Effect of dibutyryl cyclic AMP on phagocytosis and production of nitric oxide and tumor necrosis factor-α in cultured rat Kupffer cells

Hepatology Research 13 (1999) 105–119

Effect of dibutyryl cyclic AMP on phagocytosisand production of nitric oxide and tumor necrosis

factor-a in cultured rat Kupffer cells

Takashi Goto *, Masafumi Komatsu, Itaru Toyoshima,Takao Hoshino, Ken-ichiro Mikami, Kazuo Yoneyama,

Tomoo Fujii, Shigetoshi Ohshima, Kunio Nakane,Xiang Wei Meng, Osamu Masamune

First Department of Internal Medicine, Akita Uni6ersity School of Medicine, 1-1-1 Hondo, Akita,010, Japan

Received 22 May 1998; received in revised form 20 August 1998; accepted 4 September 1998

Abstract

Dibutyryl cyclic AMP (DBcAMP) is an analogue of cAMP. DBcAMP has many effects onhepatocellular carcinoma, liver cirrhosis, ischemic liver failure and endotoxin-induced inflam-matory liver injury. However, little is known about the mechanism of DBcAMP action inKupffer cells. We examined the effects of DBcAMP on phagocytic activity and productionof nitric oxide (NO) and tumor necrosis factor-a (TNF-a) in cultured rat Kupffer cellstreated with lipopolysaccharide (LPS). NO concentrations in culture supernatants weremeasured using an NO analyzer and TNF-a levels were measured with ELISA. Immu-nofluorescent staining for nuclear factor-k B (NF-k B) was visualized using an anti-NF-k Bp65 antibody. DBcAMP premedication had no effect on LPS-stimulated phagocytic activityof Kupffer cells but increased NO production and inhibited TNF-a production in adose-dependent manner. Genistein did not block, but H-89 did block, the inhibitory effect ofDBcAMP on LPS-stimulated TNF-a production in Kupffer cells. We conclude that DB-cAMP inhibits LPS-stimulated TNF-a production by activating cyclic AMP-dependent

* Corresponding author. Tel.: +81 188 341111; fax: +81 188 362611.

1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII S1386-6346(98)00086-2

T. Goto et al. / Hepatology Research 13 (1999) 105–119106

protein kinase. Using immunofluorescent staining for NF-k B, we demonstrate that the effectof DBcAMP does not involve signal transduction through NF-k B. © 1999 Elsevier ScienceIreland Ltd. All rights reserved.

Keywords: cAMP-dependent protein kinase inhibitor; H-89; Protein tyrosine kinase inhibitor;Genistein; Nuclear factor-k B

1. Introduction

The sinusoidal liver cells include four types of non-parenchymal cells: Kupffer cells,stellate cells, endothelial cells, and pit cells. These cells interact with each other [1].Kupffer cells comprise the largest resident macrophage population and have twoimportant roles. One is phagocytizing foreign bodies including endotoxin, bacteria,viruses and a wide variety of antigens [2,3]. Another is producing a variety of cytokinesincluding interleukin-1, tumor necrosis factor-a (TNF-a), and superoxides stimulatedby foreign antigens [4]. Fulminant hepatitis or acute hepatic failure after partialhepatectomy or liver transplantation are caused by a variety of interacting mediatorssuch as these cytokines [5–7].

Dibutyryl cyclic AMP (DBcAMP), colchicine, pentoxifylline, prostaglandin E2,polymyxin-B, N-acetylcysteine, and a-tocopherol have been reported to inhibitTNF-a production in Kupffer cells [8–10]. DBcAMP, an analogue of cAMP, passesthrough the cell membrane into the cytoplasm more easily than cAMP. In addition,DBcAMP has an antitumor effect on hepatocellular carcinoma [11], enhancesrecovery from liver injury [12], improves hepatic functional reserve in patients withliver cirrhosis [13], and protects against ischemic liver failure and endotoxin-inducedinflammatory liver injury [14,15]. However, little is known about the mechanisms bywhich DBcAMP has its effects on Kupffer cells.

We studied the effect of DBcAMP on phagocytosis and the production of nitricoxide (NO) and TNF-a in cultured rat Kupffer cells.

2. Materials and methods

2.1. Animals

The protocols for animal experimentation described herein have been approved bythe Animal Research Committee, Akita University School of Medicine. All animalexperiments adhered to the Guidelines for Animal Experimentation of AkitaUniversity School of Medicine.

2.2. Materials

DBcAMP and lipopolysaccharide (LPS) (from Salmonella enteritidis) were ob-tained from Sigma (St. Louis, MO, USA). Genistein and H-89 were obtained fromBiomol (Plymouth Meeting, PA, USA).

T. Goto et al. / Hepatology Research 13 (1999) 105–119 107

2.3. Kupffer cell isolation and culture

Sinusoidal cell fractions were obtained by a method employing collagenase [16].The livers of Sprague–Dawley male rats (body weight 250–350 g) were perfusedsequentially with: (1) Ca2+- and Mg2+-free Hanks balanced salt solution (HBSS)with 0.5 mM ethyleneglycol bis (2-aminoethylether) tetraacetic acid (EGTA) (37°C,20 ml/min) for 10 min; and (2) HBSS containing 0.05% collagenase (type I) (Sigma,St. Louis, MO, USA) (37°C, 10 ml/min) for 20 min. The resulting liver cell suspensionwas agitated gently in the same solution at 37°C for 15 min to suspend theparenchymal cells completely. The cell suspension was filtered through sheets of gauzeand a mesh (pore size 70 mm). The cells were suspended in HBSS and centrifugedthree times at 500×g for 5 min. The resulting pellet was resuspended in HBSS andlayered on 25, 50% Percoll (Sigma, St. Louis, MO, USA) in HBSS, and centrifugedat 4°C, 30000×g for 30 min. The layer of Kupffer cells having a density of 1.062was collected and washed with HBSS. The cells were suspended in growth factor-en-riched medium, GIT (WAKO, Osaka, Japan) with 10% fetal calf serum and platedon 24-mm cover slips. The viability of the cells was 94–96% as estimated by trypanblue exclusion. After incubation for 1 h in a 37°C, 5% CO2 incubator, the cells werewashed with GIT and reincubated for 48 h in a 37°C, 5% CO2 incubator and usedunder various test conditions.

2.4. Measurement of the cytotoxicity of Kupffer cells

The viability of Kupffer cells was tested by measuring lactate dehydrogenase (LDH)leakage. After a 3-h incubation with 10, 50, 100, and 500 mM DBcAMP, LDH levelsin the culture supernatants of Kupffer cells were determined.

2.5. Measurement of the phagocytic acti6ity of Kupffer cells

The phagocytic activity of Kupffer cells was measured according to the numberof latex particles 3 mm in diameter (Polybeads; Polyscience, Warrington, PA, USA)that were incorporated into the cytoplasm. Latex particles (1.3×106) were added toeach well of Kupffer cells. The cells on the cover slips were washed 90 min after theloading of the latex particles, and fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) at 37°C for 10 min. The number of latex particles incorporatedinto 200 Kupffer cells on the cover slips was counted under a microscope. Theprotocols for studying the effects of DBcAMP on the phagocytic activity of Kupffercells are shown in Fig. 1a.

2.6. Measurement of NO le6els in culture supernatants of Kupffer cells

The NO concentrations in culture supernatants of Kupffer cells were measuredusing a Sievers NO analyzer, model 270B (Saan, Osaka, Japan). The protocols forstudying the effect of DBcAMP or Genistein on NO production in Kupffer cells areshown in Fig. 1b.


Values represent the percent change in NO production in supernatant fromcultured Kupffer cells.

Percent change (%)= (NO levels produced by the cells after treatment with LPSplus the agent)/(NO levels produced after LPS alone)×100.

Fig. 1.


Fig. 1. The protocols for studying the effects of DBcAMP. (a) Effects of DBcAMP on phagocyticactivity. Control group: Neither LPS nor DBcAMP were added to the culture media. Experiment 1:LPS was added to the culture media at a final concentration of 1 mg/ml. After a 6-h treatment, latexparticles were applied. Experiment 2: DBcAMP was added to the culture media at final concentra-tions of 10, 50, 100, and 500 mM. After a 3-h treatment, LPS was added to the culture media at afinal concentration of 1 mg/ml. After a 6-h treatment, latex particles were applied. (b) Effects ofDBcAMP on NO production. Control group: Neither LPS nor DBcAMP were added to the culturemedia. Experiment 1: LPS was added to the culture media at a final concentration of 1 mg/ml. Aftera 24-h treatment, culture supernatants were assayed for NO. Experiment 2: DBcAMP was added tothe culture media at final concentrations of 10, 50, 100, and 500 mM. After a 3-h treatment, LPS wasadded to the culture media at a final concentration of 1 mg/ml. After a 24-h treatment, culturesupernatants were assayed for NO. Experiment 3: Genistein was added to the culture media at finalconcentrations of 5, 10, 50, and 100 mM. After a 3-h treatment, LPS was added to the culture mediaat a final concentration of 1 mg/ml. After a 24-h treatment, culture supernatants were assayed forNO. (c) Effects of DBcAMP on TNF production. Control group: Neither LPS nor DBcAMP wereadded to the culture media. Experiment 1: LPS was added to the culture media at a final concentra-tion of 1 mg/ml. After a 6-h treatment, culture supernatants were assayed for TNF-a. Experiment 2:DBcAMP was added to the culture media at final concentrations of 10, 50, 100, and 500 mM. After a3-h treatment, LPS was added to the culture media at a final concentration of 1 mg/ml. After a 6-htreatment, culture supernatants were assayed for TNF-a. Experiment 3: Genistein was added to theculture media at final concentrations of 5, 10, 50, and 100 mM. After a 3-h treatment, LPS wasadded to the culture media at a final concentration of 1 mg/ml. After a 6-h treatment, culturesupernatants were assayed for TNF-a. Experiment 4: Genistein was added to the culture media atfinal concentrations of 5, 10, 50, and 100 mM. After a 0.5-h treatment, DBcAMP was added to theculture media at a final concentration of 10 mM. After a 3-h treatment, LPS was added to the culturemedia at a final concentration of 1 mg/ml. After a 6-h treatment, culture supernatants were assayedfor TNF-a. Experiment 5: H-89 was added to the culture media at final concentrations of 5 and 10mM. After a 0.5-h treatment, DBcAMP was added to the culture media at a final concentration of 10mM. After a 3-h treatment, LPS was added to the culture media at a final concentration of 1 mg/ml.After a 6-h treatment, culture supernatants were assayed for TNF-a. (d) Effects of DBcAMP onNF-k B immunofluorescent staining. Control group: Neither LPS nor DBcAMP were added to theculture media. Experiment 1: LPS was added to the culture media at a final concentration of 1mg/ml. After a 4-h treatment, the Kupffer cells were fixed. Experiment 2: DBcAMP was added to theculture media at final concentrations of 10, 50, 100, and 500 mM. After a 3-h treatment, LPS wasadded to the culture media at a final concentration of 1 mg/ml. After a 4-h treatment, the Kupffercells were fixed. In every experiment, to adjust the pH, HEPES was added at a final concentration of20 mM.


Fig. 2. Effect of DBcAMP on the phagocytic activity of Kupffer cells. The numbers of beads in eachKupffer cell were counted as described in Section 2. Data are expressed as the mean9S.E. *PB0.05.

Fig. 3. Effect of DBcAMP on TNF-a production in Kupffer cells. TNF-a in culture supernatants wasmeasured as described in Section 2. Data are expressed as the mean9S.E. *PB0.05.


Fig. 4. Effect of Genistein on TNF-a production in Kupffer cells. TNF-a in culture supernatants wasmeasured as described in Section 2. Data are expressed as the mean9S.E. *PB0.05.

Fig. 5. Effect of DBcAMP on NO production in Kupffer cells. No in culture supernatants was measuredas described in Section 2. Data are expressed as the mean9S.E. *PB0.05.


2.7. Measurement of TNF-a le6els in culture supernatants of Kupffer cells

TNF-a levels in the culture supernatants of Kupffer cells were measured using arat TNF-a ELISA kit (Genzyme, Cambridge, MA, USA). The protocols for studyingthe effects of DBcAMP, Genistein, DBcAMP and Genistein, or DBcAMP and H-89on TNF-a production in Kupffer cells stimulated by LPS are shown in Fig. 1c. Valuesrepresent the percent inhibition of TNF-a production in the supernatant fromcultured Kupffer cells.

Percent inhibition (%)= (TNF-a levels produced by the cells after treatment withLPS plus the agent)/(TNF-a levels produced after LPS alone)×100.

2.8. Immunofluorescent staining for NF-k B

NF-k B protein in Kupffer cells was visualized using an anti-NF-k B p65 (C-20)antibody (at a 1:100 dilution in PBS) (Santa Cruz Biotechnology, Santa Cruz, CA,USA) and a tetramethylrhodamine isothiocyanate-conjugated rabbit immunoglobu-lin antibody (at a 1:100 dilution in PBS) (Dako, Glostrup, Denmark). LPS-stimulated,LPS-unstimulated, and (10, 50, 100, and 500 mM) DBcAMP pretreated andLPS-stimulated Kupffer cells were fixed with 3.7% formaldehyde in PBS for 10 min,stained using an anti-NF-k B p65 (C-20) antibody overnight at 4°C, and then stainedwith the secondary antibody for 4 h at 37°C. The protocols for NF-k B immu-nofluorescent staining of Kupffer cells are shown in Fig. 1d.

2.9. Statistical analysis

The number of latex particles incorporated into Kupffer cells was expressed as themean9S.E., and differences between samples were analyzed using one-way ANOVAand Scheffe test. The percent changes in NO and TNF-a production were expressedas the mean9S.E., and differences between measurements were analyzed using theunpaired Mann–Whitney U-test. PB0.05 was considered statistically significant.

3. Result

3.1. Cytotoxicity of DBcAMP to Kupffer cells

LDH levels in the culture supernatants of Kupffer cells incubated with DBcAMPat concentrations of 10, 50, 100, and 500 mM for 3 h at 37°C were all less than 14IU/l (data not shown). These levels of DBcAMP appear to have no cytotoxic effecton Kupffer cells.

3.2. Phagocytic acti6ity of Kupffer cells

LPS treatment significantly enhanced the phagocytic activity of Kupffer cells ata concentration of 1 mg/ml. Preadministration of DBcAMP had no effect on theLPS-stimulated phagocytic activity of Kupffer cells (Fig. 2).


Fig. 6. Effect of Genistein on NO production in Kupffer cells. No in culture supernatants was measuredas described in Section 2. Data are expressed as the mean9S.E. *PB0.05.

Fig. 7. Effect of DBcAMP with Genistein premedication on TNF-a production in Kupffer cells. TNF-ain culture supernatants was measured as described in Section 2. Data are expressed as the mean9S.E.*PB0.05.


3.3. Effect of DBcAMP or Genistein on TNF-a production in Kupffer cellsincubated with LPS

LPS treatment significantly enhanced TNF-a production in Kupffer cells at aconcentration of 1 mg/ml. The percent inhibition of TNF-a production in Kupffercells pretreated with DBcAMP at concentrations of 10, 50, 100, and 500 mMcompared to LPS treatment alone were 68.792.6%, 60.393.7%, 39.291.1% and28.391.7%, respectively. DBcAMP preadministration significantly inhibited theenhancement of TNF-a production in LPS-stimulated Kupffer cells in a dose-de-pendent manner (Fig. 3). The percent inhibition of TNF-a production in Kupffercells pretreated with Genistein at concentrations of 5, 10, 50, and 100 mMcompared to LPS treatment alone were 89.193.4%, 74.391.0%, 45.192.2% and10.790.6%, respectively. Genistein preadministration also significantly inhibitedthe enhancement of TNF-a production in a dose-dependent manner (Fig. 4).

3.4. Effect of DBcAMP or Genistein on NO production in Kupffer cells

LPS treatment significantly enhanced NO production in Kupffer cells at aconcentration of 1 mg/ml. The rates of NO production in Kupffer cells pretreatedwith DBcAMP at concentrations of 10, 50, 100, and 500 mM compared to LPS

Fig. 8. Effect of DBcAMP with H-89 premedication on TNF-a production in Kupffer cells. TNF-a inculture supernatants was measured as described in Section 2. Data are expressed as the mean9S.E.*PB0.05.


Fig. 9. Localization of the NF-k B p65 subunit using immunofluorescent staining: (A) in resting Kupffercells without LPS stimulation; (B) in Kupffer cells 4 h after LPS stimulation; (C) in Kupffer cells 4 hafter LPS stimulation by 10 mM DBcAMP preadministration; and (D) in Kupffer cells 4 h after LPSstimulation by 500 mM DBcAMP preadministration.

treatment alone were 116.391.7%, 117.792.2%, 114.092.0% and 117.693.5%,respectively. DBcAMP preadministration significantly increased the enhancementof NO production in LPS-stimulated Kupffer cells (Fig. 5). However, the rates of


NO production in Kupffer cells pretreated with Genistein at concentrations of 5,10, 50, and 100 mM compared to LPS treatment alone were 96.295.9%,86.095.2%, 58.493.4% and 29.593.4%, respectively. Genistein preadministrationsignificantly inhibited the enhancement of NO production in LPS-stimulatedKupffer cells (Fig. 6).

3.5. Effect of DBcAMP and Genistein, or DBcAMP and H-89 on TNF-a productionin Kupffer cells incubated with LPS

The percent inhibition of TNF-a production in Kupffer cells pretreated with 10mM DBcAMP and Genistein at concentrations of 5, 10, 50, and 100 mM comparedto LPS treatment alone were 39.792.3%, 27.991.7%, 17.792.1% and10.891.5%, respectively. Genistein did not block the inhibitory effect of DBcAMPon enhancement of TNF-a production in LPS-stimulated Kupffer cells (Fig. 7).However the percent inhibition of TNF-a production in Kupffer cells pretreatedwith 10 mM DBcAMP and H-89 at concentrations of 5 and 10 mM compared toLPS treatment alone were 94.591.2% and 97.792.8%, respectively. H-89 blockedthe inhibitory effect of DBcAMP on enhancement of TNF-a production in Kupffercells stimulated by LPS (Fig. 8).

3.6. Effect of DBcAMP on localization of NF-k B in Kupffer cells

The cytoplasm of resting, unstimulated Kupffer cells stained more intensely thanthe nucleus (Fig. 9A). However, the nucleus of the Kupffer cells stimulated by LPSstained more intensely than the cytoplasm (Fig. 9B). DBcAMP preadministrationhad no effect on the NF-k B p65 subunit staining pattern in Kupffer cellsstimulated by LPS (Fig. 9C,D).

4. Discussion

We studied the effect of DBcAMP on phagocytosis and production of NO andTNF-a in rat Kupffer cells incubated with LPS. DBcAMP preadministration hadno effect on LPS-stimulated phagocytic activity of Kupffer cells, but increased NOproduction and resulted in a dose-dependent inhibition of TNF-a production inLPS-stimulated Kupffer cells.

The roles of NF-k B and cAMP-dependent protein kinase in signal transductionhave been studied recently [17,18]. In the latent state, NF-k B is found in thecytoplasm bound with its inhibitory protein, inhibitor-k B (I-k B) [19]. Afterexposure to stimuli such as LPS, IL-1 or TNF-a, or radiation, I-k B is phosphory-lated by activation of protein kinase C, serine/threonine kinase or protein tyrosinekinase [20,21], and the NF-k B/I-k B complex dissociates [22–24]. This activatedNF-k B translocates to the nucleus and binds to NF-k B promoter sites on DNA,activating gene transcription of cytokines such as TNF-a and inducible NOsynthase [23,25].


The mechanism by which the second messenger cAMP amplifies extracellularsignals is by stimulating the activity of the cAMP-dependent protein kinase. Theinactive kinase increases its ability to phosphorylate specific acceptor proteins bybinding to cAMP. This protein kinase contains four subunits, two regulatorysubunits (R) and two catalytic subunits (C). When cAMP binds to the R subunits,they dissociate from the C subunits and their kinase activity increases. C subunitsare carried from the cytoplasm to the nucleus where they activate cAMP-responseelement-binding protein (CREB) by phosphorylating a single serine at position 133[26]. Phosphorylated CREB binds to the cAMP-response element (CRE) on DNA,activating gene transcription [18].

In this study, DBcAMP or Genistein preadministration significantly inhibited theenhancement of TNF-a production in LPS-stimulated Kupffer cells in a dose-de-pendent manner. However, DBcAMP preadministration significantly increased theenhancement of NO production in LPS-stimulated Kupffer cells even thoughGenistein preadministration significantly inhibited NO production. We hypothe-sized that Genistein [27,28], an inhibitor of protein tyrosine kinases, inhibitsproduction of TNF-a and NO by blocking the activation of NF-k B in signaltransduction, and that the effect of DBcAMP occurs through a signal transductionpathway which is different from NF-k B. We studied the relationship betweenDBcAMP and Genistein, or DBcAMP and H-89 on TNF-a production in Kupffercells incubated with LPS. Genistein did not block the inhibitory effect of DBcAMPon LPS-stimulated TNF-a production in Kupffer cells; however, H-89 [29,30], aninhibitor of cAMP-dependent protein kinase, blocked the inhibitory effect ofDBcAMP on TNF-a production. From these results, we conclude that DBcAMPinhibits LPS-stimulated TNF-a production by activating cAMP-dependent proteinkinase in signal transduction.

By immunofluorescent staining of NF-k B p65, we demonstrated that the effectof DBcAMP is not related to effects on NF-k B translocation. The activation ofNF-k B persists for up to 4 h after stimulation, after which it begins to decrease[31]. We examined the distribution of the NF-k B p65 subunit in Kupffer cells after4 h of LPS stimulation and found that 10, 50, 100, and 500 mM DBcAMPpreadministration results in as intense staining of the nuclei of Kupffer cells as withLPS stimulation alone. We conclude that the preadministration of DBcAMP doesnot inhibit translocation of NF-k B from the cytoplasm to the nucleus, and that theinhibitory effect of DBcAMP on TNF-a production does not involve NF-k B.

The use of DBcAMP in the prevention or treatment of endotoxin-induced liverinjury may be either contraindicated or indicated based on the finding thatDBcAMP preadministration increases NO production and inhibits TNF-a produc-tion in LPS-stimulated Kupffer cells. NO is a known cytotoxic radical andendothelium-derived relaxing factor. In the liver, NO inhibits protein synthesis inhepatocytes [32], affects sinusoidal blood flow [33], and induces relaxation ofcontracted bile canalicules [34]. The pathophysiologic significance of NO produc-tion by Kupffer cells is not fully understood. Since activation of cAMP-dependentprotein kinase by DBcAMP may be useful for the prevention or treatment ofendotoxin-induced liver injury, we would further study using DBcAMP in vivo,while manifesting for signs of NO cytotoxity.


Acknowledgements

We thank Yuki Watanabe and Takako Sasaki for their excellent technicalassistance.

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Effect of dibutyryl cyclic AMP on phagocytosis and production of nitric oxide and tumor necrosis factor-α in cultured rat Kupffer cells