Cultured Rat Kupffer Cells Research Articles

Response of cultured rat Kupffer cells to lipopolysaccharide.

The effects of Salmonella abortus equi lipopolysaccharides (LPS) on pure cultures of rat Kupffer cells (Kc) were studied. In vitro, LPS is ingested by Kc and located in vacuoles and secondary lysosomes. Culture of Kc in the presence of 1-50 micrograms LPS/ml during 24 h did not affect the viability of the Kc as measured by trypan blue exclusion, neutral red uptake, lactate dehydrogenase leakage and cell survival and spreading. LPS treatment did not influence the ultrastructure of Kc. The exposure of Kc to LPS in vitro did not change the phagocytic activity. Several biochemical processes were stimulated: glucose consumption, MTT-tetrazolium salt reduction, total protein synthesis and secretion of proteins. LPS activated Kc to tumoricidal activity against L929 mouse fibrosarcoma cells. In the light of the above observations, it is concluded that purified LPS is not cytotoxic for pure Kc in culture. On the contrary, LPS stimulates several biochemical and functional processes.

Release of lysosomal enzymes is not correlated with superoxide and prostaglandin production by stimulated rat Kupffer cells in primary culture

A substantial production of prostaglandin E2 (PGE2) was induced in primary cultures of rat Kupffer cells by zymosan, calcium ionophore A23187, phorbol ester and arachidonic acid, whereas contact with latex particles, glucan or immunocomplexes led to a minor PGE2 release only. Superoxide generation, on the other hand, was observed after administration of zymosan, glucan and the phorbol ester but not after treatment with the calcium ionophore, arachidonate, latex particles or immunocomplexes. Lysosomal enzymes like beta-glucuronidase and N-acetyl-beta-D-glucosaminidase were detected in the medium of rat Kupffer cells in primary culture after contact with zymosan or calcium ionophore A23187. Other particulate matter, e.g., latex particles, glucan and immunocomplexes, lipopolysaccharides or soluble agents such as phorbol ester, arachidonic acid and gamma-interferon did not provoke the release of lysosomal enzymes. The activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase found following prolonged exposure to zymosan or to A23187 were accompanied by the appearance of typical cytosolic enzymes like lactate dehydrogenase and glucose-6-phosphate dehydrogenase in similar proportions and with the same time course. The release of lysosomal enzymes seen after administration of zymosan or calcium ionophore is thought to be the result of unspecific leakage rather than a specific response of elicited Kupffer cells.

Bile acids reduce the bindtng and uptake of endotoxin by cultured rat kupffer cells

Bile acids reduce the bindtng and uptake of endotoxin by cultured rat kupffer cells

Prostaglandin release but not superoxide production by rat Kupffer cells stimulated in vitro depends on Na+/H+ exchange.

The release of the prostaglandins E2 and D2, induced by zymosan and phorbol ester in cultured rat Kupffer cells, was found to depend on the extracellular concentration of Na+. Eicosanoid formation following the administration of the Ca2+ ionophore A23187 or of arachidonic acid, however, did not require the presence of sodium ions in the medium. A half-maximal rate of prostaglandin release by zymosan-treated Kupffer cells was obtained between 4 mM and 5 mM Na+; and a Na+ concentration of greater than or equal to 30 mM was required to maximally stimulate prostaglandin E2 and D2 formation in the cultured liver macrophages. In contrast, the superoxide production following the administration of zymosan or of phorbol ester was quite independent of extracellular Na+. The zymosan and phorbol-ester-stimulated release of prostaglandins E2 and D2 was inhibited by amiloride. Artificial intracellular alkalization enhanced the prostanoid production of unstimulated and of zymosan-stimulated cells whereas artificial intracellular acidification inhibited the zymosan-elicited prostaglandin synthesis. In contrast, the superoxide formation was independent of the pH changes. The data presented here suggest that the prostaglandin production elicited by zymosan or phorbol ester in cultured rat Kupffer cells requires an activated Na+/H+ exchange.

Transferrin receptor on rat Kupffer cells in primary culture.

Kupffer cells may play a role in the turnover of iron in acute viral hepatitis. The transferrin receptor of rat Kupffer cells in primary culture was therefore investigated in this study. Daily specific bindings on 125I-diferric transferrin (Tf) to rat Kupffer cells in primary culture from day 3 to day 6 of culture were 1.64 +/- 0.08%, 4.16 +/- 0.05%, 4.34 +/- 0.07% and 2.63 +/- 0.07%, respectively. The specificity of the Tf binding sites was examined by competition studies showing that galactose (30 mmol x l-1) and ovalbumin (90 mumol x l-1) did not compete for the binding sites, but human lactoferrin (50 mumol x l-1) competed for the binding sites by about 30%. The affinity and capacity of Tf receptor on rat Kupffer cells in 5-day culture were analyzed according to the method of Scatchard. A single class of 125I-diferric Tf binding sites with an affinity constant of 1.65 x 10(7) l x l-1) and a capacity of 6.86 x 10(6) sites/cell was found. After zymosan (500 micrograms/ml) preincubation for 30 min, the binding capacity increased about 1.7-fold, and this increase depended upon the increase of the affinity of Tf receptor. These data suggest that Kupffer cells in the activated state accelerate the removal of elevated serum iron.

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[ 14C]choline and contained [ 3H]inulin or [ 125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 μM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 μM), the drug also reduced the internalization, while a high concentration (22 μM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro.

We investigated the intracellular metabolic fate of [Me-14C]choline-labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline-phospholipid and phosphatidylserine (molar ration 5:4:1). With both labeled compounds only small proportions of water-soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell-associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of water-soluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine. With [Me-14C]choline-labeled lysophosphatidylcholine, label was found to become cell-associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents. It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4°C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161–176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca 2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4°C and 37°C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.

培養ラットＫｕｐｆｆｅｒ細胞のｅｎｄｏｃｙｔｏｓｉｓに及ぼすエタノールとアセトアルデヒドの影響について

Pronase消化法および遠心分離法によりラット肝より分離培養したKupffer細胞のendocytosisに及ぼすethanol (EtOH), acetaldehyde (AA)の影響を種々の被食粒子を用い,光顕,電顕的に検討した.50mMおよび100mM EtOHの作用下では,培養Kupffer細胞の表面突起数の減少が観察され,phagocytosisで取り込まれる直径2.2μのlatex粒子のen-docytosisは培養液中EtOHのdose dependentに抑制をうけたが,pinocytosisで取り込まれる直径0.59μのlatex粒子およびstreptococcal preparationであるOK-432粒子のendocytosisは抑制をうけなかった.200μ AA作用下では,Kupffer細胞の表面突起数の著明な変化は認められず,上記被食粒子のendocytosisも抑制をうけなかった.これらのことより,EtOHは直接的に培養Kupffer細胞の表面突起数を減少させ,phagocytosisを抑制すると考えられた.

Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells.

Rat Kupffer cells in monolayer culture were allowed to phagocytose unopsonized zymosan granules. They responded with a strongly stimulated synthesis and release of prostanoids, mainly the immunologically determined prostaglandins PGE2 and PGF2 alpha. The same response could be obtained by treatment with the calcium ionophore A23187. The effects of the ionophore and the zymosan particles were of the same magnitude but not additive. The rapid uptake of Ca2+ after contact with phagocytosable material recently described by us [(1983) Eur. J. Biochem. 131, 539-543] appears to mediate the enhanced prostaglandin synthesis. That response was suppressed not only by indomethacin but also by trifluoperazine which does not inhibit Ca2+ entry in the Kupffer cells. Similar effects by R24571 and 4-bromophenacyl bromide support the participation of calcium-calmodulin and of phospholipase A2. The calcium channel blocker Verapamil did not influence the zymosan-provoked production of prostaglandin PGE2 nor were any indications obtained for a feedback inhibition by PGE1 or PGE2. Contact with zymosan resulted in a rapid but transient rise of the intracellular levels of cAMP and cGMP: 10 nM indomethacin completely blocked the increase of both cyclic nucleotides while trifluoperazine elicited different responses in the cAMP and cGMP levels. The stimulated release of prostaglandin E2 was inhibited in a dose-dependent manner by nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase and by FPL 55712, known as a receptor antagonist for some leukotrienes. This suggests a regulatory role for its metabolites on prostaglandin synthesis.

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert 3H-labeled inulin or the degradable 125I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4°C were effectively internalized and processed intracellulary after shifting the temperature to 37°C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37°C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37°C. From the temperature-change experiments we conclude that, at 4°C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37°C.

Calmodulin content and activity of Ca2+-ATPase and phospholipase A2 in rat Kupffer cells.

A protein resembling calmodulin was isolated from non-parenchymal and parenchymal cells of rat liver by affinity chromatography. The biological activity of the purified protein was assessed by the bovine brain cAMP phosphodiesterase assay. A highly sensitive radioimmunoassay as well as the cAMP phosphodiesterase method were employed to determine the calmodulin content of crude extracts from monolayer cultures of rat Kupffer cells and hepatocytes. An ATP-dependent, calmodulin-enhanced calcium transport was demonstrated in a membrane fraction of the non-parenchymal cells. Phospholipase A2 activity specific for 2-arachidonoyl phosphatide and with a pH optimum of 8.1 was measured in homogenized Kupffer cells; it was stimulated by agents previously shown to enhance prostaglandin synthesis in Kupffer cells, e.g. zymosan particles and lipopolysaccharide isolated from Salmonella minnesota. The increase in activity was completely prevented by pretreatment with or simultaneous addition of R 24571, a known calmodulin antagonist. However, if this inhibitor or calmodulin was added to the cell-free extract phospholipase A2 activity was not influenced. Phospholipase A1 activity could be detected at pH 5 only, showing a slight decrease in the homogenate of stimulated macrophages. Acyltransferase activity was high but independent of treatment of the Kupffer cells.

Interaction of liposomes with Kupffer cells in vitro

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14 C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4 : 5 : 1) and contained either 3 H-labelled inulin or 125 I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2–3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10 6 cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15–20%) both at 4°C and at 37°C. In the presence of metabolic inhibitors the uptake at 37°C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.

Ca2+ flux as an initial event in phagocytosis by rat Kupffer cells.

Ca2+ uptake by monolayer cultures of rat Kupffer cells was strongly stimulated immediately after contact with phagocytosable material, e.g. zymosan particles. The intensity of the luminol-mediated chemiluminescence following zymosan addition was dependent on extracellular Ca2+; addition of the Ca2+-ionophore A 23187 did not further increase the zymosan-elicited response. The superoxide-mediated chemiluminescence was not inhibited by indomethacin but could be suppressed by compounds known to inhibit Ca2+-calmodulin-dependent reactions. Analysis of their efficiency, however, suggests that the O-2 production is mediated by Ca2+ rather than Ca2+-calmodulin and that the inhibitors exert another, e.g. membrane-directed influence.

Penetration and uncoating of frog virus 3 (FV 3) in cultured rat Kupffer cells

The in vitro interactions between unenveloped frog virus 3 and Kupffer cells have been studied at 37°, a nonpermissive temperature for viral multiplication. Kupffer cells were isolated by collagenase perfusion of rat livers followed by purification of the cells by centrifugal elutriation. Electron microscopic examinations revealed peculiar modes of interaction with the cellular membranes. Two hours after infection most virus particles were present in phagocytic vacuoles or dense and only few in pinocytic vesicles. About 25% of the engulfed virions displayed fusion between the trilaminar internal core membrane and the membrane of the vacuoles resulting in an opening of the shell that allowed dense core material to be released into the cytoplasm. The empty capsids were integrated into the membrane of the vacuole and were often invaded by the endoplasmic reticulum. Some virions fused directly with the plasma membrane allowing their electrondense content to be shed into the cytoplasm from the outside. Finally, some particles came into contact with the plasma membrane through a stalk-like structure which permitted the passage of the dense core material into the cell.

Erythrophagocytosis inhibition in cultured rat Kupffer cells infected with frog virus 3 (FV3): An ultrastructural study

Resume L'infection in vitro de cellules de Kupffer de rat avec du «frog virus 3 (FV3) conduit a une inhibition de leur pouvoir de phagocytose. Des etudes en microscopie electronique a transmission et a balayage montrent que l'attachement des globules rouges de mouton opsonises aux cellules infectees n'est pas modifie. Il s'effectue comme dans les cellules non infectees, soit par des evaginations des erythrocytes qui s'engagent dans de petites invaginations de la membrane cytoplasmique, soit par la capture des erythrocytes par l'intermediaire de pseudopodes. Cependant, dans les cellules de Kupffer infectees avec le FV3, le deuxieme stade de l'erythrophagocytose, c'est-a-dire l'ingestion des globules rouges, est totalement inhibee. Les observations ont montre d'autre part que l'inhibition de l'erythrophagocytose n'est pas apparemment liee a des alterations morphologiques entrainees par l'infection virale. En effet, 1 heure apres l'infection, en dehors de quelques anomalies du noyau, les cellules de Kupffer ne presentent pas de lesions.

Lysosomal enzymes in cultured rat kupffer cells

Lysosomal enzymes were measured in rat Kupffer cells cultured for different periods of time in medium containing varying amounts of newborn calf serum. Specific activities of acid DNAse, cathepsin D and β-acetylglucosaminidase were found to increase several-fold during the first week in vitro while acid phosphatase and β-glucuronidase activities remained constant. The increases were found roughly proportional to the amount of serum available in the culture medium. Phagocytosis of opsonized sheep red cells on days 4–6 did not have any significant effect on the lysosomal enzyme activities. The possibility is suggested that endocytosis does not induce increases in lysosomal enzyme activities, and that the increases observed were part of a general positive growth response to in vitro conditions.

Uptake of macromolecules by rat Kupffer cells in vitro

Pinocytosis was studied in cultured rat Kupffer cells by using colloidal 198Au, 131I-labelled serum albumin and 3H-labelled sucrose as test substances. The kinetics of uptake as well as electron microscopic examinations indicated that colloidal gold was pinocytosed directly as part of the fluid medium without prior adsorption to the cell membrane. This process was resistant to cytochalasin B (CB), colchicine, and metabolic inhibitors at concentrations where phagocytosis would have been totally inhibited, but was reduced to less than 10% by low temperature. These data suggest that colloidal gold was taken up by micropinocytosis. Serum showed an inhibitory effect on colloidal gold and albumin uptake whereas it stimulated sucrose uptake. Sucrose uptake was also more sensitive to CB. The possibility and significance that distinct processes of macropinocytosis and micropinocytosis with separate control mechanisms may be occurring simultaneously in these cells were discussed.

On the mechanism of internalization of opsonized particles by rat Kupffer cells in vitro

The attachment and internalization of opsonized sheep red blood cells by cultured rat Kupffer cells were studied with phase-contrast and scanning electron microscopy (SEM) as well as timelapse microcinematography. We observed that sheep red cells coated with IgG attached over the entire Kupffer cell surface at random, whereas those coated with IgM and complement attached all over the cell with the exception of the extreme periphery. When the Fc and C 3 receptors were given appropriate stimuli to internalize the attached red cells, they functioned very differently. In Fc internalization, the Kupffer cell membrane rose above the main cell body and wrapped tightly around the attached red cell, eventually surrounding it entirely. In the C 3 internalization, triggered by new-born calf serum, the membrane activity was less spectacular; the folds that did sometimes rise up were coarser and did not fit tightly around the red cell, which was eventually interiorized by a sinking, deep into the cytoplasm of the Kupffer cell. These two mechanisms of internalization also showed different sensitivities to cytochalasin B (CB); the Fc internalization being far more vulnerable to this inhibitor of microfilament activities. Studies with colchicine, however, did not show any clear-cut difference in sensitivity between the two cases.