Defence mechanisms in fish

The lympho-reticular tissues in the plaice were investigated for their phagocytic properties on colloidal carbon after its intraperitoneal injection. Fish were killed at intervals ranging from 10 min to 25 days after injection. Although peritoneal macrophages constituted a large population of phagocytic cells, most of the carbon apparently gained access to the circulation as free particles and phagocytosis was performed predominantly by the ellipsoids of the spleen, the network of reticulo-endothelial (RE) cells throughout the haemopoietic tissue of the kidney, and by the RE cells occupying intermuscular spaces in the atrium of the heart. The cardiac macrophages rapidly emigrated from the organ while the carbon containing macrophages in the kidney and spleen formed aggregates in the lymphoid areas, either within or outwith pre-existing aggregates of melano-macrophages.
The possible significance of phagocyte aggregations, including melano-macrophages, in association with lymphoid elements in the kidney and spleen is discussed in the context of immune mechanisms.     

A quantitative and histochemical study on melano-macrophage centres in the spleen of the teleost fish Diplodus annularis L.

The incidence and histochemical features of melano-macrophage centres in the spleen of the teleost fish D. annularis were studied with respect to starved and fed fish. Tissue catabolism following complete starvation appears to induce formation of new centres containing melanin and lipofuscin pigments. However, a change in the amount of melano-macrophages was also recorded in fed fish, suggesting that some other factor(s), internal to fish, may affect the presence of such structures within the spleen of D. annularis .     

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.     

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.     

An electron microscopical study on the genesis of lipofuscin, melanin and haemosiderin in the haemopoietic tissues of fish

The mode of origin of the pigments within the macrophages of the haemopoietic tissues of some fish species was studied with the electron microscope. Lipofuscin appears to be derived from damaged cellular components, such as effete mitochondria, through the peroxidation of their unsaturated lipids. Haemosiderin is almost certainly derived from the breakdown of haemoglobin from effete erythrocytes. Melanin appears to be derived from phagocytosis of melanin granules or their precursor organelles from melanin-containing cells. Both lipid peroxidation and haemoglobin breakdown produce free radicals and cations which are potentially toxic. Melanin absorbs free radicals and has strong affinity for cations and it is probable that they are neutralized by the melanin in macrophages. The electron micrographs published here illustrate the association of the lysosomal apparatus with pigment formation in fish melano-macrophages. These findings appear valid for all the species examined and may apply to all fish. It has been suggested that fish melano-macrophage centres represent primitive analogues of the germinal centres of higher animals. This study reveals that melanocyte-like cells outnumber melano-macrophages in the kidney of rainbow trout, Salmo gairdneri. Moreover, like melano-macrophages, these cells increase markedly in number during starvation.     

Ligula intestinalis (L.) (Cestoda: Pseudophyllidea): plerocercoid-induced changes in the spleen and pronephros of roach, Rutilus rutilus (L.), and gudgeon, Gobio gobio (L.)

The effects of the plerocercoid of Ligula intestinalis (L.) (Cestoda: Pseudophyllidea) on the major lymphoid organs, the spleen and pronephros, of roach, Rutilus rutilus (L.), and gudgeon, Gobio gobio (L.), are described. The spleen of ligulosed roach showed a significant decrease in weight. Differential cell counts suggested this was due to a reduction in erythrocytes, despite significant increases in macrophages and vacuolated granulocytes. The spleen of gudgeon, which consisted almost entirely of erythrocytes, showed a slight reduction in weight in ligulosed fish. In both roach and gudgeon this decrease was independent of parasite burden. Differential cell counts of the pronephros from ligulosed roach revealed a significant decrease in neutrophils and increase in vacuolated granulocytes. In the pronephros of gudgeon, however, cell counts were unaffected by ligulosis. Ultrastructural observations included an apparent disintegration of vacuolated granulocytes and increased pinocytic activity in specialized endothelial cells in the spleens of ligulosed roach. Also, melano-macrophage centres and melano-macrophages increased in the spleen of ligulosed roach and gudgeon, respectively. The marked changes in spleen weight and differential cell counts in ligulosed roach and lack of such changes in ligulosed gudgeon correlate with the differential response to the parasite by these two fish species.     

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.     

Lymphomyeloid organs of the Antarctic fish Trematomus nicolai and Chionodraco hamatus (Teleostei: Notothenioidea): a comparative histological study

Lymphomyeloid organs of two common species of Antarctic fish, Trematomus nicolai and Chionodraco hamatus, were studied with the aim of analysing some morphological aspects of these organs in relation to adaptation to low environmental temperature. The thymuses of T. nicolai and C. hamatus were flattened, incompletely lobated, with numerous Hassall-like bodies, which were mainly located in the central part of the organ in C. hamatus. In T. nicolai, thymocytes, erythroid and reticular epithelial cells filled the organ. In C. hamatus, the thymocytes intermingled with reticular epithelial cells were often close to groups of melano-macrophages. In both species, the thymus did not show distinct compartmentalisation; however, the thymocytes had significantly different sizes in the outer and inner portions of the thymus. The head kidney of both species was completely filled by haematopoietic tissue, highly vascularised and mainly lymphopoietic in T. nicolai, while both erythropoietic and lymphopoietic in C. hamatus. The spleen appeared mainly erythropoietic in T. nicolai and mainly lymphopoietic in C. hamatus. Solitary melano-macrophages in T. nicolai were close to numerous small vascular ellipsoids where erythroid and lymphoid cells were intermingled without the formation of red and white pulp areas. In C. hamatus, large lymphoid areas were organised around the capillaries. The possible adaptation of lymphoid organs to the low temperature of polar water is discussed. Accepted: 8 November 1999     

Distribution of ferric iron in larval lampreys, Petromyzon marinus L

The distribution and abundance of ferric iron in larval lampreys (Petromyzon marinus L.) were investigated using light microscopy and the Prussian blue stain. Animals from various watersheds contained different concentrations of iron, although the sites of deposition were the same for all animals. A major portion of iron is within adipose tissue, while the liver, and cartilage contain predominantly low to trace amounts of iron, respectively. Iron is associated with fibrous connective tissue in several places in the body, and this association may have particular significance in the inner ear. Iron is also located in cells of the meninges. The presence of iron in the epithelial cells of the posterior intestine may reflect elimination of the metal through the extrusion of iron-loaded cells into the intestinal lumen. Iron within mucous cells of the epidermis, suggest elimination of iron during mucous secretion. Iron-loaded cells of bipolar shape are also present in the epidermis, but are particularly prominent around the branchiopore. Low concentrations of iron are observed within in melanin-containing macrophages (melano-macrophages) in regions of iron absorption, erythrophagocytosis, and haemopoiesis. High levels of iron in the epithelia and lumina of pronephric tubules are concomitant with degeneration of this organ. These data are evidence of the wide spread distribution of iron in lamprey tissues and additional evidence for the potential value of lampreys for the study of iron metabolism in vertebrates.     

Liver histopathology and accumulation of melano-macrophage centres in Hoplias malabaricus after long-term food deprivation and re-feeding

In the Neotropical traíra Hoplias malabaricus , hepatocyte surface area declined after 30 days of fasting due to reserve utilization. Changes in normal organization of liver were not confirmed until 180 days of fasting. Severe histopathological changes occurred after 240 days. Pigment accumulation in the hepatocytes and increase in number and size (surface area) of melano-macrophage centres (MMC) were also verified during long-term food deprivation. The melano-macrophages were rich in ferric compounds, probably haemosiderin. This suggests that the activity of hepatic macrophages is related to the intense erythrocyte degradation that occurred in traíra following long-term food deprivation. After re-feeding for 30 days, the liver presented a partial restoration, but the large MMC remained. When compared to the respective starved group, the hepatocytes of re-fed fish increased in size, revealing recovering cell activity and storage of some energy reserves.     

The influence of high oral doses of mercuric chloride on organ toxicant concentrations and histopathology in rainbow trout,Oncorhynchus mykiss

1. Trout were fed a pellet diet for 42 d contaminated with mercuric chloride to provide a mercury concentration of 10 g metal/kg dry weight of food.2. Sloughing of the gut epithelium and food regurgitation occurred in the last 3 weeks of the study, but no mortalities were observed.3. The kidney showed increased numbers of melano-macrophages, but gill pathology was notably absent.4. Mercury accumulated in the gill, liver, kidney, muscle and mucus. The highest toxicant concentrations were recorded in the gill and liver, while contamination in the muscle was lower than most tissues examined.5. Fish excreted mercury via the body surface mucus, a previously unknown route of mercury clearance. Mucus mercury content correlated positively with whole body burdens, suggesting mucus toxicant levels may be used to identify mercury contamination in live fish.     

Studies of a primitive mammalian spleen, the opossum (Didelphis virginiana)

Light microscopic sections of the adult opossum (Didelphis virginiana) spleen were observed to lack venous sinuses; this primitive mammalian spleen may be classified as non-sinusal in nature. In the spleen of the opossum, the capillary segments of the penicillar arteries lacked ellipsoid sheaths characteristic of certain mammalian spleens. Separating the lymphoid nodules from the surrounding red pulp was a distinct band of vascular tissue, the marginal zone. Arising from the central artery within the lymphoid nodule, vessels of capillary dimension were observed to terminate within the marginal zone and the area between lymphoid nodule and marginal zone. In addition to the vascular channels established by the terminal arterial vessels within the red pulp, the system of vessels within the marginal zone has been implicated as an important intermediate vascular channel within the spleen.     

Kinetics of CTL induction by concanavalin A.

Induction of maximal CTL activity was achieved within 12 hr of exposure to Con A in vitro in various mouse lymphoid cell populations. These included spleen cells from normal unsensitized mice, spleen cells from mice previously immunized with alloantigen, and mouse spleen cells exposed to alloantigen in long-term mixed leukocyte culture (LTMLC). Although induction of maximal incorporation of tritiated thymidine was accomplished within this same period in the cells obtained from LTMLC, a much longer period of Con A exposure (greater than 24 hr) was required for freshly prepared spleen cells from normal or previously immunized mice. These findings indicate that the increased tritiated thymidine uptake induced in freshly prepared spleen cells on continued exposure to Con A beyond 12 hr is not associated with the development of cytolytic activity, and that it probably represents stimulation of subpopulations no longer present in the LTMLC population where positive selection for cells responsive to cellular alloantigens has taken place.     

Studies of erythrocyte sequestration. I. Spleen homing of heat altered erythrocytes.

Heat altered erythrocytes are taken up by spleen macrophages without any signs of mechanical strain as revealed by light and scanning electron microscopy. There was no scintigraphic evidence for changed flow rates within the spleen after sequestration of erythrocytes.     

Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1.

We have examined the generation of CTL immunity immediately after localized footpad infection with herpes simplex virus 1 (HSV-1) using three coordinated in vivo T cell tracking methodologies. Tetrameric MHC class I containing the immunodominant peptide from HSV-1 glycoprotein B (gB) showed that after infection the proportion of Ag-specific T cells peaked at day 5 within draining popliteal lymph nodes and 2 days later in the spleen. Preferential expression of the activation marker CD25 by tetramer-positive cells in draining popliteal nodes but not spleen suggested that gB-specific T cells were initially activated within the lymph node. In vivo cytotoxicity assays showed that Ag-specific effector cells were present within the draining lymph nodes as early as day 2 after infection, with a further 2-day lag before detection in the spleen. Consistent with the very early arming of effector CTL in the draining lymph node, adoptive transfer of CFSE-labeled gB-specific transgenic T cells showed that they had undergone one to four rounds of cell division by day 2 after infection. In contrast, proliferating T cells were first detected in appreciable numbers in the spleen on day 4, at which time they had undergone extensive cell division. These data demonstrate that HSV-1-specific T cells are rapidly activated and armed within draining lymph nodes shortly after localized HSV-1 infection. This is followed by their dissemination to other compartments such as the spleen, where they further proliferate in an Ag-independent fashion.     

Ontogeny of IgE-bearing lymphocytes in the rat

IgE-bearing lymphocytes were detected by immunofluorescence in the spleen of neonatal Hooded Lister strain rats within 24 hr after birth. The same cells were detected in the bone marrow as early as the 4th day after birth. Both fetal spleen and liver obtained 1 day before birth contained IgM-bearing cells but no detectable IgE-bearing cells. The proportion of IgE-bearing cells in the spleen and bone marrow increased during the neonatal period and reached an adult level within 3 to 4 weeks after birth. In adult Hooded Lister rats, IgE-bearing cells were 3 to 6% of total spleen cells and 1.5 to 2.2% of bone marrow cells. Most of the IgE-bearing cells from bone marrow cells. Most of the IgE-bearing cells from both newborn and adult animals carried IgM determinants on their surface. Capping experiments showed that epsilon chain determinants and mu chain determinants belonged to separate molecules. IgG2a-bearing lymphocytes were detected in the neonatal spleen as early as the 4th day after birth, but a significant number of these cells was not detected in the bone marrow until the 4th week. In newborn spleen the percentage of IgE-IgM double bearing cells was higher than that of IgG2a-bearing cells.     

Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate

Summary Dichloromethylene diphosphonate can be used for temporary elimination of macrophages in the spleen when administered after entrapment in liposomes. No comparable effect on the macrophages of the spleen was observed with free dichloromethylene diphosphonate or in the case of empty liposomes. Marginal metallophils on the boundary between white pulp and marginal zone as well as macrophages in the marginal zone and red pulp disappeared from the spleen within one day and remained largely absent for about a week. After this time cells reappeared slowly, and at approximately four weeks after injection their presence in the spleen did not differ from that in control animals. Marginal metallophils and macrophages in the spleen were demonstrated by use of enzyme-histochemical methods and by their capacity to ingest carbon particles.     

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia.

A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snap-frozen. During infusion of echistatin (2.3 micrograms/kg/min), circulating platelet counts decreased from 331,000/mm3 to 167,000/mm3. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate 111Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.     

The microscopic structure of the adult raccoon (Procyon lotor) and woodchuck (Marmota monax) spleens

Histological serial sections of spleens from the adult raccoon (Procyon lotor) and woodchuck (Marmota monax) were processed for microscopic examination. Observations related to various aspects of the internal vascular pattern in the spleen of the raccoon and woodchuck demonstrated features that were characteristic of the respective animal. The spleen of the raccoon possess well-developed ellipsoid sheaths, whereas these same structures were not as prominent in the spleen of the woodchuck. The spleen of both mammals examined demonstrated the presence of an anastomosing series of venous sinuses within the red pulp tissue and may be classified as sinusal in nature.