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.     

Functional dissimilarity of melanomacrophage centres in the liver and spleen from females of the teleost fish <Emphasis Type="Italic">Prochilodus argenteus</Emphasis>

Melanomacrophage centres (MMCs) are formed by macrophage aggregates containing pigments such as hemosiderin, melanin and lipofuscin. MMCs are found in animals such as reptiles, amphibians and, mainly, fishes, in organs such as the kidney, spleen, thymus and liver. In teleost fish, several functions have been attributed to MMCs, including the capture and storage of cations, the phagocytosis of cellular debris and immunological reactions. As the use of MMCs has been suggested as a tool for the assessment of environmental impacts, our aim has been to describe the various metabolic processes performed by MMCs in diverse organs (liver and spleen) by using the teleost Prochilodus argenteus as an animal model. MMCs from the liver and spleen were assessed by histochemistry, transmission electron microscopy, scanning electron microscopy, X-ray microanalysis techniques and biochemical assay for N-acetylglucosaminidase activity. The data showed metabolic differences in MMCs between the liver and spleen of P. argenteus in their morphometric characteristics and biochemical and elemental composition. The implications of these findings are discussed, focusing on their role in organ metabolism.     

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.     

Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice

Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation.     

Immunohistochemical localization of carbamoyl-phosphate synthetase (ammonia) in adult rat liver; evidence for a heterogeneous distribution

Different fixation media have been compared in order to find one that preserves the histological structure of rat liver and allows unambiguous immunohistochemical detection of carbamoyl-phosphate synthetase (ammonia). Fixation of rat liver in a mixture of methanol, acetone, and water yields the most intense immunohistochemical staining. Using a specific antiserum raised against rat liver carbamoyl-phosphate synthetase, less than 1% of the enzyme protein is extractable after this fixation procedure, and the histological structure is similar to that after fixation in Bouin's fixative. Specific immunohistochemical staining is localized exclusively in the cytoplasm of the parenchymal cells; its granular distribution is in accordance with the mitochondrial localization of carbamoyl-phosphate synthetase. Immunohistochemical staining shows a heterogeneous distribution within the liver acinus. Staining is most intense around the portal venules, decreases slowly toward the hepatic venules and is, after an abrupt decrease, virtually absent in a limited area surrounding these venules. The possible significance of the heterogeneous distribution of carbamoyl-phosphate synthetase for ammonia metabolism is discussed.     

Location of platelet activating factor binding in rat liver.

Autoradiographs of tissue slices from livers perfused with 1 x 10(-9) M-1-O-[3H]octadecyl-2-acetyl-sn-glycero-3-phosphocholine ([ 3H]18:0-sn-3-AGEPC) indicate that binding of this agonist is localized in the portal venules in anterograde perfused livers, and in the central venules in retrograde perfused livers. The pattern of silver grains in anterograde perfused liver was not affected significantly by prior exposure to 100-fold excesses of unlabelled 16:0- or 18:0-sn-3-AGEPC, 16:0-sn-1-AGEPC, or a 1000-fold excess of U.66985. [3H]18:0-sn-3-lyso-GEPC produced the same pattern of binding as the acetylated analogue. Measurement of glucose release stimulated by 16:0-sn-3-AGEPC demonstrated that the retrograde perfused liver was nearly 1000-fold less sensitive to this compound than the anterograde perfused liver. Exposure of the livers to bovine serum albumin prior to 5 x 10(-11) M-[3H]18:0-sn-3-AGEPC resulted in inhibition of stimulated glucose release, and decreased both the amount of label retained in the livers and the amount of silver grains over the portal sinusoidal cells without affecting the amount of grains seen over all other regions of the liver. Glucose release from primary monolayer cultures of hepatocytes or suspensions of liver slices was not stimulated by 16:0-sn-3-AGEPC. The results suggest that specific binding of [3H]18:0-sn-3-AGEPC is restricted to the portal side of the liver microvasculature, the majority of binding is nonspecific, and the biological response to AGEPC requires an intact and perfused vasculature.     

Regulation of hepatic eNOS by caveolin and calmodulin after bile duct ligation in rats

In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.     

Leaky livers, portal shunting and immunity to schistosomes

In mice, concomitant immunity to schistosomes seems to be largely dependent on factors that lack immunological specificity. In this review Alan Wilson describes the anatomical changes stimulated in the host's vasculature after the formation of granulomas around schistosome eggs in the liver. The blockage of portal venules by the granulomas leads to portal hypertension and the development of anastomoses that direct blood flow, schistosomula and eggs from the liver. An intact portal vasculature is required for schistosome maturation; some strains of mice appear to be resistant to schistosome infection because they have a predisposition to form anastomoses in the absence of infection.     

Lympho-granulocytic tissue associated with the wall of the spiral valve in the African lungfish Protopterus annectens

We describe the structure of the lympho-granulocytic tissue associated with the wall of the spiral valve of the African lungfish Protopterus annectens. The study was performed under freshwater conditions and after 6 months of aestivation. The lympho-granulocytic tissue consists of nodes surrounded by reticular tissue. The nodes are formed by an outer and an inner component separated by a thin collagenous layer. The outer component is a reticular-like tissue that contains two types of granulocytes, developing and mature plasma cells and melanomacrophage centres (MMCs). The inner component, the parenchyma, contains a meshwork of trabeculae and vascular sinusoids and shows dark and pale areas. The dark areas contain diffuse lymphoid tissue, with a large number of mitoses and plasma cell clusters. The pale areas contain a small number of macrophages and lymphocytes. Macrophages and sinus endothelial cells are filled with haemosiderin granules and appear to form part of the reticuloendothelial system of the lungfish. The reticular tissue houses granulocytes, plasma cells and MMCs and might serve for the housing and maturation of cells of the white series. After aestivation, the nodes undergo lymphocyte depletion, the suppression of mitosis, granulocyte invasion and the occurrence of cell death. By contrast, few histological changes occur in the reticular tissue. Whereas the nodes appear to be involved in lymphocyte proliferation and plasma cell maturation, the function of the reticular tissue remains obscure.     

Cellular organization of normal mouse liver: a histological,quantitative immunocytochemical,and fine structural analysis

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 μm) and small (0.03 μm) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.     

Monoclonal antibodies directed against rat liver epithelial cell lines selectively recognize bile duct epithelium in livers of adult rats

Monoclonal antibodies directed against antigens on rat liver epithelial cell lines were prepared. Three antibodies, 4C3, 19C6, and 3C2, recognized surface antigens present (although in different quantities) on eight epithelial cell lines tested, irrespective of whether they were normal or transformed. For MAb 3C2, the primary antigen common to all but one cell line showed a Mr of 135 kD. In paraffin sections of liver tissue, two antibodies, 40 and 19C6, reacted exclusively with bile duct epithelium, whereas the MAb 3C2 additionally reacted with sinusoidal endothelium and the endothelium of the portal venules. In sections of livers from rats exposed to diethylnitrosamine, the MAb 19C6 selectively stained bile duct-like structures in cholangiomas, while other preneoplastic and neoplastic lesions were not stained. These results demonstrate that the monoclonal antibodies obtained may prove useful for investigating cell lineages related to propagable liver epithelial cell lines and suggest that these cells may be derived from terminal bile ductular cells.Abbreviations ABTS2 2,2azinobis(3-ethylbenzthiazolinesulfonic) acid - ARL adult rat liver - DEN diethylnitrosamine - FCS fetal calf serum - MAb monoclonal antibody - PAP peroxidase antiperoxidase     

Intrahepatic microvascular changes in carbon tetrachloride-induced cirrhotic livers in the rat.

Liver cirrhosis was produced in the rat by combined carbon tetrachloride-phenobarbitone treatment, and the microcirculation in the cirrhotic liver was observed by a quantitative in vivo transillumination technique. The total sinusoidal flow in the observed region of the cirrhotic liver did not differ significantly from that in the normal liver, despite the reduced number of sinusoids and the increased portal venous pressure. The cirrhotic liver also presented a fast-velocity population of portal and hepatic venules and sinusoids in addition to the normal slow-velocity population. The possible mechanism of these "arterialized" microvessels is discussed.     

Blood-flow route from the hepatic artery and portal vein to the sinusoid in normal human liver observed by confocal laser scanning microscopy

OBJECTIVE: To observe the microvasculature in normal human liver. STUDY DESIGN: Four autopsy livers cut into 50-micron-thick sections were observed by confocal laser scanning microscopy. Immunofluorescence was performed using anti-alpha smooth muscle actin (alpha-SMA) antibody. In addition, double immunofluorescence was performed on the other sections using antilysozyme antibody. The routes from the portal vein branches and hepatic artery branches to the sinusoids were defined as follows: portal venule, septal branch, inlet venule, hepatic arteriole and terminal hepatic arteriole. RESULTS: The reactivity of the walls of septal branches and inlet venule was positive for alpha-SMA. Lysozyme-positive cells (Kupffer cells) were dense in the sinusoids but were sparse in the septal branches and absent from the inlet venules. Terminal hepatic arterioles were observed along the septal branch, and the anastomoses between them were observed at the peripheral portion. No routes opening directly from the terminal hepatic arteriole into the sinusoids or arterioportal anastomoses in the portal tract were observed on alpha-SMA-stained sections. CONCLUSION: Regulation of the microcirculation in human liver may be performed by the smooth muscle layer of both peripheral portal and hepatic arterial routes.     

Ubiquitous perivenous smooth muscle cords in viscera of the teleost Rachycentron canadum,with special emphasis on liver

The liver of the cobia, Rachycentron canadum, was examined by gross dissection, histological, and ultrastructural procedures. Other visceral organs were examined by histological techniques only. Unique perivenous smooth muscle cords are associated with veins in these systems, but they are particularly prominent in their association with the hepatic portal veins and their numerous intrahepatic branches. The perivenous smooth muscle cords accompany tributaries of the portal veins to the junction of the venules with the hepatic sinusoids. The reciprocal contraction and relaxation of various segments of the smooth muscle cords appear to result in pooling of blood in temporary reservoirs and in its transport to various regions of the organ. This process might apply to other organ systems as well. Possibly this unique relationship of the smooth muscle cords with veins functions in a diving reflex. Triads are occasionally encountered in the cobia liver. © 1992 Wiley-Liss, Inc.     

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.     

Characteristics of melanomacrophage centers in the liver and spleen of the roach Rutilus rutilus (Cypriniformes: Cyprinidae) in Lake Kotokel during the Haff disease outbreak

Characteristics of morphology and number of melanomacrophage centers (MMCs) in the liver and spleen of the roach Rutilus rutilus and the amount of pigments in MMCs during the Haff disease outbreak and the death of fish in Lake Kotokel in relation to these parameters in the roach from Lake Baikal are described. Pathological changes in the microvasculature and parenchyma in the liver of the roach from Lake Kotokel were found. The area of melanomacrophage centers in the liver of the roach from this lake was significantly smaller, whereas the number and size of these centers in the spleen was significantly larger than in the roaches from Lake Baikal. Among the pigments studied, the strongest response to the content of this toxin in the water body was shown by hemosiderin. An increase in its amount in the spleen MMCs testifies to an enhanced degradation of erythrocytes and iron release, which may be caused by the damage of cells of the erythrocyte lineage by the toxin.     

The spleen of the African lungfish Protopterus annectens: freshwater and aestivation

We describe the structure of the spleen of the African lungfish Protopterus annectens in freshwater conditions, and after 6?months of aestivation. The spleen is formed by cortical tissue that surrounds the splenic parenchyma. The cortex is a reticulum that contains two types of granulocytes, developing and mature plasma cells, and melanomacrophage centres (MMCs). The parenchyma is divided into lobules that show a subcapsular sinus and areas of red pulp and white pulp. Red pulp contains vascular sinuses and atypical cords formed by delicate trabeculae. White pulp also contains vascular sinuses and cords. Structural data indicate that red pulp is involved in erythropoiesis, destruction of effete erythrocytes, and plasma cell differentiation. White pulp appears to be involved in the production of immune responses. Macrophages and sinus endothelial cells constitute the reticulo-endothelial system of the spleen. After aestivation, the number of MMCs increases, and spleen tissue is infiltrated by lymphocytes, granulocytes, and monocytes. Also, white pulp is reduced, and sinus endothelial cells undergo vacuolar degeneration. Lungfish spleen shares structural characteristics with secondary lymphoid organs of both ectothermic and endothermic vertebrates, but appears to have evolved in unique ways.     

Immunocytochemical detection of Ig-positive cells in blood, lymphoid organs and the gut associated lymphoid tissue of the turbot (Scophthalmus maximus)

The present study was designed to search for the sites of the B-cell lineage in the different lymphoid organs of turbot (Scophthalmus maximus) by immunoperoxidase staining with a rabbit polyclonal antiserum against deglycosylated turbot IgM (TUDG-6). A turbot immunoglobulin (Ig) fraction, isolated by protein A, was checked for purity by gel filtration and SDS-PAGE under reducing conditions. The turbot IgM was deglycosylated and used to raise an antiserum. The antiserum titre was evaluated in ELISA. It was then used to analyse turbot peripheral blood leucocytes for membrane and cytoplasmic Ig and for immunohistochemistry with turbot lymphoid tissues. Very low numbers of Ig+ cells were found in thymus sections. In sections of spleen, Ig+ cells were observed in white pulp, around ellipsoids but were mostly concentrated and associated with melanomacrophage centers (MMCs). The lymphoid Ig+ cells in the kidney tended to be dispersed among haematopoietic and granulopoietic cell populations and were in intimate association with the MMCs and blood vessels. This association between MMCs and Ig+ cells in the spleen and the kidney, is discussed with respect to the role played by these organs in the immune system of fish. Last, the lymphoid population in the gut associated lymphoid tissue (GALT) of turbot was characterised with respect to staining for Ig. Immunoreactive cells were rarely detected in the epithelial layer although many lymphocytes were present, but they were frequently observed in the lamina propria, presumably as part of the GALT and involved in mucosal immune responses.     

Long-term peripheral infusion of nociceptin/orphanin FQ promotes hyperplasia, activation and migration of mucosal mast cells in the rat gastric fundus

The endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) modulates behavioral and gastrointestinal responses to stress. Mucosal mast cells (MMCs) are primary mediators of stress-related responses in the gastrointestinal tract. We investigated the influence of N/OFQ and of the N/OFQ peptide (NOP) receptor antagonist, UFP-101, on MMCs in the rat gastric fundus. N/OFQ was infused subcutaneously for 52 h at 0.1, 1 and 10 μg/kg/h and at 1 μg/kg/h for 4 h, 52 h, 7 days and 14 days via Alzet osmotic minipumps. Density of MMCs and connective tissue mast cells (CTMCs) was assessed histochemically and immunohistochemically. Activation and location of MMCs were assessed by transmission electron microscopy. Contacts between MMCs and nerve elements were assessed by double immunofluorescence. N/OFQ (1 μg/kg/h) and UFP-101 (10 and 30 μg/kg/h) were infused subcutaneously in the absence and presence of acute cold-restraint stress and density of MMCs was assessed. Peripheral N/OFQ dose-dependently increased the density of MMCs, while not influencing CTMCs. The increasing effect was maintained up to 14 days following continuous infusion, while after termination of the 4-h infusion, the effect declined rapidly. The peptide promoted the activation of MMCs and their migration from the lamina propria toward the epithelial layer. The association between MMCs and nerve fibers was time-dependently down-regulated following N/OFQ infusion. The stress-induced hyperplasia of MMCs was not influenced by N/OFQ and abolished by UFP-101. UFP-101 alone was ineffective. The present results suggest that endogenous N/OFQ could be considered a potential component of the circuit neuropeptides-mast cells-stress.     

Pathogenesis of pipe-stem fibrosis of the liver (experimental observation on murine schistosomiasis)

Mice infected with 30 cercariae of Schistosoma mansoni developed portal and septal fibrosis due to the massive and concentrated deposition of eggs in the periportal areas which occurred following the 16th week after infection. The lesion resembled pipe-stem fibrosis seen in human hepatosplenic schistosomiasis in the following characters: portal fibrosis interconnecting portal spaces as well as portal spaces and central canals; portal inflammation; periovular granulomas; vascular obstruction and telangiectasia. The liver parenchyma maintained its normal architecture. Vascular injection techniques with Indian ink and vinylite revealed that the portal system developed numerous dilated collateral venules coming from the large and medium-sized portal branches, about 10 weeks after schistosome infection. The lodging of schistosome eggs into these collaterals resulted in granulomatous inflammation and fibrosis along all the portal tracts, thus forming the pipe-stem lesion. Although not readily demonstrable grossly, the pipe-stem fibrosis of murine schistosomiasis has many similarities with the human lesion and can be considered to have the same basic pathogenesis.