Pmtqhdins 0 Longman
Lcukotriems and Essential Group UK Ltd 1992
Fatty Acids (19!32) 46,105-110
Enhancement of Prostaglandin Ez Production by Liver Macrophages (Kupffer Cells) After Stimulation with Biological Response Modifiers N. Kawada, Y. Mizoguchi, K. Kobayashi,
T. Manna*, P. Liut and S. Morisawa+
The Third Department of Internal Medicine, *Department of Public Health, and +The First Department of Biochemistry, Osaka City University Medical School, l-5-7, Asahi-machi, Abeno-ku, Osaka 545, Japan (Reprint requests to NK) ABSTRACT. PG& production by liver macropbages (Kupffer cells) activated by biological response modiks was examined. Kupffer cells obtained from a normal rat liver possesed cyclooxygenase activity and produced TXR2, PGD2, and PGEz from (l-i4C)arachidonic acid. The major product was PGD2. When Kupffer cells were incubated in the presence of I@-polysaccharide (LPS), OK-432, or heat-kiied Propionibacterium acnes for 24 h, the amount of arachidonate cyclooxygenase products increased and the major product changed from PGDz to PGE2. When liver macrophages including Kupffer cells were prepared from rats after an injection of LPS, 0K-432, or heat-hilled P. acnes, it was noticed that the number of cells obtained and PGEz production increased compared with those of normal rat. These results suggested that PGEz production by rat liver was induced when they were treated with biological response modifiers.
INTRODUCTION When mononuclear phagocytic cells are exposed to a variety of stimuli, they synthesize and release a number of biologically active materials including arachidonate metabolites of cyclooxygenase (1, 2). Kupffer cells are known to be resident macrophages of the liver and are a part of the organism’s mononuclear phagocyte system (3). Prostaglandin (PG) production by Kupffer cells has also been demonstrated in many studies (4-8). Kupffer cells synthesize and release PGs and thromboxane Bz (TX&) in response to various stimulations such as lipopolysaccharide (LPS) 9 zymosan , calcium ionophore A23187, tumor necrosis factor (TNF) and phorbol ester. PG biosynthesis is regulated by two enzymatic reactions. One is the release of arachidonic acid (AA) from the cellular membrane mediated mainly by phospholipase A2 (9, lo), and the other is the formation of PGH2 from AA by cyclooxygenase (11, 12). Concerning the latter, recent reports have suggested that the cyclooxygenase enzyme is induced in a variety of cells after stimulation and this is attributed to the increase in amount of the enzyme protein (13-25).
Previous reports about the production of arachidonate cyclooxygenase metabolites by Kupffer cells have not clearly demonstrated any change of the products in response to stimulation. In the present study, the cyclooxygenase metabolism in Ku ffer cells was estimated by reacting the cells with (l- !I C)AA to minimize the influence of phospholipase A2 and analysed by reverse-phase high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC). As a result, we observed that the cyclooxygenase activity in Kupffer cells was elevated and PGEz production was dramatically induced in response to the activation by biological response modifiers in vitro and in vivo.
MATERIALS AND METHODS Materials Male Wistar rats (200-220 g) were obtained from Clea Japan Inc (Shizuoka, Japan). The animals were housed under constant conditions and provided with standard chow pellets and water ad libitum. Collagenase (Type IV) was purchased from Wako Pure Chemical Co Ltd (Osaka, Japan). Pronase E was from Merck (Darmstadt). Metrizamide (2-(3acetamide-5-methylacetamide-2,4,6-triiodoacetamide)2-deoxy-D-glucose), lipopolysaccharide (LPS) from
Date received 14 June 1991 Date accepted 13 November 1991 105
106 Prostaglandins Leukotrienes and Essential Fatty Acids
Salmonnella enteritidis, and calcium ionophore A23187 (A23187) were obtained from Sigma Chemical Co. (St. Louis, MO). RPM1 1640 medium was purchased from Nissui Pharmaceutical Co Ltd (Tokyo, Japan). Fetal calf serum (FCS) was from M. A. Bioproducts (Maryland, WA). (l-‘4C)arachidonic acid (AA) was obtained from International (Bucks, UK), and Amersham arachidonic acid from Nu-Chek-Prep (Elysian, USA). Authentic 6-keto-PGFi,, TXB2, PGD2, and PGE2 were kindly provided by Ono Pharmaceutical Company. OK-432 was a kind gift from Chugai Pharmaceutical Company. Propionibacterium acnes was kindly donated by the Department of Bacteriology, Osaka City University Medical School.
Preparation of liver macrophages (Kupffer cells) The rats were intravenously injected with physiological saline, LPS (100 &rat), OK-432 (5 KE/rat), or P. acnes (1 mg/rat). 1, 4, or 7 days after the injection of these BRMs, the rats were anesthetized by an intraperitoneal injection of pentobarbital (10 mg/rat). After laparotomy, non-parenchymal liver cells were separated by collagenase-pronase perfusion according to Knook’s methods with a slight modification (26). In brief, the liver was perfused in situ with Hank’s balanced salt solution (HBSS) for 2 min and then subsequently perfused with HBSS containing 0.05% collagenase and next with HBSS containing 0.05% pronase for 5 min each. After excision, the liver was minced and incubated at 37°C in HBSS containing 0.05% pronase for 30 min. Sinusoidal cells thus separated were harvested from the digested liver by centrifugation (10 min, 400 x g). Sinusoidal cell suspensions were freed of erythrocytes and cell debris by density centrifugation in Gey’s balanced salt solution without NaCl containing 17.5% metrizamide at 1400 x g for 10 min. After the cells were washed by centrifugation (10 min, 400 x g), they were suspended in RPM1 1640 medium with 20% heat-inactivated fetal calf serum and cultured in a plastic dish at 37°C in 5% CQ/95% air. After 18 h incubation, liver macrophages and Kupffer cells adhering to the plastic dish were isolated with the aid of a rubber policeman and used for the following experiments.
Activation of Kupffer cells Kupffer cells obtained from a normal rat were incubated in RPM1 1640 medium containing 5% FCS in lOO-mm plastic dishes. After 18 h incubation, LPS (10 pg/ml), OK-432 (1 KE/ml), or P. acnes (100 pg/ml) was added to each well. Kupffer cells were harvested after another 24 h incubation, and the PG synthesis was determined.
Assay of prostaglandin biosynthesis For the assay of PG biosynthesis, the whole liver macrophages or Kupffer cells were used. The prepared liver macrophages and Kupffer cells were suspended in phosphate-buffered saline (1 X lo7 cells/5 ml) and allowed to react with 25 DM (l-14C)AA (1.25 x lo6 cpm) at 37°C for 30 min. The reaction mixture was centrifuged (400 x g, 4°C 10 min), and the cell free supernatant was acidified to pH 3 with 1 N HCl. After extraction with 10 ml of ethyl acetate, the solvent was evaporated. The dried material was analysed by TLC and reversephase HPLC. The solvent for TLC was the organic phase of ethyl acetate : isooctane : acetic acid: water (110:50:20: 100, V/v). The radioactivity existing in the silica gel TLC plate (Kieselgel60 F254, Merck, Darmstadt) after 40 min development was visualized by auto-radiography. The dried materials were redissolved in methanol and applied to reversephase HPLC with a TSK-gel column (type ODS120T, 5 pm, 4.6 x 250 mm, Tosoh) connected to a dual pump model (LC-6A, Shimadzu Corporation, Kyoto, Japan) and an injector model (SILdA, Shimadzu Corporation, Kyoto, Japan). The solvent mixture was acetonitrile : water: phosphoric acid (35:65:0.1). The flow rate was 1.0 ml/min. Absorption at 195 nm was monitored for PGs and TXBz using a programmable detector (SPDdA, Shimadzu Corporation, Kyoto, Japan). The radioactivity of the organic solvent of each 1 min eluate fraction was determined by a liquid scintillation counter (LS 5801, Beckman Instrument Inc, Fullerton, CA).
RESULTS PGs and TXBz production by Kupffer cells Kupffer cells were reacted with (l-14C)AA, and the reaction products were analyzed by reverse-phase HPLC and TLC. Figure 1A shows the analysis of the authentic PGs and TXB;! by reverse-phase HPLC monitoring the column eluate at 195 nm. The retention times of 6-keto-PGFi,, TXBz, PGE2, and PGDz were 8.3 min, 20.6 min, 25.5 min and 29.8 min, respectively. The reaction products were analyzed by this system, and the radioactivity originating from (l-14C)AA was counted. As shown in Figure lB, one major and two minor radioactive peaks were detected and these peaks were coincident with the retention times of authentic TXB;!, PGl!$, and PGD2. These results suggested that non-treated rat Kupffer cells produced these PGs and TXB:! from exogenous AA and the major product was 1 x lo5 M indomethacin identified as PGDz. inhibited PG production and, when the cells were boiled at 95°C for 5 min, the production of PGs and
PGE, Production and Biological Response Modifiers
107
Fig. 2 TLC analysis of PGE, production by Kupffer cells. Non-stimulated or BRM-stimulated rat Kupffer cells (2 x 10”) were allowed to react with (l-“C)AA (25 x 10’ cpm) at 37°C for 30 min, and the reaction products were analyzed by TLC and visualized by autoradiography as described in Materials and Methods. (A), (C), and (E) Non-stimulated Kupffer cells. (B) LPS-stimulated Kupffer cells. (D) OK-432-stimulated Kupffer cells. (F) P.acnes-stimulated Kupffer cells.
Retention
Time
(mid
Fig. 1 Reverse-phase HPLC analysis of arachidonate cyclooxygenase products by Kupffer cells. Non-stimulated or BRM-stimulated rat Kupffer cells (2 x 106) were allowed to react with (l-“C)AA (25 x 10'cpm) at 37°C for 30 min, and the reaction products were analyied’by reverse-phase HPLC as described in Materials and Methods. (A) The analvsis of authentic 6-keto-PGF,,, TXB,, PGE, and PGD 2’ (B) ’ Non-stimulated Kupffer cells. (C) LPS-stimulated Kupffer cells. (D) OK-432-stimulated Kupffer cells. (E) P. acnes-stimulated Kupffer cells.
TXB;! was not detected, which suggested that the reaction from exogenous (l-14C)AA to each PG and TXB;! was the event of enzymatic procedure (data not shown). Each peak was identified as TXB2, PGEZ, and PGD2 by radioimmunoassay by using a specific antibody for each PG and TXB2 raised in rabbit serum (data not shown). We next examined the effect of BRMs on the arachidonate cyclooxygenase metabolism of Kupffer cells in in vitro treatment. Kupffer cells were incubated with LPS (10 &ml), OK-432 (1 KE/ml), or heat-killed P. acnes (100 pg/ml) for 24 h, and the cellular levels of cyclooxygenase activity were measured. Figures lC-1E shows the results and demonstrated that the production of PGE2 by Kupffer cells was enhanced by the treatment of the cells with BRMs. The production of PGE2 by BRMtreated Kupffer cells was enhanced about 4 times more than that of normal Kupffer cells. These results were confirmed by analysis performed using TLC as shown in Figure 2. The band corresponding to PGE2 increased in density, when Kupffer cells were treated with BRMs. This enhancement of
PGEz production was dose-dependent with reference to the stimulants, and the optimal doses of LPS, OK-432, and heat-inactivated P. acnes were 0.1-10 pg/ml, OS-5 KE/ml, and l-100 pg/ml, respectively (data not shown).
Effect of in vivo administration of BBMs on the production of PGs and TXBz by liver macrophages Next we examined whether in vivo administration of these BRMs has effects on the arachidonate cyclooxygenase metabolism of Kupffer cells. 1, 4, or 7 days after an intravenous administration of LPS (100 pg/rat), OK-432 (5 KE/rat), or P. acnes (10 mg/rat), liver macrophages were prepared as described in materials and methods. At first, we observed the increase in the number of obtained liver macrophages. The number of Kupffer cells from a normal rat was about 1 x lo7 cells/rat (n = 5). However, when the rats were treated with LPS, OK-432, or P. axes, the number of liver macrophages obtained increased to about 1.5 X107 (n = 3), 2 x lo7 (n = 3), or 1 x lo8 (n = 3) cells/rat, respectively. The microscopic examination of conventionally prepared tissue section showed an increase of mononuclear cells which mostly existed in the sinusoidal capillary lumina after the injection of BRMs. Especially, granuloma formation was seen in the liver of rats treated with P. acnes (Fig. 3). The cells obtained were mononuclear cells, adherent to plastic dishes, and phagocytosed latex particles (0.8 pm), which were most likely macrophages. We examined the PG production by these liver macrophages according to the methods described
108
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Fig. 3 Microscopic examination of the liver of P. acnes-injected rats. At 7 days after the injection of heat-killed P. acnes through a tail vein, the liver was fixed and prepared conventionally for microscopic examination with H-E stain. The accumulation of mononuclear cells and the formation of granulomas in the liver were seen. (X 200)
ontrol
LPS
OK-432
P.acnes
Fig. 4 The cyclooxygenase activity in liver macrophages obtained from BRM-injected rats. Liver macrophages were obtained from rats injected with normal saline, LPS (100 pg), OK-432 (5 KE), or P. acnes (1 mg) and allowed to react with (1J4C)AA. The reaction products were analyzed by reverse-phase HPLC and the amount of radioactivity corresponding to 6-keto-PGF,,, TXB,, PGE, and PGD, was determined. Data are expressed as mean + SD of 3 different examinations. The statistical significance is analyzed by Student’s t-test. *-**; p < 0.05, *-***; p < 0.01.
above. The PG production was significantly increased after the injection of LPS, OK-432, or P. axes (Fig. 4). TLC analysis of the metabolites produced by the macrophages obtained from OK-432 or P. acnes-treated rats showed that the production of PGE:! was significantly amplified (Fig. 5). We obtained similar results by reversephase HPLC analysis.
DISCUSSION In the present study, we demonstrated that the PG production by Kupffer cells was elevated in response to in vitro stimulation with LPS, OK-432,
Fig. 5 TLC analysis of arachidonate cyclooxygenase products by liver macrophages. Liver macrophages (2 x 104 from BRM-treated rats were allowed to react with (l-14C)AA (25 x lo4 cpm) at 37°C for 30 min, and the reaction products were analyzed by TLC and visualized by autoradiography as described in Materials and Methods. (A) and (C) Liver macrophages from physiological saline-injected rat. (B) Liver macrophages from OK-432-injected rat. (D) Liver macrophages from P. acnes-injected rat.
or P. acne.s and that the major product of arachidonate cyclooxygenase metabolites changed from PGDz to PGE*. In this case, as the number and the viability of Kupffer cells were not changed after stimulation with BRMs, the intracellular metabolic changes possibly caused the elevation of PGE2 production. We observed the increased production of not only PGE2 but also PGD2, TXB2, HHT (a biproduct of TXA2 synthetase (27)), and HETEs (a biproduct of cyclooxygenase (28)), which may be one of the reasons for the activation of cyclooxygenase. However, the activation of PGE;! synthetase (PGH2 isomerase) was not ruled out and we do not have the answer to the question as to why PGE2 came to be predominantly produced more than the other cyclooxygenase products. These observations were also seen when liver macrophages obtained from rats injected with BRMs in vivo were reacted with (l-%)AA. In vivo administration of BRMs to rats caused the increase of the adherent cell number in the liver per rat and
PGE, Production and Biological Response Modifiers
elevated the PGE2 production. In this case, we have to carefully discuss about the cells used in the assay. Some reports have demonstrated that Kupffer cells proliferate in response to stimulation by zymosan, OK-432, or P. awes in vivo (29, 30). On the other hand, mononuclear cells and polymorphonuclear leukocytes migrate into the liver in the case of inflammation (31, 32). Therefore, we can not declare which is the cause of the cyclooxygenase activation in liver macrophages, a functional change in Kupffer cells in accordance with proliferation or a change of cell types based on the migration of inflammatory macrophages from the other organs. However, the ability to produce PGEz in the liver was augmented after treatment of rats with BRMs. The increased production of PGE2 may originate from the activation of cyclooxygenase (33) or the induction of cyclooxygenase itself. Recent reports have suggested that cyclooxygenase is induced in many types of cells after stimulation (13-25). More recently, Masferrer and Fu have reported that the cyclooxygenase activity in human or mouse monocytes rises after they are stimulated with LPS in vivo (22) or in vitro (23). Thus, in a variety of cells, the induction of cyclooxygenase has been shown under conditions of stimulation with agents which have multipotential effects on cell functions and make changes in intracellular metabolism. Our above observation may originate from such an induction of cyclooxygenase but more detailed examination may be required for the determination. The enhancement of PGE2 production may play a role in the regulation of cell function. PGE2 has been reported to have some effects on liver cell function such as cytoprotection (34, 35) and the regulation of glyconeogenesis (36, 37) for hepatocytes, the maintenance of microcirculation (38), and modification of collagen synthesis (6). Especially, PGE;! has been known to diminish the production or release of interleukin 1 (IL-l) and tumor necrosis factor (TNF). Peters et al have clearly demonstrated that TNF production by LPS-stimulated rat Kupffer cells is regulated by the elevation of intracellular CAMP induced by PGE2 (39). We have also reported that the production of IL-l and TNF by P. acnes-elicited liver adherent cells is inhibited by PGE2 and PGI:! which are released from the cells in the autocrine or paracrine manner (40). Therefore the increased production of PGEz may be important in diminishing the enhancement of inflammatory reactions. Acknowledgements We are grateful to Mrs Haruko Ueda for preparing this manuscript. Y. M. was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, and the Ministry of Science and Technology of Japan.
References 1. Tsunawaki S, Nathan C. Release of arachidonate and reduction of oxygen: independent metabolic bursts of the mouse-peritoneal macrophage. J. Biol. Chem. 1986: 261: 11563-11570. 2. Goldyne M E, Burrish G F, Poubelle P, Borgeat P. Arachidonic acid metabolism among human mononuclear leukocytes: lipoxygenase-related pathways. J. Biol. dhem. 1985:259: 88X-8821. 3. Jones E A. Summerfield J A. Kuoffer cells. In: Arias I M,‘Popper H, Jakoby W B, Schachter D, Shafritz D A, ed. The liver: biology and pathology. 2nd ed. New York: Raven, 1988: 683-704. 4. Decker K. Eicosanoids, signal molecules of liver cells. Semin. Liver Dis. 1985: 5: 175-190. 5. Dieter P, Schulze-Specking A, Decker K. Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells. Eur. J. Biochem. 1986: 159: 451-457. 6. Bhatnagar R, Schade U, Rietschel E T, Decker K. Involvement of prostaghmdin E and adenosine 3’,5’-monophosphate in lipopolysaccharidestimulated collagenase release by rat Kunffer cells. Eur. J. Biochem. 1982: 125: 1251130. . 7. Than-Thi T A. Gvufko K. Haussineer D. Decker K. Net prostaglanhin release by pe;fused’ rat liver after stimulation with phorbol 1Zmyristate 13-acetate. J. Hepatol. 1988: 6: 151-157. 8. Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur. J. Biochem. 1990: 192: 245-261. 9. Chang J, Musser J H, McGregor H. Phospholipase A,: function and pharmacological regulation. Biochem. Pharmacol. 1987: 36: 2429-2434. 10. Hoffman T, Lizzio E F, Suissa J, Rotrosen D, Sullivan J A, Mandell G I, Bonvini E. Dual stimulation of phospholipase activity in human monocytes: role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation. J. Immunol. 1988: 140: 3912-3918. 11. Ohki S. Oeino N. Yamamoto S. Havaishi 0. Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem. 1979: 254: 829-836. 12 Yamamoto S. Purification and assay of PGH synthase from bovine seminal vesicles. Methods in enzymol. 1982: 86: 55-61. 13. Koshihara Y, Inagaki A, Murota S. Induction of prostacyclin formation by sodium n-butylate in a cloned epithelial liver cell line. Biochim. Biophys. Acta 1981: 664: 278-290. 14 Koshihara Y, Kawamura M, Senshu T. Murota S. Effect of sodium n-butylate on induction of prostaglandin synthase activity in cloned mastocytoma P-815 2-E-6 cells. Biochem. J. 1981: 194: 111-117. 15 Kondo M. Koshihara Y, Kawamura M, Murota S. Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells. Biochem. J. 1983: 212: 219-222. 16 Wu K K, Hatzakis H, Lo S S, Seong D C, Sanduja S K, Tai H H. Stimulation of de novo synthesis of prostaglandin G/H synthase in human endothelial cells by phorbol ester. J. Biol. Chem. 1988: 263: 3022-3028. 17 Raz A. Wyche A, Siegel N, Needleman P. Regulation of fibroblast cyclooxygenase synthesis by interleukin 1. J. Biol. Chem. 1988: 263: 3022-3028. 18 Casey M L. Korte K, Macdonald P C. Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in aminion cells. Induction of prostaglandin H, synthase. J. Biol. Chem. 1988: 263 : 7846-7854.
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19. Habenicht A J R, Goerig M, Grulich J, Rothe D, Gronmald R, Loth U, Schettler G, Kommerell B, Ross R. Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase. J. Clin. Invest. 1985: 75: 1381-1387. 20. Goerig M, Habenicht A J, Heitz R, Zeh W, Katus H. sn-1,2-Diacylglycerols and phorbol diesters stimulate thromboxan synthesis by de nova synthesis of prostaglandin H synthase in human oromvelocvtic leukemia cells. J. Clin. Invest. 1987: is: 963-91i. 21. Frasier-Scott K, Hatzakis H, Seong D, Jones M, Wu K K. Influence of natural and recombinant interleukin 2 on endothelial cell arachidonate metabolism. Induction of de nova synthesis of prostaglandin H synthase. J. Clin. Invest. 1988: 82: 1877-1883. 22. Yokota K, Kusaka M, Ohshima T, Yamamoto S, Kurihara N, Yoshino T, Kumagawa M. Stimulation of orostaglandin E, svnthesis in cloned osteoblastic celis of mouse (MC3T3-El) by epidermal growth factor. J. Biol. Chem. 1986: 261: 15410-15415. 23. Kusaka M, Ohshima T, Yokota K, Yamamoto S, Kumegawa M. Possible induction of fatty acid cyclooxygenase in mouse osteoblastic cells (MC3T3-El) by CAMP. B&him. Biophys. Acta 1988: 972: 339-346. 24. Masferrer J L, Zweifel B S, Seibert K, Needleman P. Selective regulation of cellular cyclooxygenase by dexamethasone and endotoxin in mice. J. Clin. Invest. 1990: 86: 1375-1379. 25. Fu J Y, Masferrer J L, Seibert K, Raz A, Needleman P. The induction and suppression of prostaglandin H, synthase (cyclooxygenase) in human monocytes. J. Biol. Chem. 1990: 265: 16737-16740. 26. Knook D L, Sleyster E C H. Separation of Kupffer and endothelial cells of rat liver by centrifugal elutriation. Exp. Cell Res. 1982: 139: 444-449. 27. Hecker M, Ullrich V. On the mechanism of prostacyclin and thromboxan A, biosynthesis. J. Biol. Chem. 1989: 264: 141-150. 28. Powell W S. Formation of 6-oxoprostaglandin FLa, 6,15-dioxoprostaglandin F1, and monohydroxyeicosatetraenoic acids from arachidonic acid by fetal calf aorta and ductus arteriosus. J. Biol.-Chem. 1982: 257: 9457-9464. 29. Bouwens L. Baekeland M. Wisse E. Imoortance of local proliferation in the expanding Kupffer cell population of rat liver after zymosan stimulation and partial hepatectomy. Hepatology 1984: 4: 213-219. 30. Bouwens L, Wisse E. Proliferation, kinetics, and
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fate of monocytes in rat liver during a zymosan-induced inflammation. J. Leuk. Biol. 1985: 37: 531-543. Pilaro A M, Laskin D L. Accumulation of activated mononuclear phagocytes in the liver following lipopoly-saccharide treatment of rats. J. Leuk. Biol. 1986: 40: 29-41. Rodriguez de Turco E B, Spitzer J A. Eicosanoid production in nonparenchymal liver cells isolated from rats infused with E. Coli endotoxin. J. Leuk. Biol. 1990: 48: 485-494. Ogino N, Ohki S, Yamamoto S, Hayaishi 0. Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. Inactivation and activation by heme and other metalloporphyrins. J. Biol. Chem. 1978: 253: 5061-5068. Statura J, Tarnawski A, Szczudrawa J, Bogdal J, Mach T, Klimczyk B, Kirchmayer S. Cytoprotective effect of 16,16’ dimethyl prostaglandin E, and some drugs on acute galactosamine induced liver damage in rat. Folia Histochem. Cvtochem. 1980: 18: 311-318. Abecassis M, Falk J A, Makoeka L, Dindzans V J, Falk R E. Levv G A. 16.16’ Dimethvl prostaglandin Er prevents the development of fluminant hepatitis and blocks the induction of monocytc/macrophage procoagulant activity after murine hepatitis virus 3 infection. J. Clin. Invest. 1987: 80: 881-889. Casteleijin E, Kuiper J, Van Rooij H C J, Kamps J A A M, Koster J F, Van Berkel ThJC. Hormonal control of glycogenolisis in parenchymal liver cells by Kupffer and endothehal liver cells. J. Biol. Chem. 1988: 263: 2699-2703. Buxton D B, Fisher R A, Briseno D L, Hanahan D J, Olson M S. Glycogenolytic and haemodynamic responses to heat-aggregated immunoglobulin G and prostaglandin E, in the perfused liver. B&hem. J. 1987: 243: 493-498. Dawiskiba J, Isaksson B, Jeppsson B, Hagerstrand I, Bengmark S. Cytoprotective effect of 16, 16-dimethyl prostaglandin (PGEJ on ischemic injuries in the rat. Eur. Surg. Res. 1984: 16: 77-83. Peters T, Karck U, Decker K. Interdependence of tumor necrosis factor, prostaglandin E,, and protein synthesis in lipopolysaccharide-exposed rat Kupffer cells. Eur. J. Biochom. 1990: 191: 583-589. Kawada N, Mizoguchi Y, Shin T, Tsutsui H, Kobayashi K, Morisawa S, Monna T, Yamamoto S. Synthesis of eicosanoids by Propionibacterium acnes-elicited liver adherent cells and their effect on production of interleukin 1 and tumor necrosis factor. Prostaglandins Leukotrienes and Essential fatty acids. 1990: 41: 187-193.
Lcukotriems and Essential Group UK Ltd 1992
Fatty Acids (19!32) 46,105-110
Enhancement of Prostaglandin Ez Production by Liver Macrophages (Kupffer Cells) After Stimulation with Biological Response Modifiers N. Kawada, Y. Mizoguchi, K. Kobayashi,
T. Manna*, P. Liut and S. Morisawa+
The Third Department of Internal Medicine, *Department of Public Health, and +The First Department of Biochemistry, Osaka City University Medical School, l-5-7, Asahi-machi, Abeno-ku, Osaka 545, Japan (Reprint requests to NK) ABSTRACT. PG& production by liver macropbages (Kupffer cells) activated by biological response modiks was examined. Kupffer cells obtained from a normal rat liver possesed cyclooxygenase activity and produced TXR2, PGD2, and PGEz from (l-i4C)arachidonic acid. The major product was PGD2. When Kupffer cells were incubated in the presence of I@-polysaccharide (LPS), OK-432, or heat-kiied Propionibacterium acnes for 24 h, the amount of arachidonate cyclooxygenase products increased and the major product changed from PGDz to PGE2. When liver macrophages including Kupffer cells were prepared from rats after an injection of LPS, 0K-432, or heat-hilled P. acnes, it was noticed that the number of cells obtained and PGEz production increased compared with those of normal rat. These results suggested that PGEz production by rat liver was induced when they were treated with biological response modifiers.
INTRODUCTION When mononuclear phagocytic cells are exposed to a variety of stimuli, they synthesize and release a number of biologically active materials including arachidonate metabolites of cyclooxygenase (1, 2). Kupffer cells are known to be resident macrophages of the liver and are a part of the organism’s mononuclear phagocyte system (3). Prostaglandin (PG) production by Kupffer cells has also been demonstrated in many studies (4-8). Kupffer cells synthesize and release PGs and thromboxane Bz (TX&) in response to various stimulations such as lipopolysaccharide (LPS) 9 zymosan , calcium ionophore A23187, tumor necrosis factor (TNF) and phorbol ester. PG biosynthesis is regulated by two enzymatic reactions. One is the release of arachidonic acid (AA) from the cellular membrane mediated mainly by phospholipase A2 (9, lo), and the other is the formation of PGH2 from AA by cyclooxygenase (11, 12). Concerning the latter, recent reports have suggested that the cyclooxygenase enzyme is induced in a variety of cells after stimulation and this is attributed to the increase in amount of the enzyme protein (13-25).
Previous reports about the production of arachidonate cyclooxygenase metabolites by Kupffer cells have not clearly demonstrated any change of the products in response to stimulation. In the present study, the cyclooxygenase metabolism in Ku ffer cells was estimated by reacting the cells with (l- !I C)AA to minimize the influence of phospholipase A2 and analysed by reverse-phase high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC). As a result, we observed that the cyclooxygenase activity in Kupffer cells was elevated and PGEz production was dramatically induced in response to the activation by biological response modifiers in vitro and in vivo.
MATERIALS AND METHODS Materials Male Wistar rats (200-220 g) were obtained from Clea Japan Inc (Shizuoka, Japan). The animals were housed under constant conditions and provided with standard chow pellets and water ad libitum. Collagenase (Type IV) was purchased from Wako Pure Chemical Co Ltd (Osaka, Japan). Pronase E was from Merck (Darmstadt). Metrizamide (2-(3acetamide-5-methylacetamide-2,4,6-triiodoacetamide)2-deoxy-D-glucose), lipopolysaccharide (LPS) from
Date received 14 June 1991 Date accepted 13 November 1991 105
106 Prostaglandins Leukotrienes and Essential Fatty Acids
Salmonnella enteritidis, and calcium ionophore A23187 (A23187) were obtained from Sigma Chemical Co. (St. Louis, MO). RPM1 1640 medium was purchased from Nissui Pharmaceutical Co Ltd (Tokyo, Japan). Fetal calf serum (FCS) was from M. A. Bioproducts (Maryland, WA). (l-‘4C)arachidonic acid (AA) was obtained from International (Bucks, UK), and Amersham arachidonic acid from Nu-Chek-Prep (Elysian, USA). Authentic 6-keto-PGFi,, TXB2, PGD2, and PGE2 were kindly provided by Ono Pharmaceutical Company. OK-432 was a kind gift from Chugai Pharmaceutical Company. Propionibacterium acnes was kindly donated by the Department of Bacteriology, Osaka City University Medical School.
Preparation of liver macrophages (Kupffer cells) The rats were intravenously injected with physiological saline, LPS (100 &rat), OK-432 (5 KE/rat), or P. acnes (1 mg/rat). 1, 4, or 7 days after the injection of these BRMs, the rats were anesthetized by an intraperitoneal injection of pentobarbital (10 mg/rat). After laparotomy, non-parenchymal liver cells were separated by collagenase-pronase perfusion according to Knook’s methods with a slight modification (26). In brief, the liver was perfused in situ with Hank’s balanced salt solution (HBSS) for 2 min and then subsequently perfused with HBSS containing 0.05% collagenase and next with HBSS containing 0.05% pronase for 5 min each. After excision, the liver was minced and incubated at 37°C in HBSS containing 0.05% pronase for 30 min. Sinusoidal cells thus separated were harvested from the digested liver by centrifugation (10 min, 400 x g). Sinusoidal cell suspensions were freed of erythrocytes and cell debris by density centrifugation in Gey’s balanced salt solution without NaCl containing 17.5% metrizamide at 1400 x g for 10 min. After the cells were washed by centrifugation (10 min, 400 x g), they were suspended in RPM1 1640 medium with 20% heat-inactivated fetal calf serum and cultured in a plastic dish at 37°C in 5% CQ/95% air. After 18 h incubation, liver macrophages and Kupffer cells adhering to the plastic dish were isolated with the aid of a rubber policeman and used for the following experiments.
Activation of Kupffer cells Kupffer cells obtained from a normal rat were incubated in RPM1 1640 medium containing 5% FCS in lOO-mm plastic dishes. After 18 h incubation, LPS (10 pg/ml), OK-432 (1 KE/ml), or P. acnes (100 pg/ml) was added to each well. Kupffer cells were harvested after another 24 h incubation, and the PG synthesis was determined.
Assay of prostaglandin biosynthesis For the assay of PG biosynthesis, the whole liver macrophages or Kupffer cells were used. The prepared liver macrophages and Kupffer cells were suspended in phosphate-buffered saline (1 X lo7 cells/5 ml) and allowed to react with 25 DM (l-14C)AA (1.25 x lo6 cpm) at 37°C for 30 min. The reaction mixture was centrifuged (400 x g, 4°C 10 min), and the cell free supernatant was acidified to pH 3 with 1 N HCl. After extraction with 10 ml of ethyl acetate, the solvent was evaporated. The dried material was analysed by TLC and reversephase HPLC. The solvent for TLC was the organic phase of ethyl acetate : isooctane : acetic acid: water (110:50:20: 100, V/v). The radioactivity existing in the silica gel TLC plate (Kieselgel60 F254, Merck, Darmstadt) after 40 min development was visualized by auto-radiography. The dried materials were redissolved in methanol and applied to reversephase HPLC with a TSK-gel column (type ODS120T, 5 pm, 4.6 x 250 mm, Tosoh) connected to a dual pump model (LC-6A, Shimadzu Corporation, Kyoto, Japan) and an injector model (SILdA, Shimadzu Corporation, Kyoto, Japan). The solvent mixture was acetonitrile : water: phosphoric acid (35:65:0.1). The flow rate was 1.0 ml/min. Absorption at 195 nm was monitored for PGs and TXBz using a programmable detector (SPDdA, Shimadzu Corporation, Kyoto, Japan). The radioactivity of the organic solvent of each 1 min eluate fraction was determined by a liquid scintillation counter (LS 5801, Beckman Instrument Inc, Fullerton, CA).
RESULTS PGs and TXBz production by Kupffer cells Kupffer cells were reacted with (l-14C)AA, and the reaction products were analyzed by reverse-phase HPLC and TLC. Figure 1A shows the analysis of the authentic PGs and TXB;! by reverse-phase HPLC monitoring the column eluate at 195 nm. The retention times of 6-keto-PGFi,, TXBz, PGE2, and PGDz were 8.3 min, 20.6 min, 25.5 min and 29.8 min, respectively. The reaction products were analyzed by this system, and the radioactivity originating from (l-14C)AA was counted. As shown in Figure lB, one major and two minor radioactive peaks were detected and these peaks were coincident with the retention times of authentic TXB;!, PGl!$, and PGD2. These results suggested that non-treated rat Kupffer cells produced these PGs and TXB:! from exogenous AA and the major product was 1 x lo5 M indomethacin identified as PGDz. inhibited PG production and, when the cells were boiled at 95°C for 5 min, the production of PGs and
PGE, Production and Biological Response Modifiers
107
Fig. 2 TLC analysis of PGE, production by Kupffer cells. Non-stimulated or BRM-stimulated rat Kupffer cells (2 x 10”) were allowed to react with (l-“C)AA (25 x 10’ cpm) at 37°C for 30 min, and the reaction products were analyzed by TLC and visualized by autoradiography as described in Materials and Methods. (A), (C), and (E) Non-stimulated Kupffer cells. (B) LPS-stimulated Kupffer cells. (D) OK-432-stimulated Kupffer cells. (F) P.acnes-stimulated Kupffer cells.
Retention
Time
(mid
Fig. 1 Reverse-phase HPLC analysis of arachidonate cyclooxygenase products by Kupffer cells. Non-stimulated or BRM-stimulated rat Kupffer cells (2 x 106) were allowed to react with (l-“C)AA (25 x 10'cpm) at 37°C for 30 min, and the reaction products were analyied’by reverse-phase HPLC as described in Materials and Methods. (A) The analvsis of authentic 6-keto-PGF,,, TXB,, PGE, and PGD 2’ (B) ’ Non-stimulated Kupffer cells. (C) LPS-stimulated Kupffer cells. (D) OK-432-stimulated Kupffer cells. (E) P. acnes-stimulated Kupffer cells.
TXB;! was not detected, which suggested that the reaction from exogenous (l-14C)AA to each PG and TXB;! was the event of enzymatic procedure (data not shown). Each peak was identified as TXB2, PGEZ, and PGD2 by radioimmunoassay by using a specific antibody for each PG and TXB2 raised in rabbit serum (data not shown). We next examined the effect of BRMs on the arachidonate cyclooxygenase metabolism of Kupffer cells in in vitro treatment. Kupffer cells were incubated with LPS (10 &ml), OK-432 (1 KE/ml), or heat-killed P. acnes (100 pg/ml) for 24 h, and the cellular levels of cyclooxygenase activity were measured. Figures lC-1E shows the results and demonstrated that the production of PGE2 by Kupffer cells was enhanced by the treatment of the cells with BRMs. The production of PGE2 by BRMtreated Kupffer cells was enhanced about 4 times more than that of normal Kupffer cells. These results were confirmed by analysis performed using TLC as shown in Figure 2. The band corresponding to PGE2 increased in density, when Kupffer cells were treated with BRMs. This enhancement of
PGEz production was dose-dependent with reference to the stimulants, and the optimal doses of LPS, OK-432, and heat-inactivated P. acnes were 0.1-10 pg/ml, OS-5 KE/ml, and l-100 pg/ml, respectively (data not shown).
Effect of in vivo administration of BBMs on the production of PGs and TXBz by liver macrophages Next we examined whether in vivo administration of these BRMs has effects on the arachidonate cyclooxygenase metabolism of Kupffer cells. 1, 4, or 7 days after an intravenous administration of LPS (100 pg/rat), OK-432 (5 KE/rat), or P. acnes (10 mg/rat), liver macrophages were prepared as described in materials and methods. At first, we observed the increase in the number of obtained liver macrophages. The number of Kupffer cells from a normal rat was about 1 x lo7 cells/rat (n = 5). However, when the rats were treated with LPS, OK-432, or P. axes, the number of liver macrophages obtained increased to about 1.5 X107 (n = 3), 2 x lo7 (n = 3), or 1 x lo8 (n = 3) cells/rat, respectively. The microscopic examination of conventionally prepared tissue section showed an increase of mononuclear cells which mostly existed in the sinusoidal capillary lumina after the injection of BRMs. Especially, granuloma formation was seen in the liver of rats treated with P. acnes (Fig. 3). The cells obtained were mononuclear cells, adherent to plastic dishes, and phagocytosed latex particles (0.8 pm), which were most likely macrophages. We examined the PG production by these liver macrophages according to the methods described
108
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Fig. 3 Microscopic examination of the liver of P. acnes-injected rats. At 7 days after the injection of heat-killed P. acnes through a tail vein, the liver was fixed and prepared conventionally for microscopic examination with H-E stain. The accumulation of mononuclear cells and the formation of granulomas in the liver were seen. (X 200)
ontrol
LPS
OK-432
P.acnes
Fig. 4 The cyclooxygenase activity in liver macrophages obtained from BRM-injected rats. Liver macrophages were obtained from rats injected with normal saline, LPS (100 pg), OK-432 (5 KE), or P. acnes (1 mg) and allowed to react with (1J4C)AA. The reaction products were analyzed by reverse-phase HPLC and the amount of radioactivity corresponding to 6-keto-PGF,,, TXB,, PGE, and PGD, was determined. Data are expressed as mean + SD of 3 different examinations. The statistical significance is analyzed by Student’s t-test. *-**; p < 0.05, *-***; p < 0.01.
above. The PG production was significantly increased after the injection of LPS, OK-432, or P. axes (Fig. 4). TLC analysis of the metabolites produced by the macrophages obtained from OK-432 or P. acnes-treated rats showed that the production of PGE:! was significantly amplified (Fig. 5). We obtained similar results by reversephase HPLC analysis.
DISCUSSION In the present study, we demonstrated that the PG production by Kupffer cells was elevated in response to in vitro stimulation with LPS, OK-432,
Fig. 5 TLC analysis of arachidonate cyclooxygenase products by liver macrophages. Liver macrophages (2 x 104 from BRM-treated rats were allowed to react with (l-14C)AA (25 x lo4 cpm) at 37°C for 30 min, and the reaction products were analyzed by TLC and visualized by autoradiography as described in Materials and Methods. (A) and (C) Liver macrophages from physiological saline-injected rat. (B) Liver macrophages from OK-432-injected rat. (D) Liver macrophages from P. acnes-injected rat.
or P. acne.s and that the major product of arachidonate cyclooxygenase metabolites changed from PGDz to PGE*. In this case, as the number and the viability of Kupffer cells were not changed after stimulation with BRMs, the intracellular metabolic changes possibly caused the elevation of PGE2 production. We observed the increased production of not only PGE2 but also PGD2, TXB2, HHT (a biproduct of TXA2 synthetase (27)), and HETEs (a biproduct of cyclooxygenase (28)), which may be one of the reasons for the activation of cyclooxygenase. However, the activation of PGE;! synthetase (PGH2 isomerase) was not ruled out and we do not have the answer to the question as to why PGE2 came to be predominantly produced more than the other cyclooxygenase products. These observations were also seen when liver macrophages obtained from rats injected with BRMs in vivo were reacted with (l-%)AA. In vivo administration of BRMs to rats caused the increase of the adherent cell number in the liver per rat and
PGE, Production and Biological Response Modifiers
elevated the PGE2 production. In this case, we have to carefully discuss about the cells used in the assay. Some reports have demonstrated that Kupffer cells proliferate in response to stimulation by zymosan, OK-432, or P. awes in vivo (29, 30). On the other hand, mononuclear cells and polymorphonuclear leukocytes migrate into the liver in the case of inflammation (31, 32). Therefore, we can not declare which is the cause of the cyclooxygenase activation in liver macrophages, a functional change in Kupffer cells in accordance with proliferation or a change of cell types based on the migration of inflammatory macrophages from the other organs. However, the ability to produce PGEz in the liver was augmented after treatment of rats with BRMs. The increased production of PGE2 may originate from the activation of cyclooxygenase (33) or the induction of cyclooxygenase itself. Recent reports have suggested that cyclooxygenase is induced in many types of cells after stimulation (13-25). More recently, Masferrer and Fu have reported that the cyclooxygenase activity in human or mouse monocytes rises after they are stimulated with LPS in vivo (22) or in vitro (23). Thus, in a variety of cells, the induction of cyclooxygenase has been shown under conditions of stimulation with agents which have multipotential effects on cell functions and make changes in intracellular metabolism. Our above observation may originate from such an induction of cyclooxygenase but more detailed examination may be required for the determination. The enhancement of PGE2 production may play a role in the regulation of cell function. PGE2 has been reported to have some effects on liver cell function such as cytoprotection (34, 35) and the regulation of glyconeogenesis (36, 37) for hepatocytes, the maintenance of microcirculation (38), and modification of collagen synthesis (6). Especially, PGE;! has been known to diminish the production or release of interleukin 1 (IL-l) and tumor necrosis factor (TNF). Peters et al have clearly demonstrated that TNF production by LPS-stimulated rat Kupffer cells is regulated by the elevation of intracellular CAMP induced by PGE2 (39). We have also reported that the production of IL-l and TNF by P. acnes-elicited liver adherent cells is inhibited by PGE2 and PGI:! which are released from the cells in the autocrine or paracrine manner (40). Therefore the increased production of PGEz may be important in diminishing the enhancement of inflammatory reactions. Acknowledgements We are grateful to Mrs Haruko Ueda for preparing this manuscript. Y. M. was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, and the Ministry of Science and Technology of Japan.
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