Immunohistochemical characteristics of chicken spleen ellipsoids using newly established monoclonal antibodies

Ellipsoids, the extra-vasculature sites surrounding penicilliary capillaries of the chicken spleen, play critical roles in the immune response and also in the clearance of pathogens or other particles. The meshwork of ellipsoids is formed by fibroblastic reticular cells. To characterize ellipsoidal reticular cells, a series of monoclonal antibodies against the chicken spleen have been developed. Of these antibodies, CSA-1 antibody reacts with fibroblastic reticular cells in ellipsoids and with endothelial cells. The reticular nature of positive cells in ellipsoids is indicated by immuno-electron microscopy, and by double-staining with anti-heat-shock protein 47 kDa (hsp47) antibody. The reaction of CSA-1 with reticular cells is limited in ellipsoids; CSA-1 does not react with reticular cells in other lymphoid organs. These findings indicate that ellipsoidal reticular cells share the antigen with endothelial cells. Ontogenic studies reveal that, on embryonic day 18, the development of ellipsoids is completed, penicilliary capillaries become fenestrated, and CSA-1 expression in ellipsoids begins. These findings suggest that CSA-1 is expressed on the cell surface of ellipsoidal reticular cells once they are exposed to blood flow.     

Ultrastructural and histochemical investigation of the terminal capillaries in the spleen of the carp (Cyprinus carpio L.)

Summary In the spleen of the carp arterial capillaries of a highly differentiated structure have been studied by light and electron microscopy. These capillaries share various structural characteristics with the sheathed capillaries (ellipsoids of Schweigger-Seidel) of higher vertebrates. The long arterial capillaries of the carp spleen are provided with cuboidal endothelial cells containing filaments approximately 7 nm in diameter. There is no basal lamina. The endothelial cells form various types of cell junctions, but there are also extensive areas without any junctions. Here, a free passage is possible between the capillary lumen and the subendothelial space. The capillaries possess a single-layered sheath of macrophages. Characteristically, the sheath macrophages possess long and slender cell processes forming a loose framework, the meshes of which are filled with lymphocytes and spindle cells. The sheath macrophages show a zone of ectoplasm rich in filaments. They also contain numerous phagolysosomes rich in hydrolytic enzymes, as identified histochemically. The sheath is sharply limited against the pulp by a thick layer of collagen fibers.     

Macrophages and Reticulum Cells in the Spleen of the Dogfish,Scyliorhinus canicula

The presence and ultrastructural features of reticulum cells and macrophages were studied in the spleen of the dogfish Scyliorhinus canicula. Three morphologically distinguishable regions of the spleen were identified: the white pulp, the red pulp and the ellipsoids. In all three, the splenic parenchyma was a meshwork supported by reticulum cells and fibres. Reticulum cells in both the white and the red pulp are irregular elements, the processes of which are joined by cell junctions and embrace developing reticular fibres. The ellipsoids of the dogfish spleen are terminal branches of the splenic arteries of the white pulp, with a sheath consisting of reticulum cells, reticular fibres, ground substance, macrophages and occasional lymphocytes. Isolated melanomacrophages also occur in the ellipsoid walls as well as in the red pulp. In both the white and the red pulp phagocytic reticulum cells, and macrophages appear frequently forming cell associations with surrounding blood cells, mainly lymphocytes. The functional significance of the ellipsoids and the cell-cell clusters of the white and the red pulp is discussed in relation to the immune capacities demonstrated in elasmobranchs.     

Vascular permeability (problem of the blood-brain barrier) in the pineal organ of the rainbow trout,Salmo gairdneri

Summary The problem of the blood-brain barrier in the pineal organ of the rainbow trout, Salmo gairdneri, was investigated following intraperitoneal or intracardial injections of several tracers and dyes with different molecular weights. As demonstrated at the light-microscopic level, repeated injections of trypan blue or horseradish peroxidase (HRP) resulted in an accumulation of these substances in the pineal epithelium (parenchyma). By use of the electron microscope, HRP was found in electron-dense bodies, probably lysosomes, in (i) the endothelial cells and perivascular macrophages 4 h after intraperitoneal injection, (ii) the supporting cells and intrapineal or luminal macrophages 8 h after injection, and (iii) the receptor cells 24 h after injection of the tracer. Ferritin particles penetrated the fenestrated endothelium of pineal capillaries. They were confined to vesicles, vacuoles and the smooth endoplasmic reticulum of the supporting cells as well as to the synaptic vesicles and the smooth endoplasmic reticulum of the pineal photoreceptors. The intercellular passage of tannic acid mixed with the fixative was blocked at the luminal junctional complex separating the pineal lumen from the basal portion of the pineal epithelium. The passive intercellular transport of substances with high molecular weight from the bloodstream to the cerebrospinal-fluid compartment is thus prevented. However, no blood-brain barrier exists for exogenously administered proteins, which are rapidly taken up by pineal cells and actively transported in a transcellular manner.The findings on the blood-brain barrier of the pineal organ of the rainbow trout are discussed with particular reference to the endocrine capacity of pineal sensory organs.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Ultrastructure and adrenergic innervation of preglomerular arterioles in the euryhaline teleost,Salmo gairdneri

Summary The opisthonephric kidney of the rainbow trout was investigated by light- and electron microscopy and a fluorescent-histochemical technique for biogenic amines was used. Preglomerular sphincters at the origin of afferent arterioles are present in this euryhaline teleost. The branching point of the afferent arteriole is characterized by (i) the formation of a right angle with the parent vessel, (ii) circularly arranged smooth muscle cells of the tunica media, (iii) additional circularly arranged smooth muscle cells intercalated between endothelium and tunica media, and (iv) a collar-like arrangement of several large endothelial cells with elaborate marginal folds and abundant myoendothelial junctions. A dense adrenergic innervation displaying specific fluorescence was found along the terminal arterioles and afferent arterioles, and conspicuously at the preglomerular sphincters. These results are suggestive of a neural participation in kidney function. They are discussed on the basis of recent evidence from pharmacological and physiological experiments for neural involvement in glomerular intermittency.     

Cytochemical demonstration of acid phosphatase activity in the pineal organ of the rainbow trout,Salmo gairdneri

Summary Activity of acid phosphatase (ACP) was investigated cytochemically in the pineal organ of the rainbow trout, Salmo gairdneri. Intense reaction product for ACP activity was observed (1) in lysosomes varying in size and shape and (2) in endoplasmic reticulum associated with the Golgi complex of (i) the pineal photoreceptor and supporting cells, (ii) vascular endothelial cells, and (iii) macrophages inhabiting pineal lumen, parenchymal epithelium and perivascular spaces. This localization of ACP is discussed with particular reference to the capacity for lysosomal digestion in a pineal organ combining photoreceptive and secretory functions, and lacking a blood-brain barrier, as holds true for the pineal of the rainbow trout. Taking advantage of its capacity for endocytotic uptake and lysosomal digestion, the pineal organ of the rainbow trout may serve as a barrier between the blood circulation and the cerebrospinal-fluid compartment. Furthermore, the macrophages may be considered as an essential component in pineal function of fish.Fellow of the Alexander von Humboldt Foundation.Fellow of the Alexander von Humboldt Foundation.     

Immune- and enzyme histochemical characterisation of leukocyte populations within lymphoid and mucosal tissues of Atlantic halibut (Hippoglossus hippoglossus)

Leukocyte populations within the kidney, spleen, posterior intestine and gills of Atlantic halibut were investigated using a panel of histological, enzyme- and immunohistochemical methods. In the kidney and spleen, a diverse population of leukocytes was associated with the extensive network of sinusoids and larger blood vessels present in these tissues. IgM+ cells (B-cells, plasma cells and IgM-bearing macrophages) and large mononuclear cells showing reactivity for non-specific esterase (NSE) and acid phosphatase (ACP), representing macrophage populations, were often associated with vessel walls that were also the site of trapping of fluorescent microspheres. In the kidney, trapping of 0.1 and 0.5 microm diameter microspheres occurred at these sites but in the spleen, the 0.1 microm diameter microspheres were retained in ellipsoids. The lymphoid tissues of the kidney and spleen possessed a spread population of 5'-nucleotidase+ (5'N+) cells but compartmentalisation of the splenic white pulp was suggested by an absence of these 5'N+ reticular cells in areas associated with melanomacrophage accumulations and in areas rich in IgM+ cells. A striking feature of the mucosal tissues was the diversity of leukocyte populations within the epithelium particularly of the posterior intestine, including IgM+ cells and NSE+, ACP+ and 5'N+ mononuclear cells. Although limited in numbers in the posterior intestine, IgM+ cells were more common in the epithelium than in the lamina propria. In the gills, leukocytes as detected by enzymatic reactivity were scarce, but IgM+ cells were very abundant in the stratified epithelium of the gill arch and filaments. The difference in distribution of these leukocyte populations between the intestines and gills suggested a compartmentalisation of the mucosal immune system and the need to assess the immunological competence of mucosal tissues in Atlantic halibut.     

Formation of virion-occluding proteinic spindles in a Baculovirus disease of Aedes triseriatus

An unusual process of inclusion formation was studied in Aedes triseriatus larvae infected with a Baculovirus (BV) similar to the nuclear polyhedrosis virus (NPV) type. In this disease virion-occluding proteinic inclusions initially developed individually. However, as the disease progressed the proteinic inclusions gradually coalesced eventually forming large rugose ellipsoids and finally, large smooth-surfaced spindles. Nuclei in late stages of infection usually contained two to five rugose ellipsoidal inclusions, frequently measuring 5 μm to 7 μm in diameter by 10 μm to 15 μm in length. The ellipsoidal forms exhibited different chemical behavior from the spindles.     

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

Morphological analysis of rat ureteric terminal arterioles in situ

Confocal imaging of Fluo‐4, Propidium iodide, and di‐8‐Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 μm in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin‐eosin, Masson's tri‐chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

Glomerular ultrastructure of the trout,Salmo gairdneri: Effects of angiotensin II and adaptation to seawater

Summary The effect of angiotensin infusion on the glomerular ultrastructure of freshwater- and seawater-adapted rainbow trout, Salmo gairdneri, has been examined by scanning and transmission electron microscopy. Adaptation of trout to seawater resulted in epithelial podocyte flattening, primary process broadening and apparent loss of foot processes in almost all glomeruli, features which were uncommon in freshwater-adapted trout. Similar changes were induced by infusion of freshwater-adapted animals with angiotensin, suggesting that the renin-angiotensin system plays a role in the modification of glomerular epithelial ultrastructure. Adaptation of trout to seawater also reduced glomerular diameter, but infusion of freshwater-adapted animals with angiotensin did not mirror this effect. Infusion of angiotensin into seawater-adapted animals increased the overall thickness of glomerular basement membrane by increasing the lamina rara interna and lamina densa. This did not occur when freshwater-adapted fish were either infused with angiotensin or adapted to seawater. These findings suggest that other humoral systems are involved in the control of glomerular diameter and basement membrane thickness as part of an integrated response to increased environmental salinity.     

Scanning- and transmission electron-microscopic study of lymphatic vessels in the splenic white pulp of the macaque monkey

Summary The fine structure of the lymphatic vessels in splenic white pulp of the macaque monkey was studied by scanning and transmission electron microscopy.Lymphatic vessels were slit-like or widened channels which extended along central arteries and their large branches. The walls of the vessels were very thin in comparison with those of nearby arteries. They were composed only of a layer of endothelium supported by underlying reticular cells. Endothelial cells were mostly ribbon-like and extended along the long axis of the vessels. Perikarya of the endothelial cells were slightly protruded into the lumen. The thin peripheral cytoplasm showed smooth surfaces, except for some tiny processes, especially at boundaries between adjacent cells. The basal surface of the endothelial cells was attached to the lattice of reticular cell processes forming the framework of the white pulp. Basal laminae in strands were intercalated between endothelial cells and reticular cells. Perforations were often seen through the endothelial cell cytoplasm. Lymphocytes or processes of macrophages seen in the perforations were considered to be in migration. Large patent openings through the endothelium were not observed. The wall structure of the lymphatic vessels in the splenic white pulp suggests that lymphocytes in the white pulp may move directly into the lymph flow, in addition to moving into the blood flow via the vascular sinuses.Supported by Research Grant-in Aid from the Ministry of Education, Japan (Grant NO. 56480081).     

Early postnatal growth of skeletal muscle blood vessels of the rat

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually. More peripherally, gradual maturation of terminal and precapillary arterioles was observed. The capillary endothelium and the pericytes showed immature features, and remained unchanged during the time studied. An intense rebuilding activity was found in the endothelial cells of the growing venules, expressed by various forms of gaps, covered by an intact basal lamina and pericytes. Numerous mast cells and macrophages were found along all vessels. Intramuscular lymphatics were not present prior to the first postnatal week.     

Non-specific cellular responses of rainbow trout to Vibrio anguillarum and its extracellular products (ECPs)

We have studied the early inflammatory response induced by Vibrio anguillarum and by its extracellular products (ECPs) in rainbow trout after intraperitoneal injection. The results showed a very similar inflammatory response which included leucopenia, mainly due to lymphopenia, neutrophilia and an increase in the number of circulating monocytes. Melanomacrophages as well as immature leucocytes were frequently observed circulating in the blood of injected rainbow trout. Monocytes often contain phagocytosed bacteria and other, altered cells including erythrocytes and leucocytes. However, neutrophils only occasionally phagocytosed bacteria. Many circulating leucocytes showed important structural alterations. Neutrophils of trout injected with bacteria and ECPs also showed stronger PAS-staining than those of control trout as well as Döhle bodies and swollen granules. A marked vasodilatation was observed in the kidney and spleen which was coincidental with a mobilization of eosinophilic granular cells and an hypertrophy of sinusoidal endothelial cells showing an increase in the number of cytoplasmic granules. An increase in the number of macrophages and melanomacrophages in the kidney and spleen as well as oedema and leucocyte infiltration in the liver and gills were also noted.     

缺血再灌后血管内皮空泡的形成与内皮的损伤

目的：在活体上探讨缺血再灌后血灌内上细胞损伤及白细胞、血小板与内皮之间粘附的变化。方法：用失血及与再回输血液造成缺血再灌流模型,在高倍显微镜下观察肠系膜微血管内皮损伤及血细胞粘附的变化。结果：缺血再灌后1－3h细静脉、集合毛细血管内出现白细胞、血小板的粘附,血管内皮水肿、管壁增厚,有的血管内皮细胞的胞浆形成圆丘形的空泡,空泡从血管内皮突入管胺、空泡直径10－30μm多出现的细动脉内,在同一根血管内可同时出现几个空泡,大的空泡几科占据血管腔的2／3。结论：缺血再灌后血管内皮水肿及空泡形成,显示内皮细胞的严重损伤。     

Modulation of fish phagocytic cells by N-terminal peptides of proopiomelanocortin (NPP)

N-terminal peptide of proopiomelanocortin (NPP, or pro-gamma-MSH) has shown to exhibit biological activity such as stimulation of adrenal mitogenesis and prolactin release-inhibiting factor activity. Structurally, studies reveal a significant difference between fish NPP from that of tetrapods, as NPPs from carp and salmonid lack gamma-MSH. Thus, fish NPP may exhibit functions different from that of mammals. The activation of phagocytic cells by NPP was analysed using rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio. Rainbow trout and carp macrophages incubated with chum salmon NPP significantly enhanced the production of superoxide anion in comparison with control macrophages (without hormones). Both rainbow trout and carp macrophages had shown increased phagocytosis when stimulated administered with NPP. The above results were complemented by in vivo studies where NPP was administered to rainbow trout and carp. NPP significantly increased superoxide anion production as well as phagocytosis in macrophages. These results show that NPP in lower vertebrates activates the function of the phagocytic cells.     

The argentophil reticular cells of the mammalian spleen. II. The argentophil reticular cell arrangement inside the red pulp and its relationship with the endothelial lining cells of the splenic sinuses

The red pulp's argentophil reticular cell network of the spleen is composed by 3 types of fixed cells: 1. the primitive reticular cell, slightly argentophil; 2. the small reticular cell; 3. the larger reticular cell, strongly argentophil and phagocytic. This latter shows the classical morphological characteristics attributed to the reticular cells of the spleen. The large argentophil reticular cell may become free, constituting a 4th cell type, the free macrophage. A 5th reticular cell type is the dendritic cell found into the lymphatic follicles of the white pulp. The argentophil reticular cells of the red pulp assemble together to form the reticular cells' network, that occurs inside the red pulp cords. The primitive and the small reticular cell form the fundamental network on which the large cells are apposed. The reticular cells of this network maitain relationship with the arterial terminal vessels of the red pulp, being responsible by the ellipsoid structure. In those arteriolar segments without ellipsoid and in those mammalian species devoid of ellipsoid, the white pulp reticular cells, that surround the blood vessel as a part of the lymphoid periarteriolar sheath, mix with the red pulp's reticular cells and both can hardly be discriminated. The ellipsoids are formed by large argentophil cells arranged in concentrical layers around its lumen that sometimes appear devoid of endothelial lining cells. The red pulp's argentophil reticular cells, either the small or the large ones, contributed to the structure of the splenic sinuses' wall; its thin processes surround the sinus wall outside the endothelial lining cell as fibrillar structures that cross the back side of the lining cells. Two or more argentophil reticular cells send fibrillar processes to a single sinus. The perisinusal reticular cells may send a process between adjacent endothelial lining, cells that insinuate and attain the sinus lumen; this process becomes thick and eventually, the reticular cell enter the sinus lumen as a free macrophage. The argentophil reticular cells of the red pulp make connection between the capsule or the trabeculae and the reticular cell network. The endothelial lining cells of the splenic sinuses are not argentophil.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

Structure of the spleen of the sea bass (Dicentrarchus labrax): A light and electron microscopic study

The spleen of sea bass (Dicentrarchus labrax) is composed mainly of red pulp, whereas the white pulp is poorly developed. The red pulp consists of clear reticular cells intermingled with blood cells, sinusoids, and melanomacrophage centers (MMCs). The MMCs are enclosed by an interrupted connective tissue capsule and show some areas in continuity with the adjacent pulp. The MMCs are formed by the association of free macrophages that have phagocytosed some blood cells. Sparse white pulp is diffuse, forming a cuff around the pulp arteries and MMCs, or occurring in small groups between the splenic cords. A longitudinal artery and vein, lying side by side, extend the length of the spleen. Frequently the capillaries are surrounded by a sheath of macrophages or ellipsoids. These macrophages may contain erythrocytes in varying degrees of degradation. Lymphopoiesis and plasmapoiesis occur in the sparse lymphold areas. Abundant plasma cell groups may indicate the presence of antibody production.