Ultrastructure and number of Kupffer cells in the mouse liver during acute CCl4 poisoning

The ultrastructure and the number of Kupffer cells in different zones of hepatic lobules of mice were studied following CCl4 injection. Migration of the Kupffer cells to necrotic zones of the hepatic lobule after CCl4 administration was detected. These data allow a tentative suggestion that the Kupffer cells are conditionally resident macrophages.     

An experimental study on the pathway of iron transfer from macrophages to erythrocytes in rat liver

In order to reveal the pathway of iron release from macrophages, a 59Fe-labelled ferric hydroxide-potassium polyvinyl sulfate complex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with activated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the 59Fe-PVS injection. Histochemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the nonparenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron compounds and that the iron is supplied for erythroid cell proliferation.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.     

Localization of brain-type fatty acid-binding protein in Kupffer cells of mice and its transient decrease in response to lipopolysaccharide

Brain-type fatty acid-binding protein (B-FABP) was localized in Kupffer cells of liver of postnatal day 10 (P10) and older mice in immunolight and electron microscopy as well as by in situ hybridization histochemistry. The immunoreaction products were localized in the cytoplasmic matrix but not within the nucleus. After peritoneal injection of lipopolysaccharide (LPS), the immunoreaction for B-FABP decreased markedly in Kupffer cells at 1 h postinjection and thereafter gradually recovered to the preinjection level by 24 h postinjection, although no decrease in the mRNA expression was detected in Northern blotting throughout the course after the injection. The specific localization of B-FABP, but not the other FABPs, in Kupffer cells, and its rapid decrease after LPS injection suggest the intimate involvement of B-FABP in Kupffer cells in the inflammatory reaction, probably through mediation of n-3 polyunsaturated fatty acids, which are strong binders of B-FABP.     

Adaptability of carp neutrophilic granulocytes to environmental temperatures: spontaneous cytotoxic activity and cell-adherent activity

This study observed the adaptability of carp neutrophilic granulocytes possessing spontaneous cytotoxic activity to different environmental temperatures. To study the adaptability of neutrophilic granulocytes, two different temperatures (25° C and 10° C) were selected, both for rearing and forin vitroassays, in which the cytotoxicity and the adherent rate against K562 target cells were measured. The cytotoxicity and adherent rate of neutrophilic granulocytes from carp kept at 25° C for 30 days were higher when assayed at 25° C than when assayed at 10° C. On the other hand, in carp acclimated from 25° C to 10° C, the cytotoxicities and adherent rates, when assayed at 25° C, decreased with increasing acclimation times, eventually becoming smaller than the values obtained when assayed at 10° C. After the fish kept at 10° C for a long period were re-acclimated to 25° C, these activities assayed at 25° C again became higher than the activities assayed at 10° C. These results indicated that carp neutrophilic granulocytes adapt their cytotoxic activity and adherent activity to different environmental temperatures. A change in cellular composition in the head kidney was also observed in carp kept at different environmental temperatures. The percentage of neutrophilic granulocytes became higher and lymphocytes became lower in carp that were kept at 10° C for a long period compared with carp that were kept at 25° C for a long period.     

Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (<Emphasis Type="Italic">op</Emphasis>/<Emphasis Type="Italic">op</Emphasis>) mice after macrophage depletion

We previously reported that macrophage colony-stimulating factor (M-CSF, CSF-1) played important roles in the process of the repopulation of Kupffer cells after their elimination by administration of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP). In this study, we examined the repopulation of Kupffer cells and splenic red pulp macrophages in osteopetrotic (op/op) mice defective in the production of functional M-CSF and their littermate mice by using the lipo-MDP model. In untreated op/op mice, numbers of F4/80-positive Kupffer cells in the liver and F4/80-positive splenic red pulp macrophages were reduced. Repopulation of Kupffer cells and splenic macrophages was observed in littermate (op/+) mice liver by 14 days after depletion. However, in op/op mice, repopulation of Kupffer cells was not observed in Kupffer-cell-depleted op/op mice until 56 days after depletion, whereas splenic red pulp macrophages repopulated and recovered to the level of control op/op mice by 10 days after depletion. Single injection of M-CSF was effective for the induction of the repopulation of Kupffer cells, and daily administration of M-CSF induced remarkable repopulation and maturation of Kupffer cells and proliferation of macrophage precursor cells in the liver of Kupffer-cell-depleted op/op mice. These results suggest that Kupffer cells are completely M-CSF-dependent tissue macrophages, whereas splenic red pulp macrophages are composed of M-CSF-dependent macrophages and M-CSF-independent macrophages. This mouse model provides a useful tool for the study of effects of growth factor on Kupffer cell differentiation in vivo. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, NIH grant CA20408, and a Tsukada Memorial Grant (2000).     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

Structural and functional characteristics of Kupffer cells at different periods of the development of a liver response in mice to injury

The number of Kupffer cells increased up to necroses and processes of liver regeneration following carbon tetrachloride-induced injury. Mouse Kupffer cells migrated to the necrotic centrolobular zones 48 h after CCl4-induced necroses. During regeneration, Kupffer cells migrated to the sinusoids from the necrotic centrolobular zones. Study of the ultrastructure of Kupffer cells indicates activation of their function during all observation periods.     

The role of bile acids in the reduction in lipopolysaccharide uptake by cultured rat Kupffer cells

The influence of bile salts on the binding and uptake of Salmonella abortus equi lipopolysaccharide by cultured Kupffer cells was studied. In control preparations, the percentage of cell-associated lipopolysaccharide increased with time and reached a plateau after about 2 h incubation at 37 degrees C. About 1.2 micrograms lipopolysaccharide was associated with 10(6) Kupffer cells at this time interval. In the presence of 0.3, 0.6 and 1 mumol bile salts/ml the cell-associated lipopolysaccharide was respectively, about 5%, 13% and 29% lower than in control cultures. In the presence of 1 mumol bile salts/ml, the association of lipopolysaccharide to cells at 0 degrees C was about 25% lower than in controls. Preincubation of Kupffer cells with 1 mumol bile salts/ml, with or without lipopolysaccharide, did not affect cell-associated lipopolysaccharide after removal of the bile salts. The rate of secretion of radioactivity by Kupffer cells was not influenced by the presence of bile salts during the uptake or the secretion periods. Bile acids proved to inactivate lipopolysaccharide. From these observations it was concluded that low concentrations of bile salts influence the binding and uptake of lipopolysaccharide by Kupffer cells. It was, therefore, considered likely that, in patients with obstructive jaundice, the high serum bile acid level accounts for spill-over of portal lipopolysaccharide into the systemic blood.     

The effect of kupffer cell stimulation or depression on the development of liver metastases in the rat

Summary Tumour cells from a squamous carcinoma (approximately 2.5×10⁵) were injected intraportally into a syngeneic strain of rats to produce liver metastases 14 days later. Kupffer cells were stimulated by Corynebacterium parvum (7 mg or 1 mg i.v.) and zymosan (10 mg intraportally). Kupffer cell activity was depressed by the administration of silica, gadolinium chloride or human red cells. The animals in each group were sacrificed at 14 days, the livers removed and the number of visible surface metastases counted and compared. (Mann-Whitney U-test).Kupffer cell stimulation significantly reduced the number of surface liver metastases in all animals (P0.0048). In contrast depression of Kupfer cell activity significantly increased the number of metastases in all animals (P0.0045), suggesting that the activity of these cells has an important effect on the development of liver metastases.     

KINETICS OF NEUTROPHILIC GRANULOCYTES IN THE BLOOD OF RATS

The kinetics of neutrophilic granulocytes in the blood of rats were investigated using in vivo ³H-TdR labelling and autoradiography. The radioactive precursor was administered by single injection, repeated injections at 5 hr intervals and continuous infusion. The appearance of labelled granulocytes in the blood and in the sputum was recorded up to 120 hr after tracer application. In contrast to results after a single injection of ³H-TdR, complete labelling of the blood granulocyte pool was achieved when the DNA precursor was given by continuous infusion or four repeated injections at 5 hr intervals. In the latter experiments, an exponential replacement of unlabelled blood granulocytes by labelled granulocytes could be demonstrated, the mean intravascular half-life being 5·7 hr. This figure is in good agreement with values obtained by isotopic techniques in other mammalian species.     

Granulocyte and macrophage proliferation after injection of antitumor active and inactive strains of Corynebacterium parvum

Summary Corparvax, a strain of Corynebacterium parvum with strong antitumor activity, had a greater and more prolonged effect of increasing the production of granulocytes and macrophages than did a weak antitumor strain, CN5888. Following the injection of Coparvax to mice, there was a prompt and sustained increase in serum granulocyte/macrophage colony-stimulating activity, an increase in the number of spleen granulocyte/macrophage progenitor cells, an increased rate of proliferation of the bone marrow granulocyte/macrophage progenitor cells and an increase in the number of blood granulocytes and monocytes. The time courses of the increased rates of proliferation of granulocyte/macrophage progenitor cells following the injection of Coparvax were different in the bone marrow and the spleen, suggesting that local microenvironmental factors were also important.If immunostimulants such as C. parvum are to be used in chemoimmunotherapy programs, the kinetics of the increased proliferative rate of the granulocyte/macrophage progenitor cells may be important, since the more rapidly proliferating cells will be more affected by cell cycle-active chemotherapeutic agents.with the technical assistance of Beverly M. Dunne and L. Atherton     

Ultrastructural changes in the histohematic barrier of the liver in the early period after endotoxin administration

Investigation of the changes of the mouse liver cells revealed early reaction of endothelial sinusoid cells and Kupffer cells after endotoxin application. During first 60 min. after endotoxin injection there were certain bulge of nuclear zone, changes of nuclear shape, appearance of microfilaments in the cytoplasm of endothelial cells. An increase in the number of the pores and fenestrae grouped in sieve plates was discovered in the endothelial cells. Their luminal surface formed the bubbles. Such changes of the endothelial cells can be described as their activation. Reaction of the endothelial cells of hepatic sinusoids and activation of the Kupffer cells were revealed at the same time.     

Functional and morphological characteristics of the digestive reaction of gastric mucosa

Morphological state of connective tissue (stromal) cells of the stomach mucous membrane has been studied in healthy persons, having a habitual regime of feeding. During digestive period in the stomach mucous membrane, certain changes develop, which are considered as a digestive reaction. Three stages of the digestive reaction, having strict morphological signs are determined, their connections being stated by means of morphometry and mathematical analysis. I stage (preparatory) is characterized with a moderate vascular reaction, degranulation of mast cells under the superficial++ epithelium of the mucous membrane, with a moderate neutrophilic leukopedesis and a moderate lymphocytic infiltration; II stage (developed) is distinguished as a definitely demonstrated reaction of the microcirculatory bed, intensive degranulation of mast cells at all levels of the mucous membrane, massive discharge of neutrophilic granulocytes and lymphocytes into stroma; III stage (restorative) is characterized with a predominance of fibroblasts and fibrocytes, with reparation of mast cells, with decreasing saturation of stroma with neutrophilic granulocytes, lymphocytes, an increased number of eosinophilic granulocytes takes place. The data obtained widen our knowledge on functional morphology of the stomach mucous membrane, normal and at gastroduodenal pathology.     

Generation of new macrophages after injection of killed Brucella abortus organisms

Summary The kinetics of macrophage formation after intravenous injection of inactivated Brucella organisms was investigated in mice. Newly formed macrophages sampled from the peritoneal cavity were identified by their adherence to glass and their recent uptake of tritiated thymidine ( ³ H-thymidine). Radiolabelled cells increased in number from the fifth day after bacterial injection. On the seventh day they were about 20 times as numerous as in the controls. The injection of Brucella also increased the number of Kupffer cells in the liver. The variations in radioactivity in this organ were parallel to those in peritoneal adherent cells.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30–60 min of incubation in cells from controls and at 10 min in cells from treated mice 12–24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Phagocytic properties of hepatic endothelial cells and splenic macrophages compensating for a decreased phagocytic function of Kupffer cells in the chronically ethanol-fed rats

The balance of phagocytic function among Kupffer cells, hepatic endothelial cells and splenic macrophages in the chronically ethanol-fed rats has been investigated. Clearance of latex particles in the blood was measured to estimate the function of the reticuloendothelial system. Phagocytosis of latex particles by Kupffer cells, hepatic endothelial cells or splenic macrophages in vivo was measured by counting the number of ingested particles in a cell after isolation of hepatic nonparenchymal cells or spleen cells following injection of different amounts of latex particles. Latex particle clearance was suppressed in the ethanol-fed rats, demonstrating a decreased phagocytic capacity of the reticuloendothelial system. Markedly decreased phagocytic function was found in 40% of Kupffer cells of the chronically ethanol-fed rats. In contrast, the number of latex particles in hepatic endothelial cells and in splenic macrophages was increased after injection of a triggering dose of latex particles. From these results it may be concluded that an increased phagocytosis of hepatic endothelial cells and splenic macrophages could compensate for the decreased phagocytic function of Kupffer cells.     

Plasmodium yoelii sporozoites modulate cytokine profile and induce apoptosis in murine Kupffer cells

Plasmodium sporozoites traverse Kupffer cells on their way into the liver. Sporozoite contact does not elicit a respiratory burst in these hepatic macrophages and blocks the formation of reactive oxygen species in response to secondary stimuli via elevation of the intracellular cAMP concentration. Here we show that increasing the cAMP level with dibutyryl cyclic adenosine monophosphate (db-cAMP) or isobutylmethylxanthine (IBMX) also modulates cytokine secretion in murine Kupffer cells towards an overall anti-inflammatory profile. Stimulation of Plasmodium yoelii sporozoite-exposed Kupffer cells with lipopolysaccharide or IFN-γ reveals down-modulation of TNF-α, IL-6 and MCP-1, and up-regulation of IL-10. Prerequisite for this shift of the cytokine profile are parasite viability and contact with Kupffer cells, but not invasion. Whilst sporozoite-exposed Kupffer cells become TUNEL-positive and exhibit other signs of apoptotic death such as membrane blebbing, nuclear condensation and fragmentation, sporozoites remain intact and appear to transform to early exo-erythrocytic forms in Kupffer cell cultures. Together, the in vitro data indicate that Plasmodium possesses mechanisms to render Kupffer cells insensitive to pro-inflammatory stimuli and eventually eliminates these macrophages by forcing them into programmed cell death.     

Isolation and characterization of Kupffer and endothelial cells from the rat liver

Non-parenchymal cell suspensions were prepared from rat livers by three different methods based on a collagenase, a pronase and a combined collagenase-pronase treatment. The highest yield of Kupffer and endothelial cells was obtained with the pronase treatment. Attempts were made for a further purification of these cells by Metrizamide density gradient centrifugation after preferentially loading lysosomal structures in Kupffer cells with Triton WR 1339, Jectofer^®, Neosilvol^®, Zymosan or colloidal carbon. After loading with Triton WR 1339 or Jectofer^®, highly purified endothelial cell suspensions were obtained, but the final Kupffer cell preparations were contaminated with about 20% of endothelial cells. Kupffer and endothelial cells purified in this way showed an altered ultrastructure and contained increased activities of the lysosomal enzymes acid phosphatase, arylsulphatase B and cathepsin D. As an alternative procedure for the purification of Kupffer and endothelial cells, a method based on centrifugal elutriation was employed. With this procedure, highly purified preparations of Kupffer or endothelial cells with a well preserved ultrastructure were obtained. Compared with endothelial cells, purified Kupffer cells had a three times higher cathepsin D activity, whereas the arylsulphatase B activity was three times higher in endothelial cells. The high cathepsin D activity in Kupffer cells could be nearly completely inhibited by the specific cathepsin D inhibitor pepstatin, which excludes a possible contribution to this activity by proteases endocytosed during the isolation of the cells.