INTRODUCTION
Kupffer cells (KCs) belong to the mononuclear phagocyte system and play an important role in host defense. They are located in the sinusoids of the liver and share many functions with macrophages. Upon inflammatory stimuli such as lipopolysaccharide (LPS), KCs as well as macrophages trigger signals for the production of a variety of bioactive substances. Among these are tumor necrosis factor alpha (TNFα), interleukin-1, interferon-α and -β, prostaglandins, platelet-activating factor, and nitric oxide (1–3
Phagocytosis is a major task of KCs because phagocytotic activity is the primary mechanism through which the immune system eliminates most extracellular pathogenic microorganisms but also apoptotic host cells. In situations where apoptosis contributes to pathophysiologic mechanisms, such as sepsis (4 5 phagocytosis of KCs represents an important protective mechanism (6
Atrial natriuretic peptide (ANP) is a 28 amino acid cyclic peptide with a disulfide bond forming the circular structure. The circulatory hormone is released mainly by the atria of mammalian hearts in response to volume expansion or cardiac hypoxia (7 7 8,9 10 11–13 10 14 phagocytosis and production of reactive oxygen species in macrophages (15 13,16,17 18 11,12 16 19
Recently we have shown that ANP protects the liver against ischemia–reperfusion-induced cell damage after warm and cold ischemia (20,21 21 22–24
Therefore, we investigated whether functional ANP receptors are present on KCs and whether ANP influences the activation of KCs, such as the production of inflammatory mediators or their phagocytotic activity.
MATERIALS AND METHODS
Materials
Rat ANP 99-126 (ANP) was purchased from Calbiochem/Novabiochem (Bad Soden, Germany). TNFα cDNA was obtained from Dr. Decker, University of Freiburg, Germany, and iNOS cDNA probe was provided by Dr. Kleinert, University of Mainz, Germany. Cell culture medium (M 199), fetal calf serum (FCS), penicillin/streptomycin, and TRIzol™ were from Gibco/BRL Life Technologies, Inc. (Eggenstein, Germany). Coumarin-labeled latex particles were from Polysciences (Warrington, PA). cGMP radioimmunoassay kit (cGMP 125 I-assay system), and ECL-detection system were from Amersham-Pharmacia (Freiburg, Germany). [α32 P]-UTP (800 Ci/mmol) was from Hartmann Analytic (Braunschweig, Germany). SP6 polymerase was obtained from Boehringer Ingelheim Bioproducts (Heidelberg, Germany), and monoclonal antibodies against macrophage iNOS and COX-2 were obtained from Transduction Laboratories (Lexington, KY), antibody against TNFα was from Santa Cruz Biotechnology (Heidelberg, Germany). T3/T7 RNA polymerase transcription system was obtained from Stratagene (Heidelberg, Germany). All other materials were purchased from either Sigma (Deisenhofen, Germany) or ICN Biomedicals (Eschwege, Germany).
Animals
Male Sprague–Dawley rats (SAVO, Kisslegg, Germany) with a body weight of 250–280 g were kept under standard conditions with free access to food and water under a 12-h light/dark regimen. We followed existing guidelines for the care and use of laboratory animals. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals and the study was approved by our Institutional Animal Care and Use Committee.
Preparation and culture of cells
Rat KCs were isolated according to the method of Knook and Sleyster (25 26 in situ after cannulation of the portal vein with 100 mL of Gay's balanced salt solution then digested by perfusion with pronase and pronase/collagenase-solutions. The obtained cells were suspended in a pronase/collagenase-solution, shaken carefully for 30 min, and passed through a 100-μm sieve. The hepatocytes were separated by differential centrifugation, and the remaining nonparenchymal cells were separated by a Nycodenz gradient. The density gradient centrifugation was performed at 1500 g for 15 min. The cells of the interphase were collected and separated according to size by counterflow elutriation using a Beckman-centrifuge (J 2-21, JE-6B rotor, Beckman Instruments, Munich, Germany). The obtained KCs were sedimented, resuspended in culture medium (M 199, 15% FCS, 100 U penicillin/mL, 100 μg streptomycin/mL), and counted in a Fuchs-Rosenthal chamber after Trypan Blue-staining. Cells were then seeded at a density of 500 000 cells/well in 24-well or 2.25 × 106 cells/well in 6-well tissue plates and cultivated for 1 to 3 days. Two hours after plating, the cultures were washed to eliminate non-adherent cells. Cultures were kept in a 5% CO2 atmosphere and saturated humidity at 37°C. KC purity was determined using a fluorescent isothiocyanate (FITC)-labeled antiserum against ED2 and fluorescence microscopy and by measuring phagocytosis of coumarin-conjugated latex beads by FACS analysis (FACScan, Becton Dickenson, San Jose, CA). Preparations of KCs were found >90% pure as judged by flow cytometry.
Measurement of cGMP
KCs (set in 24-well tissue plates) were washed three times and pretreated with 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM) in serum-free cell culture medium for 10 min at 37°C. Various stimuli were added for 30 min. Thereafter, medium was aspirated, and cGMP was extracted immediately by the addition of HCl (0.1 N). After 10 min of incubation on ice the cell extracts were transferred to fresh tubes, lyophilized, and assayed for cGMP content by radioimmunosassay using a commercially available kit according to (16
Nitrite accumulation and measurement of PGE2
KCs (24-well plates, 200 μL) were treated with LPS (Escherichia coli , serotype 055:B5, 1 μg/mL, Sigma, Deisenhofen, Germany) in the presence or absence of two concentrations of ANP 99-126 (200 nM, 1 μM). After 20 h the stable metabolite of NO, nitrite, was measured in the medium by the Griess reaction (27 3 PO4 and 90 μL of 0.1%N -(1-naphthyl)ethylenediamine dihydrochloride in H2 O was added, followed by spectrophotometric measurement at 550 nm (reference wavelength 620 nm). In addition, after a 20-h incubation period, PGE2 was measured in the medium by enzyme-linked immunosorbent assay using a commercially available kit.
TNFα bioassay
KCs (24-well plates, 200 μL) after 1–3 days in culture were untreated, treated with ANP alone or treated with LPS (E. coli , serotype 055:B5, 1 μg/mL) in the presence or absence of ANP (1 μM). ANP was added to the cells simultaneously with LPS. After 4 h, TNFα was measured in the supernantant by a L929 cytotoxicity bioassay. This assay is based upon quantitation of the cytotoxic activity of TNFα on L929 cells in the presence of actinomycin D. The mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan was determined as an indicator of cell viability (28 4 cells per well into a 96-well microtiter plate. After incubation for 24 h at 37°C in a humidified atmosphere with 5% CO2 , the medium in the wells was replaced with fresh medium containing actinomycin D at a final concentration of 1 μg/mL. After 1 h of pre-incubation with actinomycin D serial dilutions of supernatants from KCs untreated or treated with test-substances in the presence or absence of LPS for 4 h were added. For quantification of TNFα production, a standard curve was prepared by the addition of recombinant TNFα (0.75 to 50 pM) to the cells. The plates were then incubated for an additional 24 h at 37°C. After removing the supernatant, cells were incubated with MTT (0.5 mg/ml) for 1 h at 37°C and solubilized in DMSO followed by spectrophotometric measurement at 550 nm. The following control experiments were performed: first, in some experiments, positive samples, in which TNFα was neutralized with TNFα antiserum, were assayed in parallel. The addition of the anti-TNFα antibody completely abolished the TNFα activity in supernatants of LPS-activated KCs as well as of recombinant TNFα, indicating that the assay was highly specific for rat TNFα. Second, to exclude a potential direct effect of ANP (1 μM) on L929 viability, the substance was added to actinomycin D-treated L929 cells for 24 h and formazan production was determined as described above. ANP showed no significant alterations of L929 viability.
Western blot analysis
KCs (24-well plates) were either untreated, treated with LPS (1 μg/mL), or a combination of LPS (1 μg/mL) plus ANP (1 μM) for 12 h. Cells were washed with ice-cold phosphate-buffered saline (PBS) and stored at −70°C. Western blot analysis was performed according to Ref. 29
Northern blot analysis
Detection of TNFα mRNA
Northern blot analysis with total RNA (20 μg) was performed as described previously (16 32 P-labeled cRNA probes (2 × 106 cpm/mL). The cDNA probe was a Sma I linearized rat TNFα cDNA fragment subcloned in a pSPT18 vector (from Dr. Decker, University of Freiburg, Germany). The TNFα probe was labeled with [32 P]-UTP (50 μCi, Amersham, Braunschweig, Germany) and Sp6 RNA polymerase. Signal intensities were evaluated by densitometric analysis (Herolab, E.A.S.Y. plus system, Wiesloch, Germany).
Detection of iNOS mRNA
KC treatment and RNA isolation was performed as described above. Membranes were hybridized to a 32 P-labeled murine macrophage iNOS cRNA probe (2 ×106 cpm/mL). iNOS cDNA (558 base pairs) was subcloned into a pBluescript SK(+) vector, linearized (Hin dIII), and labeled with [32 P]-UTP (50 μCi) using a T3 RNA polymerase transcription system. Signal intensity was evaluated by densitometric analysis.
Phagocytosis assayCoumarin-labeled latex particles (No. 098847, carboxylated, 2 μm diameter, Polysciences, Warrington, PA) were opsonized by incubating beads (4 × 109 beads/mL) with 10% rat serum. KCs were seeded in 6-well plates at a density of 2.25 × 106 cells/well, cultivated for 3 days, and incubated in the presence or absence of rat ANP (1 and 0.1 μM), 8-Br-cGMP (1 mM), CNP (1 μM), or the NPR-C agonist cANF (1 μM) for 2 h. Latex beads were sonicated (5 s) before addition to the cells at a final ratio of 100 beads per cell. Incubation was conducted for 30 min. Thereafter the monolayers were washed 4–5 times with PBS, and the absence of considerable amounts of undigested latex particles was controlled microscopically. Cells were harvested with a rubber policeman, cell suspensions transferred to plastic tubes, and centrifuged at 250 g for 20 min. The cell pellet was washed in PBS, resuspended in buffer, and subjected to flow cytometric analysis. Flow cytometry using a FACScan (Becton-Dickinson, Heidelberg, Germany) was used to measure the number of particles ingested per cell. Analysis was performed on 104 cells. The green fluorescence from coumarin-labeled latex beads was collected through a 530-nm bandpass filter. The data acquired were processed using Lysis II software. The fluorescence distribution was displayed in single histograms. The phagocytotic activity was assessed by monitoring the mean fluorescence of phagocytozing cells as well as by calculating the phagocytic index (PI). Mean fluorescence of total ingested particles was determined by recording the fluorescence intensity within the region between cells having ingested at least one bead, two beads, three beads, and cells having ingested more than three beads. The PI was defined as the average number of particles ingested per KCs and was calculated as described in detail (30
Statistical analysis
All data are expressed as the mean ± standard error of mean (SEM) (n = number of cell preparations from different animals). Unless stated otherwise, all experiments were at least performed three times. Statistical significance between groups was determined with the unpaired Students t test. A P value of below 0.05 was considered statistically significant. The statistical analyses were performed with SPSS software, release 6.1.3 (SPSS Inc., Chicago, IL).
RESULTS
Demonstration of functional ANP receptors in KCs
KCs were kept in culture for 1–3 days, untreated or treated with ANP, and cGMP production was determined. Intracellular cGMP concentrations were significantly elevated in KCs exposed to ANP (1 μM) for 30 min compared with untreated cells (Fig. 1 ). This significant increase was even more distinct when KCs were kept in culture for 2 or 3 days. To exclude that enhanced cGMP levels were due to enzymatic activity of soluble guanylate cyclase (sGC), cells were incubated with sodium nitroprusside (SNP), which releases NO, a known activator of sGC (30,31 Fig. 1 ). These data demonstrate the presence of functional guanylate-cyclase-coupled A-receptors for ANP on isolated KCs.
Fig. 1: ANP treatment of KCs significantly elevates intracellular cGMP levels demonstrating the presence of functional guanylate-cyclase-coupled A-receptors for ANP. Intracellular cGMP levels of KCs, kept in culture for 1 to 3 days, were determined in untreated controls (Co), cells treated with ANP (1 μM), or after incubation with sodium nitroprusside (SNP, 100 μg/mL) for 30 min. cGMP was extracted with ice-cold 0.1 N HCl, and cGMP levels were determined by radioimmunoassay. Results are expressed as the mean ± SEM of at least three experiments, each performed in triplicate. * P < 0.005 compared to control.
Effect of ANP on TNFα production and TNFα protein and mRNA levels
The effect of ANP on TNFα production in KCs kept in culture for 1–3 days was investigated (Fig. 2 ). KCs were stimulated with LPS (1 μg/mL) to evoke TNFα production, which was measured in the supernatant by L929 cytotoxicity bioassay. ANP (1 μM) alone showed no effect on basal TNFα production compared to LPS treatment alone (Fig. 2 ). However, coincubation of KCs with ANP and LPS resulted in a significant reduction of TNFα production after 2 and 3 days of culture (Fig. 2 , P < 0.01). Northern blot analysis was performed to determine whether ANP (200 nM and 1 μM) inhibits TNFα mRNA accumulation when added simultaneously with LPS (1 μg/mL). In unstimulated cells no TNFα mRNA was detectable (Fig. 3A ). LPS-induced TNFα mRNA expression was not reduced by ANP (Fig. 3A ), suggesting an influence of ANP on posttransscriptional processing of LPS stimulated TNFα. Indeed, when the cell-associated 32 kDa TNFα, a precursor of TNFα release, was visualized employing Western blot, we observed an increase in LPS-stimulated KCs by ANP (Fig. 3B ). Thus, ANP seems to inhibit TNFα processing in KCs.
Fig. 2: ANP inhibits LPS-induced TNFα production. KCs were untreated (Co), treated with ANP alone, or treated with LPS (1 μg/mL) in the presence or absence of ANP (1 μM) after 1 to 3 days duration of culture. After 4 h, TNFα was measured in the supernantant by a L929 cytotoxicity bioassay. Bars represent percentage of TNFα production compared with LPS-treatment only (100%). Results are expressed as the mean ± SEM of three independent experiments, each performed in triplicate. ** P < 0.01 compared to LPS activated cells.
Fig. 3: Northern blot analysis of TNFα mRNA (A) and Western blot analysis of cell-associated precursor of TNFα (B). In (A), ANP does not affect LPS-induced TNFα mRNA expression. Northern blot analysis of TNFα mRNA. Total RNA was isolated from KCs, which were either unstimulated (Co) or treated with LPS (1 μg/mL) in the presence or absence of ANP (200 nM or 1 μM) for 4 h. Total RNA (15 μg) was loaded per lane and hybridized to a 32 P-labeled cRNA probe for TNFα mRNA. A representative autoradiograph out of three independent experiments is shown. In (B), ANP elevates cell-associated TNFα. Western blot analysis of cell-associated precursor of TNFα (32 kDa) in cell lysates. Samples were loaded on an SDS/PAGE gel, electroblotted, and TNFα protein was detected using an anti-TNFα monoclonal antibody and the ECL detection system. KCs were either unstimulated (Co) or treated with LPS (1 μg/mL) in the presence or absence of ANP (200 nM or 1 μM) for 4 h. A representative autoradiograph out of three independent experiments is shown.
Effect of ANP on NO synthesis and iNOS
Northern blot analysis was performed to determine whether ANP inhibits iNOS mRNA accumulation when added simultaneously with LPS (1 μg/mL). In unstimulated cells, no iNOS mRNA was detectable (Fig. 4C ). ANP (200 nM and 1 μM) caused no significant reduction of LPS-induced iNOS mRNA steady-state levels (Fig. 4C ). To evaluate if ANP treatment reduces iNOS protein expression, iNOS protein was measured by immunoblot with a monoclonal anti-iNOS antibody. No iNOS was detected in unstimulated KC (Fig. 4B ). Upon stimulation with LPS (1 μg/mL, 12 h), a band corresponding to iNOS appeared. The expression of iNOS protein was not significantly changed by ANP (200 nM and 1 μM) (Fig. 4B ). In addition, KCs were exposed to LPS (1 μg/mL) for 20 h to stimulate NO synthesis, measured by determining the concentration of nitrite in the supernatant. A distinct nitrite accumulation was observed in LPS-activated KC compared to untreated cells (Fig. 4A ). Coincubation of KCs with ANP (1 μM) and LPS (1 μg/mL) resulted in no significant change of nitrite accumulation in cells precultured from 1 to 3 days (Fig. 4A ). ANP (1 μM), in the absence of LPS, did not alter the basal nitrite accumulation (data not shown). Taken together, ANP does not affect NO production in KCs.
Fig. 4: Treatment with ANP does not affect nitrite accumulation, iNOS protein expression or iNOS mRNA accumulation. (A) nitrite accumulation: KCs were cultured for 1 to 3 days. Then experimental conditions were established with KCs cultured in either medium alone (Co) or medium containing LPS (1 μg/mL) or a combination of LPS (1 μg/mL) and ANP (1 μM). Culture supernatants were assayed for nitrite accumulation using the Griess reaction. Data are expressed as percentage of nitrite concentration accumulated in the supernatant of LPS-activated KCs (100%) and represent the mean ± SEM of n=3 wells of three independent experiments. (B) Western blot analysis of iNOS in LPS-activated KCs: Detection of iNOS was performed with a specific monoclonal antibody in lysates of KCs either untreated (Co), stimulated with LPS (1 μg/mL), or co-treated with LPS (1 μg/mL) and ANP (200 nM or 1 μM) for 12 h. A representative blot out of three independent experiments with similar results is shown. (C) Northern blot analysis of iNOS mRNA: Total RNA was isolated from KCs, which were either unstimulated (Co) or treated with LPS (1 μg/mL) in the presence or absence of ANP (200 nM or 1 μM) for 6 h. Total RNA (15 μg) was loaded per lane and hybridized to a 32 P-labeled cRNA probe for iNOS mRNA. A representative autoradiograph of three independent experiments is shown.
Effect of ANP on PGE2 synthesis and COX-2 protein expression
To evaluate whether ANP treatment reduces COX-2 protein expression, COX-2 protein was measured by immunoblot with a monoclonal anti-COX-2 antibody. ANP (200 nM and 1 μM) did not alter LPS-induced expression of COX-2 protein (Fig. 5B ). When PGE2 , the most prominent product of COX-2 activity, was measured in the supernatant after 20 h of treatment with LPS (1 μg/mL), ANP showed no effect on PGE2 accumulation (Fig. 5A ). The data clearly indicated that ANP does not influence COX-2 expression.
Fig. 5: Treatment with ANP does not affect PGE2 accumulation or expression of COX-2 protein. (A) PGE2 : KCs were cultured for 12 h. Then experimental conditions were established with KCs cultured in either medium alone (Co) or medium containing LPS (1 μg/mL) or a combination of LPS (1 μg/mL) and ANP (200 nM or 1 μM). Data represent the mean ± SEM of three independent experiments performed in triplicates. (B) Western blot analysis of COX-2 in LPS-activated KCs : Detection of COX-2 was performed with a specific monoclonal antibody in lysates of KCs either untreated (Co), stimulated with LPS (1 μg/mL), or co-treated with LPS (1 μg/mL) and ANP (200 nM or 1 μM) for 12 h. A representative blot out of three experiments with similar results is shown.
Effect of ANP on phagocytosis
The phagocytic activity of KCs was measured by the use of coumarin-labeled latex beads, followed by flow cytometric analysis. Preincubation of KCs with ANP for 2 h increased their phagocytotic activity as shown in the fluorescence histogram of Figure 6A . Treatment of KCs with ANP significantly increased the portion of phagocytozing cells 2.48 ± 0.54 fold and 1.34 ± 0.1 fold (1 μM and 0.1 μM ANP, P < 0.05). The phagocytic index was elevated in ANP-treated cells compared to controls by 2.69 ± 0.6 fold and 1.40 ± 0.15 fold (P < 0.05, Fig. 6B ). Moreover, in the presence of ANP the mean fluorescence of phagocytosing cells was enhanced by 1.51 ± 0.21 fold and 1.27 ± 0.15 fold (P < 0.05, Fig. 6C ), determined as described in (30 phagocytosis .
Fig. 6: Effect of ANP on phagocytosis of latex beads by KCs. Phagocytotic activity of KCs was assessed by measuring the uptake of Coumarin-labeled latex beads by flow cytometry ( see Materials and Methods ). The respective histograms show fluorescence (FL1) of latex beads. Representative histograms display a fluorescence shift to the right of ANP (1 μM, 2 h) exposed cells (ANP, dashed line) in comparison to control cells (Co, solid line) (A). (B) and (C) display the increase of phagocytotic indexes and mean fluorescence intensities after incubation with ANP (1 μM, n = 10; 0.1 μM, n = 5, 2 h each), respectively. Data are means ± SEM given as percentage of phagocytosis activity of control cells (Co, 100%, n = 5). * P < 0.05 compared to control.
NPR-A-mediated effect on phagocytosis
The receptor specificity of the effect of ANP on phagocytosis was determined by investigating the second messenger analogue 8-Br-cGMP, the NPR-B agonist CNP, or the NPR-C agonist cANF. 8-Br-cGMP (1 mM) significantly increased the rate of phagocytozing cells 2.15 ± 0.3 fold (P < 0.05), the phagocytosis index was enhanced by 2.21 ± 0.31 fold (P < 0.05, Fig. 7A ), as well as the mean fluorescence of phagocytosing cells by 1.17 ± 0.11 fold (P < 0.05, Fig. 7B ), whereas neither CNP nor cANF showed any significant alterations on phagocytotic activity (Fig. 7A and B ).
Fig. 7: Receptor specificity of the effect of ANP on phagocytosis in KCs. Phagocytotic activity of KCs treated with either 8-Br-cGMP (1 mm), CNP (1 μm), or cANF (1 μm) was assessed by measuring the uptake of coumarin-labeled latex beads by flow cytometry (see Materials and Methods). (A) and (B) display the phagocytosis indexes and mean fluorescence intensities of three independent experiments performed at least in triplicates. Data are means ± SEM given as percentage of phagocytosis activity of control cells (co, 100%). * P < 0.05 compared to control.
DISCUSSION
The present study shows that KCs represent target cells for the cardiovascular hormone ANP because functional cGMP-dependent ANP receptors were demonstrated in isolated KCs. Furthermore, we investigated for the first time mechanisms by which ANP selectively influences TNFα production by KCs, and we showed that ANP markedly increases phagocytic activity of KCs.
Prior to investigating possible effects of ANP on KC activation, the presence of functional ANP receptors had to be determined. KCs have been shown previously to express NPR-A mRNA (20 31
Our functional studies concerning a potential effect of ANP on KC activation showed a significant decrease of TNFα production after 2 and 3 days of culture, i.e., in the presence of functional ANP receptors. TNFα inhibition after one day of culture was not significant most likely because of impaired receptor function or insufficient receptor density early after the isolation. The data are in line with our recent demonstration of cGMP-mediated inhibition of TNFα production by ANP in murine bone marrow-derived macrophages (18 32,33 34 34 13 16 in vivo relevance of the inhibitory action of ANP on TNFα production: ANP significantly reduced the production of this inflammatory inhibitor in a model of whole human blood (18 18
The fact that the TNFα inhibitory action of ANP is strongest at day 2 is not in accordance with the maximal increase of intracellular cGMP on day 3. We suggest that a decline in certain cell functions, such as signal transduction after cGMP production, may intervene recovery and viability at day three of culture.
To obtain information on the underlying mechanism of ANP-mediated TNFα inhibition, TNFα mRNA levels were determined by Northern blot analysis. LPS-induced TNFα mRNA expression was not significantly affected by ANP, suggesting an influence of ANP on posttransscriptional processing of LPS-stimulated TNFα production. Both transcriptional and posttranscriptional regulation of TNFα synthesis have been described (35,36 37 38 39 18 38,40
In contrast to the well-described inhibitory action of ANP on LPS-induced NO production by iNOS in murine macrophages (13,16,17
Because eicosanoid production by KCs plays a major role in the pathogenesis of septic shock and because KCs release PGE2 in response to in vitro stimulation with LPS, we were interested in the effect of ANP on COX-2 activity and PGE2 synthesis. However, the expression of COX-2 protein and the PGE2 accumulation in LPS-activated KCs was not significantly changed by ANP. These data suggest that ANP may not be a factor in the regulation of COX-2 expression and prostanoid production in activated rat liver macrophages under our experimental conditions.
Our study provides the interesting evidence that ANP influences another important function of KCs, i.e., phagocytosis . ANP significantly increased the phagocytic activity of KCs, measured as an increase in phagocytic index by flow cytometry. Thus, our results demonstrate for the first time that ANP affects phagocytosis in isolated rat KCs, thereby influencing one of the most representative functions of cells of the mononuclear macrophage system. It has previously been described that ANP stimulates phagocytosis and production of reactive oxygen species in bone marrow derived-macrophages and the murine macrophage cell line J774 (15 phagocytosis by ANP are not defined, the regulatory pathways may be similar in rat KC and murine macrophages. This is supported by the observation that in both cases NPR-A mediated cGMP seems to represent the responsible second messenger for the phagocytosis stimulating property of ANP. Besides this common effect of ANP on macrophage activity, ANP influences various other immunological functions in macrophages: it stimulates migration of human neutrophils (41 42,43
Taken together, by affecting phagocytosis of KCs, ANP may influence cellular defense mechanisms against invading microorganisms. Furthermore, because a depressed phagocytotic activity of KCs was observed after warm ischemia-reperfusion of the liver (44 20,21 5 phagocytosis can increase IRI (6 45 5,46
In summary, we could demonstrate that (1) isolated rat KCs express functional NPR-A, that (2) ANP specifically interacts with TNFα production of LPS-stimulated KCs, and that (3) treatment with ANP increases the phagocytotic activity of KCs via NPR-A. These effects of ANP on liver macrophages differ from those on peritoneal or bone marrow-derived macrophages. These data further support a specific regulatory action of ANP, originally described as a cardiovascular hormone, on inflammatory processes.
ACKNOWLEDGMENTS
We thank Prof. Dr. Ramadori, Goettingen, Germany for his advice regarding the isolation of Kupffer cells. The excellent technical assistance of Ursula Rüberg, Susanne Reiter, Ingrid Liβ and Brigitte Weiss is gratefully acknowledged. We thank Tobias Gerwig for performing liver perfusions. We also thank Dr. Kleinert, Mainz, Germany for the iNOS cDNA probe, and Prof. Dr. Decker, Freiburg, Germany for the TNFα cDNA probe. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ge 576/14-1 and Vo 376/8-2) and by the Friedrich-Baur-Stiftung, Munich, Germany. A.K.K. is supported by the “Bayerischer Habilitationsförderpreis.”
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