The fate of intraperitoneally injected carbon particles in cyprinid fish

Summary The lymphoid organs of rosy barb (Barbus conchonius) and carp (Cyprinus carpio) were investigated for their phagocytic uptake of carbon, after its intraperitoneal injection. Carbon handling was similar in both species. It was first detected in the lymphoid organs at 30 min after injection. During the first day, carbon was phagocytized by macrophages situated in the spleen within the ellipsoids and in the red pulp. In head and trunk kidney, carbon was found in macrophages scattered throughout the haemopoietic parenchyma, and in cells lining the blood sinuses. In the spleen, macrophages replete with carbon left the ellipsoidal structures and formed aggregates with pigment-containing macrophages from day 6 onwards. In all lymphoid organs, almost all carbon was ultimately concentrated in the melano-macrophage centres.     

Structural characterization of the melano-macrophage centres (MMC) of goldfish Carassius auratus

In the present work we have studied the organization of melano-macrophage centres (MMCs) in the peripheral lymphoid organs, including spleen, pro- and mesonephros, of the goldfish, Carassius auratus, in an attempt to clarify their cellular composition, origins and functional relationships. Histological analysis demonstrated a similar organization in the three organs on the basis of closely packed phagocytic cells containing abundant pigment. The MMCs of Carassius auratus are found throughout the parenchyma of spleen and kidney and show a close association with the vascular system, i.e. splenic ellipsoids, sinusoids of red pulp and renal blood sinuses. They exhibit distinct degree of development from small groups of actively phagocytic macrophages to large, totally or partially encapsulated centres, where effete phagocytic cells are filled by cell debris. Ultrastructural and histochemical data suggest that the main inclusion observed in the MMCs of Carassius auratus is lipofuscin. Haemosiderin occurs in lesser amounts and melanin is almost restricted to kidney MMCs,--mainly mesonephros--. Our results suggest various non-specific physiological roles for the teleost MMCs, including tissue breakdown and erythrocyte catabolism.     

The development of the lymphoid organs of flounder, Paralichthys olivaceus, from hatching to 13 months

The growth of the lymphoid organs, such as head kidney, spleen and thymus were studied in flounder, Paralichthys olivaceus Temminck & Schlegel, from hatching to 13 months of age. Except for the thymus, all organs grew as the fish grew. By 2 months of age the lymphoid organs attained their maximum relative weight. The organ weight showed a closer correlation to body weight than they did to age. The total number of leucocytes in the lymphoid organs increased with age, but the number per milligram of lymphoid organ remained constant. A micro and ultrastructural study of the lymphoid organs showed that the full development of the lymphoid organs was not achieved until the juvenile stage. The spleen and head kidney had mixed populations of "red" and "white" cells. The head kidney was more lymphoid than the spleen. The thymus involuted quickly during the first 6 months. The blood components had no obvious relationship with age or season during the period studied.     

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.     

Localisation of Ig heavy chain mRNA positive cells in Atlantic cod (Gadus morhuaL.) tissues; identified byin situhybridisation

Localisation of immunoglobulin heavy chain (IgH) producing cells was determined in sections from head kidney, spleen, thymus, gills, gut, skin, heart and liver from the Atlantic cod. In general, IgH mRNA positive cells were detected in all organs examined and were mainly located to the connective tissue surrounding the vascular system in these organs. In the head kidney and spleen, IgH mRNA positive cells appeared as single distributed cells or as dense clusters, whereas in the thymus only single distributed positive cells were observed. The percentage of Ig heavy chain mRNA positive (plasma) cells in the head kidney, spleen and thymus was estimated at about 1% of the total cell mass. The number of IgH mRNA positive cells was lower than this in all the other organs examined.     

Melano-macrophage centres in liver and spleen of ruffe(Gymnocephalus cernua) from the Elbe Estuary

Macrophages with yellow or black deposits of melanin occur singly or in groups in the melano-macrophage centres are functionally the primitive analogues of lymph nodes in birds and mammals. From 1980 to 1982, tumours and pretumorous tissue changes in ruffe from the Elbe Estuary were studied. Pathological alterations occur mainly in the liver and spleen. In connection with both, neoplastic abnormalities in liver and spleen, and fatty degenerative processes in the liver, an obvious increase in number and size of the melano-macrophage centres was observed. The colour, structure and some histochemical properties of melano-macrophage centres in the liver differ somewhat from those in the spleen. The majority of splenic centres were filled with large amounts of haemosiderin, whereas many hepatic macrophages contained fatty inclusions. Possible differences in the functions of splenic and hepatic melano-macrophage centres are discussed.     

Localization of iodopsin and rod-opsin immunoreactivity in the retina and pineal complex of the river lamprey,Lampetra japonica

Melano-macrophages in the head-kidney, spleen and liver of sea bass and gilthead seabream have been investigated by means of light and electron microscopy, histochemistry and phagocytic assays. The results demonstrate the presence of both free and clustered melano-macrophages (melano-macrophage centres), with similar ultrastructural features. These large cells are PAS-, hemosiderin-and melanin-positive, and contain large, alkaline-and acid phosphatase-positive lysosomes, whose reaction intensity depends on the amount of accumulated pigment. The relationship between the cytochemical features of these lysosomes and the capacity of the melano-macrophages to phagocytose bacteria and latex beads, has been studied. The large melanomacrophage centres have a capsule of flattened cells, whose ultrastructural and cytochemical features are similar to fibroblast-like reticular cells. Melanin is the main accumulated pigment. A subpopulation of head-kidney mononuclear phagocytes engulfs melanin associated with cell debris. The relationship between the origin of the melano-macrophage pigment and the ability of monocytes/macrophages to phagocytise the melanin from melanocytes, is considered. The origin and possible function of melano-macrophage centres are discussed.     

White pulp compartments in the spleen of the turtle Mauremys caspica

Summary The aim of the present study was to analyze the nature of lymphoid and non-lymphoid cellular components occurring in distinct histological compartments of the splenic white pulp of the turtle, Mauremys caspica, in order to define their possible correlations with those of the spleen of higher vertebrates, principally mammals. The white pulp of M.caspica consisted of 3 clearly distinguishable regions: (1) the periateriolar lymphoid sheath, and (2) the inner and (3) the outer zones of the periellipsoidal lymphoid sheath. Reticular cells intimately associated with reticular fibres constituted an extensive meshwork in the periarteriolar lymphoid sheath which housed principally Ig-negative lyphoid cells, mature and immature plasma cells, and interdigitating cells. A few Ig-positive cells were also present in the peripheral region of the periarteriolar lymphoid sheath. The inner and outer zones of the periellipsoidal lymphoid sheath were separated by a discontinuous layer of reticular cell processes. In the inner zone, surface Ig-positive lymphoid cells predominated as well as dendritic cells, resembling ultrastructurally the mammalian follicular dendritic cells, although no germinal centres were found in the turtle spleen. Macrophages, some cytoplasmic Ig-positive cells, and Ig-negative lymphoid cells appeared in the outer zone of the periellipsoidal lymphoid sheath. These results allow us to speculate on a phylogenetic relationship between the periarteriolar lymphoid sheath and the inner and the outer zones of the periellipsoidal lymphoid sheath of the spleen of M. caspica and the periarteriolar lymphoid tissue, the lymphoid follicles and the marginal zone, respectively, of the mammalian splenic white pulp.     

鲤鱼和鲫鱼脾脏显微结构和亚显微结构的研究

鲤鱼和鲫鱼的脾脏中都没有小梁,其红髓和白髓是混合的。脾脏中都有椭圆体,其末端向脾髓开放。脾髓的纤维网眼中缺乏血管壁,椭圆体毛细血管中的血液可直接流入脾髓网状结缔组织的腔隙中。腹腔注射墨汁的鲫鱼,2小时后即可在椭圆体的巨噬细胞中看到碳粒的积累,可见脾脏的过滤作用主要在椭圆体中进行。在注射墨汁的鲫鱼脾脏中,黑色素巨噬细胞中心比未经注射鱼的脾脏明显,且数量增多,它和哺乳动物淋巴结和脾脏中的生发中心结构和功能都不相同。       

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.     

The effects of splenectomy and subsequent starvation on the storage of haemosiderin by the melano-macrophages of rainbow trout Salmo gairdneri Richardson

In clinically normal, cachectic and diseased teleost fish, haemosiderin is observed mostly in the melano-macrophage centres of the spleen and to a much lesser extent in the centres of the kidney and liver. When rainbow trout that had been splenectomised were starved, accumulation of haemosiderin was diverted to the kidney melano-macrophages.     

Composition of main haematopoietic compartments in normal and bled channel catfish

Differential cell counts showed that the head and trunk kidney of control and bled channel catfish Ictalurus punctatus had myeloid characteristics. They contained lymphoid and granuloid cells, thrombocytes, erythroid and agranular cells in decreasing order of abundance (%). Among the blast and precursor cells, the most numerous erythroid ones were followed by granuloid, lymphoid and agranular ones. The main changes after blood withdrawal were the decrease of thrombocytes and the increase of precursor cells in both kidney parts. In the group examined 7 days after bleeding the head kidney had a higher percentage of erythroid cells and lymphocyte precursors than the trunk kidney while the latter had more granuloid cells and their precursors. Basophils were present ( c . 1%) in both regions of the kidney of all groups. The spleen was predominantly a lymphatic organ. It contained c . 80% lymphoid cells, a higher incidence of granulated lymphocytes than in kidneys, 15% thrombocytes and 1.4% agranular cells. Blood withdrawal caused an increase of thrombocytes, a decrease of lymphoid cells and an increase of erythroid precursors in the spleen. The last probably stemmed from the circulation. While haematocrit values failed to indicate the anaemic state in the bled groups, the differential red blood cell count showed dramatic differences between the control and bled groups as well as between the two groups in different stages of recuperation from the blood loss.     

Effects of sublethal concentrations of potassium dichromate on the occurrence of splenic melano-macrophage centres in juvenile plaice, Pleuronectes platessa, L.

Plaice were exposed to 0.5 and 2.0 ppm potassium dichromate (K₂Cr₂O₇) concentrations for 27 days. In both water concentrations chromium was accumulated up to a level of 400 ppb in the carcass of the fish. At the same time the number of splenic melano-macrophage centres (MMC) tripled, whereas their average size was reduced to one third. The percentage area of MMC in the splenic tissue remained unchanged. Histological alterations following exposure to K₂Cr₂O₇ included increases both in melanin content of the MMC and in the number of melanin-containing cells within blood vessels; also, the splenic ellipsoids became less clearly demarcated. Splenic melano-macrophages show a decreased ability to aggregate to form large MMC under the influence of K₂Cr₂O₇, which leads to the assumption that the turnover rate of macrophage settlement in, and macrophage elimination from, the spleen is increased.     

Evidence for melano-macrophage centres of teleost as evolutionary precursors of germinal centres of higher vertebrates: an immunohistochemical study

The melano-macrophage centres (MMCs) of the haemolymphopoietic organs of teleost fish trap and retain antigens and are closely associated with immunoglobulin-secreting cells. The hypothesis that they are the phylogenetic precursors of the germinal centres of higher vertebrates has been questioned due to their apparent lack of organising cells. In this study the immunoreactivity of MMC cells from spleen and kidney of the teleosts Cyprinus carpio, Odontesthes bonariensis and Solea senegalensis to CNA-42, an antibody usually employed for labelling follicular dendritic cells of higher vertebrates was investigated. Free melano-macrophages and MMCs in the spleens of all three species were labelled by the antibody. This finding adds new evidence to the hypothesis that an evolutionary relationship exists between the MMCs of fish and the germinal centres of many birds and mammals.     

Morphogenesis of blood cell lineages in channel catfish

The morphogenesis of blood cell lineages in channel catfish Ictalurus punctatus , from head and trunk kidney and spleen imprints as well as from blood smears of bled and control fish, showed that early maturation stages resembled those in higher vertebrates. The erythroid lineage consisted of the proerythroblast, erythroblasts (basophilic, polychromatic, orthochromic), young erythrocyte and erythrocyte. The rare bilobed erythrocyte seemed to be a cell in apoptosis while old erythrocytes and erythroplastids represented remnants of this process. Maturation stages of neutrophils and basophils encompassed the granuloblast, young progranulocyte, progranulocyte and metagranulocyte. The basophilic lineage was regularly present in kidneys, rare in spleen and absent from blood. It contained large Sudan Black and PAS-negative, water soluble granules and small PAS-positive ones. Lymphocytes with azurophilic granulation occured regularly in kidneys and spleen. Monoblasts and promonocytes in kidneys preceded monocytes. A phagocytic lineage devouring apoptotic blood cell remnants was present in kidneys and spleen. Its youngest identified stage (promacrophage) resembled more a granuloid cell without granules than a monocytoid one. The larger, young macrophages contained a few to several ingestions and the very large mature macrophages were loaded with them. The latter two stages corresponded to cells in melano-macrophage centres (macrophage aggregates). Precursor stages of the thrombocyte were not identified.     

Distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) from Aeromonas salmonicida in Atlantic salmon, Salmo salar L.

The distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) purified from the outer surface of the fish pathogen Aeromonas salmonicida, was studied in Atlantic salmon. Radiolabelling was achieved by conjugating the antigens to tyramine cellobiose (TC) or fluorescein isothiocyanate (FITC) which were radioiodinated either before or after conjugation. Since both TC and FITC are trapped intralysosomally at the cellular site of uptake, the ligands are advantageous in studies on tissue distribution of antigens. Injection of TC-A-layer protein and TC-LPS resulted in high specific radioactivity (cpm g⁻¹tissue) in both head kidney and trunk kidney. In contrast, only low specific radioactivity was recovered in spleen, heart and liver. Surprisingly, use of FITC-LPS as the antigen changed the uptake to be high in both spleen and head kidney. Radiolabelled (¹²⁵I-TC-) LPS and A-protein, administered by a dorsal aorta catheterisation technique, were cleared from the blood within 24 h. In immunised fish, the antibody activity against the A-layer protein was diminished even within 10 min after administration, in contrast to the level of anti-LPS antibodies which remained high. These results suggest that immune-complex formation took place at least with the A-layer protein, but the uptake of A-layer protein in the various tissues did not differ significantly in vaccinated (A. salmonicida bacterin) and non-vaccinated fish.     

Induction of enhanced alkaline phosphatase activity in the melano-macrophage centres of Oreochromis aureus (Steindachner) through starvation and vaccination

Employing histochemical methods, alkaline phosphatase activity was studied in the melano-macrophage centres of the spleen of the cichlid fish Oreochromis aureus (Steindachner). Enzyme activity was observed to be very low in normal fish. Prolonged starvation induced an enhanced enzyme response. Starvation followed by antigenic stimulation through an intraperitoneal injection of a bacterial vaccine further elevated the levels of alkaline phosphatase activity. The marked response of the pigment-bearing macrophages to bacterial antigen provides further evidence of the lymphoreticular nature of these pigmented cell aggregates. The association of alkaline phosphatase activity and lipofuscin (the most common pigment in fish melano-macrophage centres) with phagocytic cells has been documented in higher animals including     

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.