells occur in the most anterior portion of the oviduct, the pars recta; they are sparsely distributed in the p. convoluta I, but abundant in the p. convoluta II and III. Non-ciliated cells comprise several small gland cells, restricted to the p. convoluta I, II, III, and undifferentiated cells both provided with microvilli, but difficult to be discerned from their surface appearance. The p. convoluta I, II, III is characterized by three types of secretory cells forming tubular glands, each type confined to a given zone. The secretory cells have slender microvilli at their surfaces. In freeze-cracked glands details of their secretory products can be visualized. The findings are compared to previously published TEM-investigations and discussed with regard to some functions of the oviduct during reproduction.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. III. Plasmalemma, "membranosomas".

Ultrastructure of the plasma membranes of cytolytical T lymphocytes (CTL) in their interaction with target cells (TC) was studied. Thirty to sixty minutes after the beginning of interaction shedding of the CTL plasma membrane was observed: its fragments shedded from a local (50–100 nm in diam.) area on the lymphocyte surface at the site opposite to the CTL contact region with TC. Oval structures of high electron density 10 to 40 nm in diam. were detected on the CTL surface. We designated them as “membranosomas” (MS). MS were in close apposition to the inner surface of the plasma membrane and showed projections of 2 to 3 nm in diam. and 5 to 6 nm long towards the outer surface of the plasma membrane. MS were separated from the CTL surface during clasmatosis or as component parts of “shedding” plasma membranes.     

Quantitative ultrastructure of cytolytic lymphocytes mediating allograft rejection in the mouse. I. Cellular alterations in T lymphocytes during specific target cell lysis

A quantitative ultrastructural analysis of cytolytic T lymphocytes (CTL) is presented which allows both the distinction of these cells from normal T lymphocytes and permits the demonstration of ultrastructural alterations of putative CTL following interaction with target cells (TC). Alloreactive CTL were generated in C57BL/10 mice receiving intraperitoneal fibroblastic allografts and target-binding splenic lymphocytes (TBSL) were concentrated by specific immunoadsorption on fibroblast monolayers. TBSL were subjected to ultrastructural quantification either at the onset of TC interaction or following 30 or 60 min incubation at 37 degrees C. By means of simple stereological relationships it was shown that, in comparison with normal, non-cytolytic splenic T lymphocytes, TBSL were slightly larger cells, displaying around 60% more cytoplasm, a similarly-sized nucleus and approximately triple the volume of Golgi apparatus. During the first 30 min of interaction with TC, the target binding surface of the TBSL plasma membrane decreased in area. This change was accompanied by a polarization of the TBSL towards the target. Incubation of lymphocytes with TC for a further 30 min resulted in a general polarization of lymphocytic cellular constituents away from the TC. These results were only attainable by objective quantitative analysis and are discussed in relation to possible mechanisms of CTL-mediated lysis.     

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.     

Colocalization of cytokeratin 18 and villin in type III alveolar cells (brush cells) of the rat lung

Alveoli of the rat lung are lined by three different cell types, the flat type I cells and the cuboidal type II and type III cells. Type III cells differ from type II cells by the presence of an apical tuft of microvilli and the absence of lamellar type secretory granules. In the present study we show by double immunolabelling that type III cells of the rat lung can be identified at the light-and electron microscope level by antibodies against both cytokeratin 18 and the actin-crosslinking protein villin. At the ultrastructural level, microvilli and their rootlets in the apical cytoplasm were labelled by the anti-villin antibodies, whereas a monoclonal antibody against cytokeratin 18 (Ks18.04) labelled bundles of intermediate filaments. In conclusion, antibodies against villin and certain monoclonal antibodies specific for cytokeratin 18 can be used as tools for selective visualization of type III cells in the rat lung.     

Fine structure of the accessory glands of the female genital tract of the ichneumonid Pimpla turionellae (Insecta,Hymenoptera)

Summary The female accessory glands include the tubular poison gland, the paired, lemon-shaped uterus glands, and Dufour's gland, an unbranched tubular organ. They consist essentially of a single layer of epithelium cells surrounded by a basement membrane. The lumen is lined by cuticle. The proteinaceous secretion of the poison gland is released into intracellular ducts provided with microvilli, each connected to a channel lined with cuticle which leads to the central lumen of the gland. The channel is formed by special canal cells. Nerve endings are interspersed among the gland cells. The uterus gland consists of four cell types derived from a single type of precursor cell found in newly hatched wasps. Type I cells are covered by type II cells and are thus without contact to the luminal surface of the gland. They contain stacks or whorls of mitochondria and smooth cisternae in an alternating arrangement. Vesicles with a secretory product are found in cells of types II and III. Deep anastomosing infoldings of the plasmalemma, stabilized by microtubules and dense material at the branchings, are characteristic for type II cells. Most secretory vesicles are found in type III cells, the prevalent cell type which is thought to be the source of the lipoprotein secretion. Coated vesicles are present at deep infoldings of the plasmalemma. The greatly enlarged apical surface area of type IV cells and the presence of mitochondria in slender outgrowths is suggestive of an osmoregulatory function. In Dufour's gland, two cell types appear in succession, the first with a very dense cytoplasm, the second with dense inclusions and many seemingly empty vesicles of smooth endoplasmic reticulum. The secretion products, lecithin and a cholesterol ester, are thought to be formed by the second cell type. The dense inclusion might be lecithin, which reacts with osmium tetroxide. The cholesterol ester could have been washed out of the empty vesicles by the embedding procedure.     

Spatial distribution of L-selectin (CD62L) on human lymphocytes and transfected murine L1-2 cells

Summary We have examined the topographical distribution of L-selectin on surface membrane domains of human lymphocytes and murine L1-2 cells transfected to express human L-selectin. L-selectin was immunolocalized using murine monoclonal DREG 200 Fab antibody and a 12 nm colloidal gold-conjugated secondary antibody. Cell surface morphology and surface distribution of gold-labelled L-selectin were visualized using backscatter electron images obtained by high-resolution, field emission scanning electron microscopy. The topographical morphologies of lymphocytes of both types were complex. The surface of human lymphocytes was composed of both microvilli and ruffles; that of the murine cells was composed of long microvilli and few, if any, ruffles. L-selectin on human lymphocytes was observed primarily as focal clusters on the apical surfaces of ruffles and microvilli. Similarly, on the transfected murine cells, L-selectin was detected predominantly on the apical surface of microvilli. We conclude that L-selectin has a common spatial distribution and clustered organization on all leukocytes examined to-date, and that these features of receptor expression likely facilitate rolling of circulating leukocytes on the endothelial surface.     

Ultrastructure of antibody dependent cellular cytotoxicity in HSV 1 infected and uninfected Chang liver cells

The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Ultrastructural characteristics of the salivary glands of the taiga tick, Ixodes persulcatus (Ixodidae). I. The granulosecreting alveoles of the fasting female

Two types of granulosecreting alveoles were found in salivary glands of hungry females by means of electron microscopy of ultrafine sections. Alveoles of the IInd type occur in the anterior helf of the gland. They are not numerous and consist of three types of secretory cells (A, B, C) surrounding the inneralveolar cavity. The secretory cells are separated from each other and from the basal membrane by the strands of the epithelial cells P. Three types of spherical inclusions were found in the secretory cells. They differ in size, electron density and intensity of staining of half-fine sections with toluidin blue. The apical cytoplasmatic membrane of secretory cells bears numerous microvilli. Alveoles of the IIIrd type, which constitute the main mass of the gland tissue, have a narrow slit-like inneralveolar cavity. The basal part of the alveole is formed by 3--4 large cells filled with large spherical electron-transparent vacuoles of the secretion. The apical part of the alveole is occupied by 9 to 11 cells E, whose cytoplasm is filled with numerous flat cisternae of granular endoplasmatic reticulum and small and medium secretory vacuoles of different electron density. Alveoles of the IInd and IIIrd type of I. persulcatus are not identical with those of Hyalomma asiaticum, Boophilus microplus and other members of the subfamily Amblyomminae.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Cell shape and plasma membrane alterations after static magnetic fields exposure

The biological effects of static magnetic fields (MFs) with intensity of 6 mT were investigated in lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs by transmission (TEM) and scanning (SEM) electron microscopy. Lectin cytochemistry of ConA-FITC conjugates was used to analyze plasma membrane structural modifications. Static MFs modified cell shape, plasma membrane and increased the level of intracellular [Ca++] which plays an antiapoptotic role in both cell types. Modifications induced by the exposure to static MFs were irrespective of the presence or absence of apoptotic drugs or the cell type. Abundant lamellar-shaped microvilli were observed upon 24 hrs of continuous exposure to static MFs in contrast to the normally rough surface of U937 cells having numerous short microvilli. Conversely, lymphocytes lost their round shape and became irregularly elongated; lamellar shaped microvilli were found when cells were simultaneously exposed to static MFs and apoptosis-inducing drugs. In our experiments, static MFs reduced the smoothness of the cell surface and partially impeded changes in distribution of cell surface glycans, both features being typical of apoptotic cells. Cell shape and plasma membrane structure modifications upon static MFs exposure were time-dependent. Lamellar microvilli were clearly observed before the distortion of cell shape, which was found at long times of exposure. MFs exposure promoted the rearrangement of F-actin filaments which, in turn, could be responsible for the cell surface modifications. Here we report data that support biological effects of static MFs on U937 cells and human lymphocytes. However, the involvement of these modifications in the onset of diseases needs to be further elucidated.     

Morphology of rabbit collecting duct.

Recently the assumed structural and functional homogeneity of the collecting duct (CD) has been questioned. The objective of this study was to determine if heterogeneity occurs in luminal surface membrane structure or in cytoplasmic configuration of cells in the collecting duct or both. Straight segments of cortical and medullary CD were examined in perfusion-fixed rabbit kidneys with scanning electron microscopy (SEM), light (LM) and transmission electron microscopy (TEM). Principal cells were the most abundant cells in all CD regions; intercalated cells comprised 37% of the cell population on the cortex, 18% in the outer medulla, and less than 1% in the inner medulla. SEM revealed two surface patterns among the ciliated principal cells: 1, located in the cortex and outer medulla, with few surface microvilli, and 2, located in the inner medulla, with abundant microvilli. Intercalated cells exhibited four distinctive luminal surface configurations: I, numerous short microvilli; II, both short and elongate microvilli; III, microplicae alone; and IV, both microvilli and microplicae. Intercalated cells with patterns I and II were predominant in the cortex, while cells with patterns III and IV were most common at the corticomedullary junction. TEM confirmed that marked variation existed in cytoplasmic structures of both principal and intercalated cells. These findings may either indicate the presence of several specific types of principal and intercalated cells or reflect different functional states of the principal and intercalated cells. Regardless of their significance, their presence must be considered in studies seeking to establish precise structural-functional relationships in this region of the rabbit renal tubule.     

Contact of Chlamydophila pneumoniae with type II cell triggers activation of calcium-mediated NF-kappa B pathway

Nuclear factor-kappa B (NF-kappa B) plays an important role in inflammation, proliferation and regulation of apoptosis. The purpose of the present study on type II cells was to investigate whether Chlamydophila pneumoniae contact induces (I) a Ca2+ release, that (II) disrupts F-actin/beta-tubulin cytoskeletal association with NF-kappa B/I kappa B alpha, leading to (III) a subsequent NF-kappa B activation. Incubation of rat type II pneumocytes with C. pneumoniae caused an intracellular calcium release within seconds. Confocal laser scanning microscopy (CLSM) revealed that bacterial contact with cell surface leads to a disappearance of the microvilli and disturbs the co-localization between F-actin and NF-kappa B (p65). Using semi-quantitative CLSM, we show that at 10-30 min I kappa B alpha was decreased and p65 or p50 was simultaneously translocated from cytoplasm to the nucleus, resulting in a 19-fold and 17-fold increase versus control cells. During this time no bacteria were internalized into type II cells. The pre-treatment of cells with BAPTA-AM inhibited C. pneumoniae-mediated calcium release. BAPTA-AM or SN50 prevented the C. pneumoniae-induced changes in F-actin cytoskeleton and inhibited NF-kappa B activation. Paclitaxel reduced C. pneumoniae-mediated changes of beta-tubulin cytoskeleton and activation of NF-kappa B. These results suggest that calcium-mediated cytoskeleton reorganization is involved in C. pneumoniae-induced NF-kappa B activation in type II cells.     

Ultrastructural study on cocoon formation in the freshwater oligochaete,Tubifex hattai

The clitellar epithelium of the freshwater oligochaete, Tubifex hattai, is composed of four types of gland cells (Type I, II, III, and IV), in addition to the cells generally found in the epidermis of this worm. The possible function of these gland cells in cocoon formation was studied with the electron microscope. Type I cells discharge their secretory granules by means of compound exocytosis and provide the materials for the future cocoon membrane. Immediately after completion of the discharge from Type I cells, Type II and III cells simultaneously discharge their secretory granules by means of compound exocytosis. The secretions from Type II cells constitute a colloid in the cocoon lumen and probably cause structural modifications in the future cocoon membrane. The secretory products from Type III cells form the cocoon plug. Although the process of discharge of secretory granules from Type IV cells was not observed, the contribution of these cells to the cocoon formation, producing hoops on the outer surface of the future cocoon membrane and fixing its anterior ends on the clitellum, is inferred from a morphological comparison of the hoop and the structure of the secretory granules.     

Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL- TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.     

An ultrastructural study of the clitellar epithelium of the earthworm Metaphire posthuma (vail.)

The ultrastructure of clitellar epithelium of Metuphire posthuma revealed mainly three types of secretory cells. Most prominent among these are the large slender granular cells which contain a large number of secretory granules filling in the entire columncr region of the cell. The secretory granules are 2-4mu in diameter with a limiting membrane and containing numerous tiny vesicles in a matrix of varying electron density. Basolateral rough endoplasmic reticulum and extensive Golgi cisternae were seen interspersed with the secretory granules. The Golgi cisternae in these cells were quite prominent extending all around the secretory granules. The secretory granules of type 2 cells are spheroid bodies with motley appearance due to varying electron density of the matrix. The immature granules contain fibrillar material. Type 3 cells contained electron lucent membrane-bound mucous like secretory granules which are reticulated with filamentous materials. All the three cell types open to the exterior at the cuticular region which is characterised by the presence of numerous microvilli.     

The role of physical forces on cytotoxic T cell-target cell conjugate stability

Theoretical considerations suggest that external forces play a significant role in cell-cell conjugate formation and may lead to the misinterpretation of adhesion data. To test this, the stability of conjugates formed between CTL and fibroblast target cells (TC) was examined in the controlled shear environment of a parallel plate flow chamber. Murine fibroblast targets expressing class I maternally transmitted Ag Mtaa or Mtab were grown on a glass slide that formed one wall of the flow chamber and were used in conjunction with anti-Mtaa and anti-Mtab specific mouse CTL clones to establish a panel of Ag-reciprocal targets and lymphocytes. Although cytolysis assays indicated that lymphocytes recognized and destroyed appropriate but not inappropriate targets, the stability of some CTL/TC conjugates was Ag independent. In all cases, the conjugate stability was shear dependent over a 100-fold range (0.04 to 4.0 dynes/cm2). For some clones, the ratio of the stabilities of Ag-specific CTL/TC conjugates to nonspecific conjugates was significantly enhanced with increasing shear. This implies that the role of Ag specificity in CTL/TC adhesion may be misinterpreted if the shear environment of CTL/TC conjugates is unknown or uncontrolled. Kinetic analysis revealed that conjugate stability was dependent on the exposure time to external forces and that there existed two populations of conjugates; weak associations that disengaged within the first 30 s of flow, and strong associations that remained attached even after a 5-min exposure to a steady shear stress. The stability of Ag-specific CTL/TC conjugates at 0.04 dynes/cm2 was enhanced by 50% as the temperature was increased from 25 to 37 degrees C, whereas the stability of nonspecific CTL/TC associations was not affected. This result indicates that significant Ag-specific strengthening may occur at physiologic temperatures. This work suggests the importance of attention to role of fluid mechanical shear stress in standard adhesion assays.     

Fine structure of the midgut of Sinopanorpa tincta (Navás) (Mecoptera: Panorpidae)

Fine structure of the midgut and degeneration of the midgut epithelium of the scorpionfly Sinopanorpa tincta (Navás) adults were investigated using light microscopy and scanning and transmission electron microscopy. The results show that the tubular midgut lacks gastric caeca and is composed of an outer longitudinal and an inner circular muscle layer, a basal lamina, an epithelium and a lumen from the outside to inside. A peritrophic membrane was not found in the lumen. A mass of nodules was observed on the surface of the basal lamina. Three types of cells were recognized in the epithelium: digestive, secretory, and regenerative cells. The digestive cells contain irregular-shaped infoldings in the basal membrane and two types of microvilli in the apical membrane. The secretory cells are characterized by irregular shape and large quantities of secretory granules in the basal cytoplasm. The regenerative cells are triangular in shape and distributed only in the nodules. The epithelial cells are degenerated through programmed cell-death mechanisms (apoptosis and necrosis). The type, function, and degeneration of the epithelial cells of the midgut are briefly discussed.