The effect of calmodulin on the active calcium-ion transport and (Ca2+ + Mg2+)-dependent ATPase in microsomal fractions of smooth muscle compared with that in erythrocytes and cardiac muscle.

Kupffer cells isolated from the rat liver are able to bind neuraminidase-treated rat erythrocytes via a D-galactose-specific receptor on the cell surface. Binding of desialylated erythrocytes was inhibited by several mono- and oligo-saccharides related to D-galactose, but not by unrelated sugars. However, after phosphorylation at position 6 D-glucose was as good an inhibitor as D-galactose. Two synthetic glycoproteins, D-galactosyl-albumin and, at a higher concentration, D-glucosyl-albumin, strongly inhibit cell contacts. Lectin-mediated binding of desialylated erythrocytes is dependent on the presence of Ca2'ons, but independent of ATP formation and cell motility. It is concluded that binding of desialylated erythrocytes by rat Kupffer cells is mediated by a Ca2-dependent D-galactosyl/D-glucosyl-recognition system.     

Determination of Kupffer cell Fc receptor function in vivo following injury

This study was carried out to determine whether Kupffer cell Fc receptor function is depressed after injury. Three approaches to the determination of Fc receptor function were evaluated: IgG-coated erythrocytes (EIgG) were used as the receptor probe with a perfused liver system, EIgG were used as the receptor probe in vivo, and small aggregates of IgG (AIgG) were used as the receptor probe in vivo. Nearly half of the injected dose of EIgG was taken up by the perfused liver (nonrecirculating, serum-free system). In contrast, only 2.6% of erythrocytes not coated with IgG were taken up, and only 5.6% of erythrocytes coated with IgM were taken up by the perfused liver. Thus, there was little nonspecific or complement-dependent uptake of EIgG by the liver. The uptake of EIgG by the perfused liver was depressed following thermal injury, endotoxemia, and the phagocytosis of EIgG. These results were interpreted as indicating that Kupffer cell Fc receptor function was depressed under these conditions. The results obtained with the hepatic uptake of EIgG in vivo were very similar to those with EIgG in the perfused liver. However, since it was found that complement receptors as well as Fc receptors were probably involved in the in vivo clearance of EIgG, these results could be due to a depression of one or both of these receptors. The hepatic uptake of AIgG was not depressed by complement depletion, but was decreased by the injection of large aggregates of IgG. However, the hepatic uptake of AIgG was not depressed following thermal injury, endotoxemia, or the phagocytosis of EIgG. Thus, AIgG was not sensitive to the effects of injury on Kupffer cell function, whereas the uptake of EIgG by the perfused liver may provide an indication of Kupffer cell Fc receptor function. The depression of Kupffer cell Fc receptor function following injury may contribute to the impairment of host defense caused by injury.     

Erythrocyte aging: a more than superficial resemblance to apoptosis?

In physiological circumstances, erythrocyte aging leads to binding of autologous IgG followed by recognition and removal through phagocytosis, mainly by Kupffer cells in the liver. This process is triggered by the appearance of a senescent erythrocyte-specific antigen. The functional and structural characteristics of senescent erythrocytes strongly suggest that this antigen originates on band 3, probably by calcium-induced proteolysis. Generation of vesicles enriched in denatured hemoglobin is an integral part of the erythrocyte aging process. These vesicles are also removed by Kupffer cells, with a major role for exposure of phosphatidylserine. Moreover, senescent erythrocyte-specific antigens are present on vesicles. Thus, vesicles and senescent erythrocytes may be recognized and removed through the same signals. These and other, recent data support the theory that erythrocyte aging is a form of apoptosis that is concentrated in the cell membrane, and provide the context for future studies on initiation and regulation of the erythrocyte aging process. Insight into the normal aging mechanism is essential for understanding the fate of erythrocytes in pathological circumstances and the survival of donor erythrocytes after transfusion.     

Carcinoma autoantigens T and Tn and their cleavage products interact with Gal/GalNAc-specific receptors on rat Kupffer cells and hepatocytes

We studied interactions of isolated Thomsen-Friedenreich (T)- and Tn-specific glycoproteins with the Gal/GalNAc-specific receptors on rat Kupffer cells and compared them to those with rat hepatocytes. Immunoreactive T and Tn are specific pancarcinoma epitopes. Electron microscopy of gold-labelled T and Tn antigens revealed their specific binding to Kupffer cells, followed by their uptake via the coated pit/vesicle pathway of receptor-mediated endocytosis. Preincubation of Kupffer cells with GalNAc and GalNAc-BSA, but not GlcNAc or GlcNAc-BSA specifically inhibited binding of the T and Tn glycoproteins. Desialylated, isologous erythrocytes (T RBC) are known to bind to the Gal/GalNAc receptors of rat Kupffer cells and hepatocytes. This attachment was specifically inhibited by T and Tn in a concentration-dependent manner: 50% T RBC-Kupffer cell contacts were inhibited at 8.5.10(-6) mM T and 8.5.10(-5) mM Tn antigen concentrations, respectively. The corresponding figures for hepatocytes were 6.10(-6) mM T and 1.2.10(-6) mM Tn antigen. Amino-terminal cleavage products of the T glycoprotein, possessing clusters terminating in non-reducing Gal/GalNAc, inhibited T RBC binding to Kupffer cells and hepatocytes usually at 10(-2) to 10(-5) mM concentrations, whereas GalNAc, galactose and galactose glycosides inhibited at millimolar concentrations. Galactose-unrelated carbohydrates were inactive at concentrations greater than or equal to 50 mM.     

Cell surface antigens on osteoclasts and related cells in the quail studied with monoclonal antibodies

The properties of five monoclonal antibodies raised against isolated osteoclasts are described. Osteoclasts were isolated from medullary bone of egg-laying female quails. Mice were immunized with cell preparations consisting for about 10% of multinucleated osteoclasts. A large number of monoclonal antibodies against cell surface antigens were obtained, five of which were extensively characterized by their interactions with different tissues of the quail and their cross-reactivity with other species. Two monoclonals (OC 5.3 and OC 6.8), recognize surface antigens present on osteoclasts, monocytes, granulocytes and endothelial cells, but not on osteoblasts, osteocytes, fibroblasts, lymphocytes, erythrocytes and others. The three other monoclonal antibodies are specific for multinucleated osteoclasts in bone tissue but recognize some cell surface structures in other tissues. Antibody OC 6.9, which in bone tissue stains primarily the surface area of the osteoclast that is adjacent to the resorbing bone surface, also interacts with bile capillaries in the liver and with specific, but not yet identified parts of the nephron. The antibodies OC 6.1 and OC 6.3 interact with Kupffer cells in the liver and tissue macrophages of small intestine. In view of the possible fallacies inherent to the use of cell surface markers for the demonstration of cell relationship and origin, definite conclusions can not yet be made. The fact that the osteoclast, the Kupffer cell and the intestine macrophage are the only cells in bone, bone marrow, liver, kidney and intestine, that share the same surface antigen recognized by monoclonals OC 6.1 and OC 6.3, suggests, however, a common origin for osteoclasts and a number of well described tissue macrophages.     

Cell surface antigens on osteoclasts and related cells in the quail studied with monoclonal antibodies

Summary The properties of five monoclonal antibodies raised against isolated osteoclasts are described.Osteoclasts were isolated from medullary bone of egglaying female quails. Mice were immunized with cell preparations consisting for about 10% of multinucleated osteoclasts. A large number of monoclonal antibodies against cell surface antigens were obtained, five of which were extensively characterized by their interactions with different tissues of the quail and their cross-reactivity with other species. Two monoclonals (OC 5.3 and OC 6.8), recognize surface antigens present on osteoclasts, monocytes, granulocytes and endothelial cells, but not on osteoblasts, osteocytes, fibroblasts, lymphocytes, erythrocytes and others. The three other monoclonal antibodies are specific for multinucleated osteoclasts in bone tissue but recognize some cell surface structures in other tissues. Antibody OC 6.9, which in bone tissue stains primarily the surface area of the osteoclast that is adjacent to the resorbing bone surface, also interacts with bile capillaries in the liver and with specific, but not yet identified parts of the nephron. The antibodies OC 6.1 and OC 6.3 interact with Kupffer cells in the liver and tissue macrophages of small intestine. In view of the possible fallacies inherent to the use of cell surface markers for the demonstration of cell relationship and origin, definite conclusions can not yet be made. The fact that the osteoclast, the Kupffer cell and the intestine macrophage are the only cells in bone, bone marrow, liver, kidney and intestine, that share the same surface antigen recognized by monoclonals OC 6.1 and OC 6.3, suggests, however, a common origin for osteoclasts and a number of well described tissue macrophages.     

TRANSCELLULAR BIOSYNTHESIS OF CYSTEINYL LEUKOTRIENES BY KUPFFER CELL—HEPATOCYTE COOPERATION IN RAT LIVER

We previously proposed that an enzymatic cooperation between Kupffer cells and hepatocytes may play an important role in cysteinyl leukotriene (LT) production in rat liver. Anin vitrotranscellular synthesis cysteinyl LTs by a Kupffer cell—hepatocyte coculture system was characterized here. Kupffer cells alone, with A23187 stimulation, did not generate cysteinyl LTs until supplemented either with isolated hepatocytes or with LTC₄synthase and glutathione, indicating that Kupffer cells can synthesize LTA₄but not convert it into LTC₄. In contrast, hepatocytes converted the LTA₄into cysteinyl LTs and further degraded the cysteinyl LTs. Cysteinyl LT production by the Kupffer cell—hapatocyte coculture system was optimized by addition of 1–3% serum albumin to the culture and by bringing the cell—cell distance closer to less than 3μ. Tumour necrosis factor also stimulated cysteinyl LT production by the coculture system. From these results, it is expected that the Kupffer cell—hepatocyte transcellular system for cysteinyl LT production actually functionsin vivo.     

Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localization of lectin-mediated endocytosis

Direct evidence is given for the presence of an N-acetyl-D-galactosamine-specific lectin on the Kupffer cell surface by visualization of ligand binding in electron microscopy. When freshly isolated Kupffer cells are incubated with asialofetuin adsorbed onto colloidal gold particles (ASF-gold), binding and endocytosis of ligand are seen. Recognition of ASF-gold by Kupffer cells is completely abolished in the presence of N-acetyl-D-galactosamine (25 mM) or EGTA (3 mM), but is not significantly reduced by N-acetyl-D-glucosamine or D-mannose (25 mM). ASF particles are endocytosed via the coated pit/vesicle pathway and appear to be transported to the secondary lysosomes by coated vesicles, as shown by the occurrence of coated areas in the secondary lysosome membrane. These observations demonstrate the presence of an asialoglycoprotein receptor on Kupffer cells; therefore, the hepatocyte is not the only cell in the rat liver with D-galactose receptor activity.     

Malaria circumsporozoite protein inhibits the respiratory burst in Kupffer cells

After transmission by infected mosquitoes, malaria sporozoites rapidly travel to the liver. To infect hepatocytes, sporozoites traverse Kupffer cells, but surprisingly, the parasites are not killed by these resident macrophages of the liver. Here we show that Plasmodium sporozoites and recombinant circumsporozoite protein (CSP) suppress the respiratory burst in Kupffer cells. Sporozoites and CSP increased the intracellular concentration of cyclic adenosyl mono-phosphate (cAMP) and inositol 1,4,5-triphosphate in Kupffer cells, but not in hepatocytes or liver endothelia. Preincubation with cAMP analogues or inhibition of phosphodiesterase also inhibited the respiratory burst. By contrast, adenylyl cyclase inhibition abrogated the suppressive effect of sporozoites. Selective protein kinase A (PKA) inhibitors failed to reverse the CSP-mediated blockage and stimulation of the exchange protein directly activated by cAMP (EPAC), but not PKA inhibited the respiratory burst. Both blockage of the low-density lipoprotein receptor-related protein (LRP-1) with receptor-associated protein and elimination of cell surface proteoglycans inhibited the cAMP increase in Kupffer cells. We propose that by binding of CSP to LRP-1 and cell surface proteoglycans, malaria sporozoites induce a cAMP/EPAC-dependent, but PKA-independent signal transduction pathway that suppresses defence mechanisms in Kupffer cells. This allows the sporozoites to safely pass through these professional phagocytes and to develop inside neighbouring hepatocytes.     

Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages.

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes. When expressed as rates of hydrolysis per 10(6) cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2-4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.     

Glucose-6-phosphate dehydrogenase and mouse Kupffer cell activation: an ultrastructural dual staining enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in Kupffer cell function, especially in phagocytosis activity. Although it was suggested that Kupffer G6PD may be upregulated in Kupffer phagocytosis/activation, direct morphological evidence has been lacking. Acid phosphatase (ACP), a representative lysosomal enzyme, can be used as a cytochemical marker for phagocyte activation. Using an ultrastructural enzyme-cytochemical dual staining method, I simultaneously localized G6PD and ACP activity in mouse Kupffer cells on a cell-by-cell basis, and examined whether or not cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Glucose-6-phosphate dehydrogenase labelings were observed in the cytoplasm and on the cytosolic side of the endoplasmic reticulum, and ACP labelings were seen in the lysosomes. In phagocytosing Kupffer cells, in which ACP deposits were observed not only in the lysosomes but also on the phagosomal membranes and phagosomal contents, G6PD labelings were denser than dormant Kupffer cells. Enzyme-cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Kupffer cell G6PD, activated in phagocytosing Kupffer cells, may play an important role not only in liver defense but also in liver disease pathogenesis/pathophysiology.     

The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.     

Kupffer cell-expressed membrane-bound TNF mediates melphalan hepatotoxicity via activation of both TNF receptors

Isolated hepatic perfusion of nonresectable liver cancer using the combination of TNF and melphalan can be associated with a treatment-related hepatotoxicity. We investigated whether, apart from TNF, also melphalan is cytotoxic in primary murine liver cells in vitro and investigated mediators, mode of cell death, and cell types involved. Melphalan induced a caspase-dependent apoptosis in hepatocytes, which was not seen in liver cell preparations depleted of Kupffer cells. Neutralization of TNF prevented melphalan-induced apoptosis and liver cells derived from mice genetically deficient in either TNFR 1 or 2, but not from lpr mice lacking a functional CD95 receptor, were completely resistant. Cell-cell contact between hepatocytes and Kupffer cells was required for apoptosis to occur. Melphalan increased membrane-bound but not secreted TNF in Kupffer cells and inhibited recombinant TNF-alpha converting enzyme in vitro. Melphalan induced also severe hepatotoxicity in the isolated recirculating perfused mouse liver from wild-type mice but not from TNFR 1 or 2 knockout mice. In conclusion, this study shows that melphalan elicits membrane TNF on Kupffer cells due to inhibition of TNF processing and thereby initiates apoptosis of hepatocytes via obligatory activation of both TNFRs. The identification of this novel mechanism allows a causal understanding of melphalan-induced hepatotoxicity.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

The effect of cholesterol oleate treatment of mice on the rosette forming cell response against sheep erythrocytes.

Mice injected with a single dose of 60 mg cholesterol oleate emulsion showed substantial blockade of the monoclear phagocyte system measured by the rate of vascular clearance of radio-labelled sheep erythrocytes. The labelled rythrocytes, in lipid treated mice, localized mainly in the spleen, contrasting with control mice in which localization was mainly in the liver. Treatment with this lipid, 24 hr before the intravenous of two different doses of sheep erythrocytes, resulted in significant depression of the rosette forming cell response in the spleen, whereas the responses in the lymph nodes of both control and lipid treated mice were at a low level and not significantly different. Intravenously administered cholesterol oleate emulsion is known to localize mainly in the Kupffer cells and in splenic red pulp macrophages. Cultured macrophages treated with this lipid show inhibition of antigen-binding and depressed phagocytosis of heterologous erythrocytes. The lipid does not affect lymphocytes. These findings are in keeping with the hypothesis that macrophages play a direct role in the induction of an immune response against a particulate antigen.     

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.     

Kupffer cell-initiated remote hepatic injury following bilateral hindlimb ischemia is complement dependent

Intravital fluorescence microscopy was applied to the livers of male Wistar rats to test the hypothesis that complement mobilization stimulates Kupffer cells and subsequently initiates hepatic injury after hindlimb ischemia/reperfusion (I/R). Following 3 h of limb reperfusion, hepatocellular viability (serum levels of alanine transaminase and cell death via propidium iodide labeling) decreased significantly from levels in sham-operated animals. Inhibition of complement mobilization with soluble complement receptor type 1 (20 mg/kg body wt) and interruption of Kupffer cell function with GdCl(3) (1 mg/100g body wt) resulted in significant hepatocellular protection. Although the effects of hindlimb I/R on hepatic microvascular perfusion were manifest as increased heterogeneity, both complement inhibition and suppression of Kupffer cell function resulted in marked improvements. No additional hepatocellular protection and microvascular improvements were provided by combining the interventions. Furthermore, inhibition of complement mobilization significantly depressed Kupffer cell phagocytosis by 42% following limb reperfusion. These results suggest that the stimulation of Kupffer cells via complement mobilization is necessary but is not the only factor contributing to the early pathogenesis of hepatic injury following hindlimb I/R.     

Biphasic control of polymorphonuclear cell migration by Kupffer cells. Effect of exposure to metabolic products of ethanol

In order to investigate the role of the Kupffer cells in the regulation of the inflammatory reaction seen in alcoholic hepatitis, rat liver Kupffer cells were cultured and exposed to products of ethanol metabolism. The resultant supernatants were tested to study their ability to stimulate or inhibit polymorphonuclear cell chemotaxis. Kupffer cells produced increased chemokinetic activity for human polymorphonuclear leukocytes (84 +/- 6 vs. 61 +/- 4 randomly migrating cells per 5 high power fields; p less than 0.01); when incubated with soluble products of microsomal peroxidation, the Kupffer cells engendered more chemokinetic activity than that produced by untreated Kupffer cells (106 +/- 6 vs. 84 +/- 6 cells per 5 high power fields; p less than 0.05). When Kupffer cells were incubated with acetaldehyde, the chemokinetic activity that appeared in the supernatant did not differ from control (51 +/- 3 vs. 61 +/- 4 randomly migrating cells per 5 high power fields; p = NS). Chemotaxis of polymorphonuclear cells was not observed when the Kupffer cell supernatants were tested by checkerboard analysis. Kupffer cells released a factor which, at different concentrations, inhibited the response of polymorphonuclear cells to the synthetic polypeptide chemotactic factor f-met-leu-phe by 47% (p less than 0.001). This effect was unchanged when the cells were exposed to acetaldehyde or to soluble products of microsomal peroxidation. Our results demonstrate that Kupffer cells are capable of stimulating or inhibiting polymorphonuclear cell chemotaxis and that some of these effects may be influenced by the products of ethanol metabolism, suggesting that Kupffer cells may play an important role in the regulation of the inflammatory reaction seen in alcoholic hepatitis.     

Pseudomonas aeruginosa delays Kupffer cell death via stabilization of the X-chromosome-linked inhibitor of apoptosis protein

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-alpha. We hypothesized that early during infection, Kupffer cells are protected against TNF-alpha-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-alpha, or both. At early time points, TNF-alpha induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-alpha-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-alpha-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-alpha-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-alpha-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-alpha-induced Kupffer cell death via stabilization of XIAP.     

Quantification and regulation of apolipoprotein E expression in rat Kupffer cells

Apolipoprotein E (apoE) is synthesized by a wide variety of cells including cells of the monocyte-macrophage lineage. In order to assess the quantitative significance of apoE synthesis in a mature tissue macrophage, apoE synthesis was compared in Kupffer cells and hepatocytes isolated from rat liver. Immunoreactive apoE synthesized by both cell types exhibited identical isoform patterns when examined by high-resolution two-dimensional gel analysis. ApoE synthesis was not detected in hepatic endothelial cells. Northern blot analysis using a rat apoE cDNA probe demonstrated a single mRNA species of approximately 1200 nucleotides in freshly isolated hepatocytes and Kupffer cells. The absolute content of apoE mRNA in each cell type was determined with a DNA-excess solution hybridization assay. The apoE mRNA content (pg/microgram RNA) for Kupffer cells and hepatocytes was 35.7 and 98.8, respectively. Accounting for cellular RNA content and the population size of each cell type in the liver, Kupffer cells were calculated to contain about 0.7% of liver apoE mRNA; hepatocytes account almost quantitatively for the remainder. These results suggest that Kupffer cells are not major contributors to the plasma apoE pool. After intravenous injection of bacterial endotoxin, apoE mRNA was decreased in freshly isolated Kupffer cells whereas whole liver showed no change in apoE mRNA. Endotoxin treatment had no effect on the apoE mRNA content in several peripheral tissues. These results indicate that apoE expression in vivo is differentially regulated by endotoxin in Kupffer cells as compared to hepatocytes or apoE-producing cells in peripheral tissues.