Rat Liver Macrophages Research Articles

Transcriptome and proteome profiling reveal complementary scavenger and immune features of rat liver sinusoidal endothelial cells and liver macrophages

BackgroundLiver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body’s most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics.ResultsWe observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation.ConclusionsOur findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.

An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

Studies on liver macrophages have elucidated their key roles in immunological, fibrotic and regenerative responses, and shown that macrophages are not a homogeneous population. In the rat, two sets of liver macrophages coexist, identified by ED1 and ED2 antibodies. Those sets have different quantitative responses in liver injuries and may have different tasks throughout the injury and recovery phases. Nevertheless, the total number (N), number per gram (N g(-1)) and proportion of those macrophages in relation to other liver cells has never been quantified using design-based stereology. Thus, we combined immunocytochemistry with those tools to produce an unbiased estimate of the N of ED1(+) and of ED2(+) cells. A smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 months old), to obtain systematic uniform random sections (30 microm thick); these were immunostained with the monoclonal antibodies: ED1, a pan-macrophagic marker; and ED2, which identifies the completely differentiated macrophages, i.e. Kupffer cells. The N of ED1(+) cells was 340 x 10(6), estimated with a coefficient of error (CE) of 0.04, and that of ED2(+) cells was 283 x 10(6), with a CE of 0.05. These figures correspond to 10.7% and 8.9%, respectively, of the total liver cells. The new data constitute reference values for correlative inferences. Also, the methodological strategy, by its accuracy and precision, is valuable for future investigations on the liver cell composition in various models of disease, and especially for studying the more subtle variations that occur during the injury and recovery phases.

Binding of lipopolysaccharide and complexes of lipopolysaccharide to serum low density lipoproteins to liver macrophages

Binding of [3H]-lipopolysaccharide toxin (LPS) and complexes of LPS with serum [125I]-labeled low density lipoproteins (LDL) with primary culture of rat liver macrophages (Kupffer cells) has been studied. Total, specific and nonspecific binding was determined. The receptor interaction was shown to dominate for both LPS and LDL-LPS complexes, amounting to 70-77% and 80-85%, respectively. The Scatchard plot was essentially non-linear for LPS binding but linear for LDL-LPS complexes. At the LPS Scatchard graph, however, two regions approximately fitting linear regression could be identified. Those regions correspond to two different types of specific binding sites: the first is for lower toxin concentrations of 0.25-0.50 microg/ml with K(d) = 0.75 microg/ml; while the second is for higher LPS concentrations of 7.5-15 microg/ml with K(d) = 5.39 microg/ml. For LDL-LPS complexes only K(d) equal to 2.80 microg/ml was ascertained. The LDL-LPS complexes significantly blocked the LPS binding (-40%) while acetylated or oxidized LDLs exerted a less pronounced effect. LPS inhibited binding of LDL-LPS complexes (-60%), while acetylated or oxidized LDLs suppressed interaction of LDL-LPS complexes with Kupffer cells insignificantly. It is suggested that, while binding to the Kupffer cell surface, a substantial portion of both LPS and LDL-LPS complexes share the same scavenger receptors with which however, modified LDLs interact weakly. The LDL-LPS complexes can interact, apart from receptors common with LPS, with other receptors exhibiting similar binding parameters, with the apo-B/E receptors playing an inessential role.

PGE 2 exerts its effect on the LPS-induced release of TNF-α, ET-1, IL-lα, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-α, endothelin (ET)-1, interleukin (IL)-lα, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E 2 inhibits the release of the fibrogenic mediators TNF-α, ET-1 and IL-lα, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE 2 is mimicked by specific agonists for the PGE 2 receptors EP2 and EP4; whereas, agonists for the PGE 2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE 2 receptors EP2 and EP4 but not the PGE 2 receptors EP1 and EP3. These data suggest that PGE 2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-α, ET-1 and IL-lα, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages.

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: Regulation by Group IV cytosolic phospholipase A 2, but not by Group V and Group IIA secretory phospholipase A 2

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2–4 h) of arachidonic acid (AA) and prostaglandin (PG) D 2 in rat liver macrophages. Group IV cytosolic phospholipase A 2 (cPLA 2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA 2 shows an enhanced in vitro activity. The Ca 2+ dependence of cPLA 2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA 2 mRNA (after 2–4 h) and an enhanced expression of cPLA 2 protein (after 8 h). The cellular cPLA 2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA 2 (sPLA 2). LPS has no effect on the levels of Groups V and IIA sPLA 2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA 2 protein and sPLA 2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA 2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA 2.

INTERFERENCE OF MACROPHAGES WITH IMMUNOTARGETING OF LIPOSOMES

ABSTRACTWe investigated the binding, uptake and intracellular degradation of immunoliposomes by isolated rat liver macrophages in vitro. Immunoliposomes were prepared either by coupling a randomly thiolated anti-CC531 rat colon adenocarcinoma monoclonal antibody to bilayer-incorporated MPB-PE by means of a thioether linkage or by attaching it through its Fc moiety to the distal terminus of hydrazide-modified PEG-DSPE. The two immunoliposome preparations clearly differ in their interaction with the tumor target cells, as well as with the macrophages. At comparable antibody densities both cell types show 1.5–2-fold higher levels of association for the Hz-PEG-immunoliposomes than for the MPB-PEG-immunoliposomes. We provide evidence that immunoliposome macrophage-interaction is both Fc-receptor and scavenger receptor mediated to about equal extents. At low antibody density the hydrazide immunoliposomes favor interaction with the tumor cells to that with macrophages. At higher antibody densities, on the other hand, interaction of these liposomes with the macrophages is increasingly favored, mostly due to enhanced scavenger receptor mediated uptake. The rate of intracellular degradation of (immuno)liposomes internalized by liver macrophages is barely influenced by the presence of either PEG or immunoglobulins on the liposomal surface.

Expression of Glutamine Synthetase in Macrophages

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.

LPS and TNFα induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages

Recent findings indicate that cytokine signaling can be modulated by other mediators of simultaneously activated signal transduction pathways. In this study we show that LPS and TNFα are potent inhibitors of IL-6-mediated STAT3 activation in human monocyte derived macrophages, rat liver macrophages and RAW 264.7 mouse macrophages but not in human hepatoma cells (HepG2) or in rat hepatocytes. Accordingly, LPS and TNFα were found to induce the expression of SOCS3 mRNA in each of the investigated type of macrophages but not in HepG2 cells. Using a specific inhibitor, evidence is presented that the p38 MAP kinase might be involved, especially for the inhibitory effect of TNFα.

Trypanosoma cruzi: Detection of a Surface Antigen Cross-Reactive to Human C-Reactive Protein

Coutinho, C. M. L. M., Cavalcanti, G. H., Bonaldo, M. C., Mortensen, R. F., and Araújo-Jorge, T. C. 1998.Trypanosoma cruzi: Detection of a surface antigen cross-reactive to human C-reactive protein.Experimental Parasitology90, 143–153. C-reactive protein (CRP) is an acute phase protein secreted by liver hepatocytes, and is also found on the surface of lymphocytes and as a membrane-associated protein expressed on rat liver macrophages and human monocytes. C-reactive protein levels increase in the sera of children infected withTrypanosoma cruzi, during the acute phase of Chagas' disease, but its role in the course of this infection is unknown. Experiments designed to detect the binding of CRP to circulating forms ofT. cruzifailed to observe it because anti-human CRP antibodies bind to the parasite. The present work intended to further clarify this novel question related to the anti-CRP cross-reactivity with the parasite. Indirect immunofluorescence, immunoenzymatic, flow cytometry, and Western blot assays showed that three different polyclonal anti-human CRP antibody preparations bind toT. cruzisurface. This binding is dose-dependent, saturable, and is inhibited when anti-CRP antibodies from different species were allowed to compete, indicating the specificity of the reactivity. The antibodies recognized a protein band below 23 kDa in Western blot analysis of parasite extracts. The divalent cation chelators EDTA and EGTA impaired the antigen recognition by the antibodies. The binding to parasite surface was also observed with some available monoclonal antibodies raised against human CRP. A polyclonal anti-human CRP presented an inhibitory effect on invasion of heart muscle cells byT. cruzi. Our results indicate that a molecule antigenically related to CRP, a possible CRP-like molecule, is expressed on the surface ofT. cruzi.

Osmolyte Strategy in Human Monocytes and Macrophages: Involvement of p38MAPKin Hyperosmotic Induction of Betaine and Myoinositol Transporters

Betaine and myoinositol are compatible organic osmolytes which are specifically accumulated by cells exposed to hyperosmotic medium. A role for compatible organic osmolytes in the regulation of immune function for rat liver macrophages has been described recently. This report describes an osmolyte strategy in human peripheral blood monocytes and human peripheral blood-derived macrophages. Hyperosmotic (405 mOsm) exposure of monocytes and macrophages led to an upregulation of betaine/γ-amino-n-butyric acid (GABA) transporter BGT-1 and sodium-dependent myoinositol transporter SMIT in mRNA levels within 6 to 12 h. Induction of BGT-1 and SMIT mRNA occurred regardless of whether hyperosmolarity was induced by addition of NaCl (50 mM) or raffinose (100 mM). Betaine (5 mM) inhibited upregulation of BGT-1 as well as SMIT mRNA. After hyperosmotic (405 mOsm) exposure uptake of betaine and myoinositol was increased up to 10-fold compared to normoosmotic conditions. Hypoosmotic exposure led to a rapid efflux of betaine and myoinositol. Treatment of cells with the pyridinyl imidazole SB 203580 (10 μM), a specific inhibitor of p38 MAP kinase, inhibited the hyperosmolarity-induced increase in BGT-1 and SMIT mRNA as well as betaine and myoinositol uptake by 45–70%. The data show that human peripheral blood monocytes and human peripheral blood-derived macrophages use betaine and myoinositol are compatible organic osmolytes when exposed to osmotic stress and that p38MAPKis involved in hyperosmolarity-induced upregulation of osmolyte transporters BGT-1 and SMIT.

Activation of mitogen-activated protein kinases and IL-6 release in response to lipopolysaccharides in Kupffer cells is modulated by anisoosmolarity

Background/Aims: The influence of anisoosmolarity on the activation of the extracellular signal-regulated kinases-1 and -2 and on interleukin-6 release was studied in lipopolysaccharide-stimulated rat liver macrophages. Methods: Experiments were performed with rat liver macrophages. Activation of the extracellular signal-regulated kinases was determined by kinase shift assay and immune complex kinase assay. Interleukin-6 mRNA was measured by Northern blot analysis and interleukin-6 production by enzyme-linked immunosorbent assay. Results: Lipopolysaccharide-induced activation of the extracellular signal-regulated kinases-1 and -2 was enhanced in hypoosmotic media (205 mosm/l) and diminished by hyperosmotic (405 mosm/l) exposure when compared to normoosmotic (305 mosm/l) conditions. These effects were paralleled by changes in lipopolysaccharide-stimulated interleukin-6 mRNA expression, when determined after 4 h and interleukin-6 release after 18 h. The mitogen-activated protein kinase-kinase inhibitor PD 098059 abolished phosphorylation of the extracellular signal-regulated kinases-1 and -2 in response to lipopolysaccharide, irrespective of the medium osmolarity, and diminished lipopolysaccharide-induced interleukin-6 mRNA expression and interleukin-6 production under normo- and hypoosmotic conditions by about 50%; it also resulted under hyperosmotic conditions in an about 80% inhibition. SB 203580, a specific inhibitor of p38 largely abolished interleukin-6 mRNA expression and interleukin-6 production, irrespective of medium osmolarity, whereas phosphorylation of the extracellular signal-regulated kinases was not affected. Conclusions: The data indicate a modulation of lipopolysaccharide-induced interleukin-6 production by ambient osmolarity and an involvement of both p38 and the extracellular signal-regulated kinases-1 and -2 in the stimulation of interleukin-6 production by lipopolysaccharide.

Betaine as an osmolyte in rat liver: metabolism and cell-to-cell interactions.

Betaine was recently identified as an osmolyte in rat liver macrophages (Kupffer cells [KCs]) and sinusoidal endothelial cells (SECs). Betaine interferes with KC functions, such as phagocytosis, cytokine, and prostaglandin syntheses. As betaine is derived from choline, the present study was undertaken to evaluate osmosensitivity and cell heterogeneity of choline metabolism in rat liver. In the perfused rat liver after in vivo prelabeling with [14C]-choline, hypoosmotic stress induced a radioactivity release into the perfusate which was identified as [14C]-betaine by high-performance liquid chromatography (HPLC) analysis and which was inhibited by the anion exchanger inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Choline metabolism was studied in cultured liver parenchymal cells, (PCs), KCs, and SECs. Choline was taken up by all but betaine formation from choline was only detectable in PCs and not in KCs and SECs. Betaine formation in PCs was not stimulated by hyperosmolarity; rather, betaine has a role as an osmolyte in KCs and SECs but is of minor importance in PCs, as evidenced by only minor hyperosmolarity-induced betaine uptake. Thus, liver PCs can produce and release betaine derived from choline, and, thereby, possibly supply the osmolyte important for KC and SEC cell function. This may be another example for cell-to-cell interaction in the liver.

AFM IMAGING AND ELASTICITY MEASUREMENTS ON LIVING RAT LIVER MACROPHAGES

The authors investigated the morphology and the elastic properties of living cultured rat liver macrophages (Kupffer cells) with an atomic force microscope (AFM). Continuous imaging and elasticity mapping of individual cells in physiological buffer was carried out for several hours without damaging the cells as judged by their persistent undisturbed morphology. Dynamic events such as protrusive activity were observed in time course. The importance of the cytoskeleton for the mechanical properties of the cell has been investigated by measuring the cell's elasticity as a function of position. Chemical disassembly of the actin network by applying 10μg/ml cytochalasin B decreased the cell's average elastic modulus seven-fold within less than 40 minutes. Treating the cells with 0.1μg/ml latrunculin A resulted in a two-fold decrease in the elastic modulus merely in the perinuclear region after 40 minutes, whereas other parts of the cell were not affected.

Myo-Inositol is an osmolyte in rat liver macrophages (Kupffer cells) but not in RAW 264.7 mouse macrophages.

The role of myo-inositol as an osmolyte was studied in cultured rat liver macrophages (Kupffer cells). Hyperosmotic exposure of Kupffer cells stimulated myo-inositol uptake and led to an increase in the mRNA levels for the sodium/myo-inositol cotransporter (SMIT). Conversely, hypo-osmotic (205 m-osM) exposure diminished myo-inositol uptake when compared with normo-osmotic (305 m-osM) control incubations. The hyperosmolarity-induced SMIT mRNA increase was counteracted by added myo-inositol or betaine. In contrast with Kupffer cells, there was only a slight hyperosmotic stimulation of myo-inositol uptake in RAW 264.7 mouse macrophages, and the myo-inositol transporter (SMIT) mRNA was not detectable. Further, a slight stimulation of taurine uptake and an increase in taurine transporter (TAUT) mRNA level by hyperosmolarity was observed in RAW 264.7 cells, whereas hypo-osmolarity led to a decrease in taurine uptake and TAUT mRNA level. When Kupffer cells were preloaded with myo-inositol, hypo-osmotic exposure led to a rapid efflux of myo-inositol from the cells. Myo-inositol efflux was also stimulated by phagocytosis of latex particles; however, latex was without effect on the hyperosmolarity-induced increase of SMIT mRNA levels. The results suggest a role of myo-inositol as an osmolyte in rat Kupffer cells but not in RAW 264.7 mouse macrophages. The functional relevance of this osmolyte strategy might lie in the maintenance of cell volume homeostasis during phagocytosis in Kupffer cells; however, the interplay with the other osmolytes betaine and taurine remains to be established.

Taurine is an osmolyte in rat liver macrophages (Kupffer cells)

Background/Aims: The availability of betaine as an osmolyte was recently shown to interfere strongly with important cell functions of liver macrophages (Kupffer cells), such as eicosanoid and tumor necrosis factor-α production or phagocytosis. We therefore investigated whether taurine is also used as an osmolyte by Kupffer cells and whether it is involved in the control of Kupffer cell functions. Methods/Results: Hyperosmotic (hypoosmotic) exposure of cultured rat liver macrophages (Kupffer cells) for 6–12 h led to an increase (decrease) in the mRNA levels of the taurine transporter (TAUT) and an increase (decrease) in taurine transport into the cells. The hyperosmolarity-induced increase in TAUT-mRNA levels was diminished by 37±10% upon addition of taurine, but not upon addition of betaine. When Kupffer cells were preloaded with taurine, hypoosmotic exposure led to a rapid efflux of taurine from the cells, which was significantly delayed in the presence of the anion exhanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). Taurine efflux was also stimulated during phagocytosis of Latex particles; however, Latex was without effect on the hyperosmolarity-induced increase of TAUT mRNA levels. Lipopolysaccharide (LPS) led to an induction of cyclooxygenase-2, which was markedly enhanced during hyperosmotic conditions. Taurine diminished the induction of cyclooxygenase-2 and inhibited the LPS/hyperosmolarity-induced stimulation of prostaglandin E 2 formation. Conclusions: The data suggest that, in addition to betaine, taurine also acts as an osmolyte in Kupffer cells, and that taurine availability may be an important modulater of Kupffer cell functions such as eicosanoid synthesis.

Osmoregulated taurine transport in H4IIE hepatoma cells and perfused rat liver.

The effects of aniso-osmotic exposure on taurine transport were studied in H4IIE rat hepatoma cells. Hyperosmotic (405 mosmol/l) exposure of H4IIE cells stimulated Na+-dependent taurine uptake and led to an increase in taurine transporter (TAUT) mRNA levels, whereas hypo-osmotic (205 mosmol/l) exposure diminished both taurine uptake and TAUT mRNA levels when compared with normo-osmotic (305 mosmol/l) control incubations. Taurine uptake increased 30-40-fold upon raising the ambient osmolarity from 205 to 405 mosmol/l. When H4IIE cells and perfused livers were preloaded with taurine, hypo-osmotic cell swelling led to a rapid release of taurine from the cells. The taurine efflux, but not taurine uptake, was sensitive to 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), suggestive of an involvement of DIDS-sensitive channels in mediating volume-regulatory taurine efflux. Whereas in both H4IIE rat hepatoma cells and primary hepatocytes TAUT mRNA levels were strongly dependent upon ambient osmolarity, mRNAs for other osmolyte transporters, i.e. the betaine transporter BGT-1 and the Na+/myo-inositol transporter SMIT, were not detectable. In line with this, myo-inositol uptake by H4IIE hepatoma cells was low and was not stimulated by hyperosmolarity. However, despite the absence of BGT-1 mRNA, a slight osmosensitive uptake of betaine was observed, but the rate was less than 10% of that of taurine transport. This study identifies a constitutively expressed and osmosensitive TAUT in H4IIE cells and the use of taurine as a main osmolyte, whereas betaine and myo-inositol play little or no role in the osmolyte strategy in these cells. This is in contrast with rat liver macrophages, in which betaine has been shown to be a major osmolyte.

Toxicity of doxorubicin entrapped within long-circulating liposomes

We studied the effect of doxorubicin entrapped within long-circulating liposomes (Dox-LCL) on the phagocytic capacity and bacterial blood clearance capacity of rat liver macrophages. Dox-LCL (125 nm in diameter) were composed of egg phosphatidylcholine (PC), cholesterol (CH) and poly(ethyleneglycol)distearoylphosphatidylethanolamine (PEG-PE) (55:45:5 molar ratio; MW PEG 1900), and loaded with doxorubicin by means of a trans-membrane pH gradient. The doxorubicin/lipid ratio was 0.36:1 (mol/mol). At different time-points after one, two or three intravenous injections of Dox-LCL, radiolabeled negatively charged test liposomes (egg PC, CH, and phosphatidylserine in a 4:5:1 molar ratio) were injected. After 2 h, liver macrophages were isolated and the amount of macrophage-associated radioactivity was determined. Twenty-four hours after a single injection of 5 mg/kg Dox-LCL, no significant effect was observed. However, 48 h after injection, phagocytic activity was reduced significantly (49%). Recovery of phagocytic capacity of the liver macrophages took 8 days after two injections of Dox-LCL (2×5 mg/kg). Seventy-two hours after the last of two injections of Dox-LCL, bacterial blood clearance was significantly reduced as compared to clearance in control rats and in rats injected twice with doxorubicin combined with placebo liposomes. When comparing these Dox-LCL data with previous data on the effects of Dox-L, Dox-LCL appears less toxic than Dox-L for the liver macrophage population following i.v. administration both with respect to specific phagocytic activity and cell numbers. Due to the delay in onset of toxic effects and the faster recovery from Dox-LCL treatment as compared to Dox-L treatment, it is conceivable that therapeutic protocols can be designed with Dox-LCL that circumvent long-term impairment of the liver macrophage population.

Regulation of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages.

The liberation of arachidonic acid (AA) is thought to be the rate-limiting step in prostanoid synthesis of rat-liver macrophages (RLM)1. The best known pathway for AA-liberation is elicited by the activation of a phospholipase (PL)A2 hydrolysing the ester-bond in sn-2-position of phospholipids2. It has been shown that RLM express a cPLA2 very similar to cPLA2 recently identified in U937 cells2: the enzyme has an apparant molecular weight of 100 kDa in SDS-polyacrylamide-gelelectrophoresis (PAGE), an alkaline pH-optimum, is strictly dependent on the presence of Ca2+ in the range from 10−7–10−6 M and shows a Ca2+-dependent translocation to cellular membranes1,2. cPLA2 phosphorylation by mitogen activated protein (MAP) kinase is also discussed as a mechanism for cPLA2 activation3. Another pathway leading to AA-release in RLM involves the activation of a PLC leading to free diacylglycerol (DAG), which serves as substrate for DAG-lipase to liberate free AA4.KeywordsArachidonic AcidU937 Cells2Kinase AssayExtracellularly Regulate KinaseArachidonic Acid ReleaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Modulation of tumor necrosis factor-α release by anisoosmolarity and betaine in rat liver macrophages (Kupffer cells)

Hypoosmotic exposure (205 mosmol/l) of rat liver macrophages together with lipopolysaccharide (LPS) inhibited the LPS-induced tumor necrosis factor-α (TNF-α) release by about 60% and markedly diminished the LPS-induced increase of TNF-α mRNA levels. Hyperosmotic exposure (405 mosmol/l) had no effect on total TNF-α release, however, both TNF-α accumulation in the medium and the LPS-induced increase of TNF-α mRNA levels were significantly delayed under these conditions. This delay was abolished upon addition of betaine, which acts as an osmolyte in Kupffer cells. When LPS was added to Kupffer cells that had been preexposed to hyperosmotic medium for 24 h, the LPS-induced TNF-α release was inhibited by 90% when compared to normoosmotic conditions. Likewise, the LPS-induced increase in TNF-α mRNA levels was largely abolished. Inhibition of TNF-α release and of the increase in the TNF-α mRNA level in response to hyperosmolarity/LPS, however, was largely overcome when indomethacin or betaine was present during the hyperosmotic preincubation period. Because betaine has recently been shown to inhibit the hyperosmolarity-induced induction of cyclooxygenase-2 and stimulation of prostaglandin production, these findings suggest that the effect of betaine in restoring the LPS-induced TNF-α response in hyperosmotically exposed Kupffer cells is mediated by an inhibition of prostaglandin synthesis. The findings point to a regulatory role of cell volume and betaine for TNF-α production by liver macrophages, suggesting a new role of osmolytes in modulating immune function.

Modulation of phagocytosis by anisoosmolarity and betaine in rat liver macrophages (Kupffer cells) and RAW 264.7 mouse macrophages

Hypoosmotic exposure (205 mosmol/l) of rat liver macrophages (Kupffer cells) for 12 h stimulated phagocytosis of latex particles by about 20%, whereas hyperosmotic exposure (405 mosmol/l) resulted in 30–40% inhibition. Inhibition of phagocytosis by hyperosmolarity was fully prevented in the presence of betaine, which acts as an osmolyte in liver macrophages. When hyperosmotically exposed Kupffer cells were preloaded with betaine, induction of phagocytosis by addition of latex particles led to the stimulation of betaine efflux from the cells. Stimulation of phagocytosis also inhibited the hyperosmolarity-induced cumulative uptake of betaine into Kupffer cells, but did not prevent the hyperosmolarity-induced increase in BGT1-mRNA levels. Whereas these findings suggest an involvement of cell volume and betaine in the regulation of phagocytosis in Kupffer cells, betaine transport was not affected upon induction of phagocytosis in RAW 264.7 mouse macrophages. The findings are compatible with a role of betaine in maintaining cell volume homeostasis during phagocytosis in Kupffer cells, but not in RAW 264.7 mouse macrophages. This may be relevant for the maintenance of liver hemodynamics, since volume changes of liver macrophages following ingestion of phagocytozable material might otherwise impair sinusoidal perfusion.