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Identification of prostaglandin D2 as the major eicosanoid from liver endothelial and Kupffer cells
143
Biochimica et Biophysics Acta 959 (1988) 143-152 Elsevier
BBA 52114
Identification of prostaglandin D, as the major eicosanoid from liver endothelial and Kupffer cells
Johan Kuiper a, Freek J. Zijlstra b, Jan A.A.M. Kamps a and Theo J.C. van Berkel a a Division of Biopharmaceutics, and b Department
Center for Bio-Pharmaceutical Sciences, University of kiden, Leiden of Pharmacology, Erasmus University, Rotterdam (The Netherlands)
(Received 17 August 1987) (Revised manuscript received 7 December 1987)
Key words: Icosanoid release; Prostaglandin D,; Endothelial cell; Kupffer cell; Parenchymal cell; (Rat liver)
The capacity of freshly isolated endothelial, Kupffer and parenchymal rat liver cells to produce eicosanoids from [l-‘4C]arachidonic acid was investigated in order to determine the relative importance of these cells to total liver eicosanoid production. Based upon the total formation of [l-‘4C]arachidonate metabolites in the liver, it can be calculated that Kupffer and endothelial cells are responsible for 65 and 234, respectively, of the total amount of eicosanoids produced by the liver. Consequently, parenchymal liver cells, representing 92.5% of the total liver mass, contribute only 12% to the total liver production of eicosanoids. The main product of Kupffer cells was prostaglandin D, (PGD,), representing 55% of the total amount of eicosanoids produced. Liver endothelial cells produced about 44imes less eicosanoids (per mg cell protein) than Kupffer cells, and PGD, was also the main product of these cells (44%). The production of eicosanoids by parenchymal cells was lower by a factor of 180 (per mg cell protein) than that in Kupffer cells. Besides the ability to form eicosanoids from added “C-labeled arachidonic acid, Kupffer and endothelial liver cells were also able to produce significant amounts of PGD, (the main liver prostaglandin) from endogenous arachidonic acid, as determined by a radioimmunoassay. It is concluded that inside the liver, Kupffer cells together with endothelial cells are of major importance in the production of eicosanoids, while the parenchymal cells may be considered metabolic target cells for these products, as indicated by the finding that the major liver prostalgandin, PGD,, could stimulate the glucose output in isolated parenchymal cells.
Abbreviations: PG, prostaglandin; di-HETE, 5,12dihydroxy5,8,10,14-eicosatetraenoic acid; HHT, 12-hydroxy-5,8,10heptadecatetraenoic acid; 15-HETE, 15-hydroxy-5,8,11,13eicosatetraenoic acid; HPLC, high-pressure liquid chromatography. Correspondence: J. Kuiper, Division of Biopharmaceutics, Center for Bio-Pharmaceutical Sciences, University of Leiden, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The Netherlands. 0005-2760/88/$03.50
Introduction The liver is an important organ in the metabolism of prostaglandins [1,2]. For example, the liver is the main organ for the processing of PGI,, which is converted to 6-keto-PGE,,, a vasodilat& and potent inhibitor of platelet aggregation [3]. Also, PGDz can be converted by a microsomal enzyme from liver, 11-keto-reductase, to PGF,, [4]
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
144
and more recently, PGD, has been described to be transformed in the liver to 9,,lls-PGF,, a newly discovered prostanoid which is a very potent vasoconstrictor and an inhibitor of platelet aggregation [5,6]. Besides the processing of prostaglandins, the liver is able to synthesize several prostaglandins and leukotrienes. Hepatocytes, the main cell type in liver, can produce thromboxane Bz when stimulated with vasopressin [7]. Kupffer cells, which form the tissue macrophages of the liver, have been described to produce, in culture, PGE,, PGD,, PGF,,, PGI, and leukotrienes [8-lo]. In addition to parenchymal and Kupffer cells, the liver also contains endothelial cells, and although extrahepatic endothelial cells have been described to produce a variety of eicosanoids, such as PGI, [11,12], 15-HETE [13,14], PGEi and PGF,, [15], the capacity of the liver endothelial cells to produce eicosanoids is unknown, probably because adequate culture conditions for liver endothelial cells were unavailable until recently [16]. In our laboratory, we developed a cell isolation and purification procedure using collagenase, which allows a direct comparison of the ability of parenchymal, Kupffer and endothelial liver cells to perform receptor-dependent uptake of ligands [17]. In the present experiments, we have compared quantitatively and qualitatively the capacity of the various liver cell types to synthesize both cyclooxygenase and lipoxygenase products from arachidonic acid. Materials and Methods Materials Collagenase type I was obtained from Sigma (St. Louis, MO); Ca2+ ionophore A23187 was from Hoechst (Calbiochem-Behring, U.S.A.); ail other radiolabeled compounds, [l- l4 Clarachidonic acid, 5-D-[5,6,8,9,11,12,14,15(n)-3H]-HETE, 12-L[5,6,8,9,11,12,14,15(n)-3H]-HETE, 15-L-[5,6,8,9, 11,12,14,15(n)-3H]-HETE, [5,6,8,9,11,12,14,15(n)3H]leukotriene B4, 6-keto[5,8,9,11,12,14,15(n)-3H]prostaglandin Fi,, [5,6,8,9,11,12,14,15(n)-3H]thromboxane &, [5,6,8,11,12,14,15(n)-3H]prostaglandin E,, [5,6,8,9,11,12,14,15(n)-3H]prostaglandin Fza and [5,6,8,9,12,14,15(n)-3H]prostaglandin D, and a radioimmunoassay kit for specific PGD,
determination were from Amersham International (U.K.). Sep Pak C,,, silica cartridges and HPLC filters HA (0.45 PM), FH (0.5 PM) and Millex (0.45 pm) were obtained from Waters/ Millipore (The Netherlands). Prepacked HPLC columns, Nucleosil5C,, and Zorbax C,, were obtained from Chrompack Middelburg (The Netherlands). Liver cell isolation The three types of liver cell (i.e., parenchymal, endothelial and Kupffer cells) were isolated and purified from livers of male Wistar rats (3 months old, weighing about 200 g) by in situ perfusion of the rat liver with 0.05% collagenase, as described previously [ 171, with one modification, whereby hepatocytes were removed by centrifugation (2 min, 75 x g), and not by elutriation. Cell viability was checked during the incubations by measuring the ATP content in the three cell types. The ATP content appeared to be constant, indicating no loss of cell viability. Purity of the cell fractions was examined by peroxidase staining with diaminobenzine. The parenchymal and endothelial cell fractions were pure and contained no other type of liver cell. The Kupffer cell fraction contained 91 k 5% (S.E.) (n = 3) peroxidase-positive cells, the remainder being (large) endothelial cells. The calculation of the relative contribution of the various liver cells to total liver eicosanoid production was performed as follows. According to Blouin et al. [18], hepatocytes contribute 92.5% to the total liver cell volume, the endothelial 3.3%, and the Kupffer cells 2.5%. The remaining 1.7% is contributed by fat-storing cells and pit cells. Since the protein concentration (in mg per ml of cell volume) in the parenchymal, Kupffer and endothelial cells is identical [19,20], these percentages can be directly related to protein. The validity of this assumption was demonstrated earlier by enzyme distribution studies with peroxidase (a specific marker for Kupffer cells), lysosomal enzymes and pyruvate kinase [21]. Cell incubations Liver cells were incubated for 10 min in 10 ml Krebs-Henseleit buffer (cell concentrations: Kupffer and endothelial cells, 0.1-0.2 mg protein/ml and parenchymal cells, 0.5-0.8 mg pro-
145
tein/ml). Tubes were placed in a water-bath (37 o C) and the cell suspensions were continuously gassed through a thin pipette with 95% 0,/5% CO,. The addition of 2.5 or 5 PCi [l“C]arachidonic acid (59.6 mCi/mmol) was followed by the addition of glutathione (final concentration, 2 mM) and the calcium ionophore A23187 (10 PM). At the end of a 10 or 20 mm 3H-labeled leukotrienes and 3Hincubation, labeled prostaglandins were added, and the liver cells were centrifuged (10 min, 1400 x g, 4O C). The supernatants of each cell type were then applied to two Sep Pak C,, and silica cartridges (the cartridges were prewashed with 10 ml ethanol and 10 ml distilled water). The samples were eluted with 5 ml ethanol and the eluates were evaporated to dryness with a Speed Vat concentrator (Savant) at 4O’C. Thereafter, the dried samples were dissolved in 0.3 ml solvent A, centrifuged and purified by a Millex filter, and kept in an HPLC microvial (Weichmann, Switzerland). Volumes of 100 ~1 were injected onto the columns and chromatographed using a 1082B HPL chromatograph (Hewlett Packard, Brussels, Belgium). Fractions were collected with a Superrac fraction collector (LKB, Sweden) and counted in a 3255 Tricarb liquid scintillation counter (Hewlett Packard). The percentage of arachidonic acid incorporated in the lipid fraction was assessed by a Folch extraction. Lipids were separated by thin-layer chromatography.
radioactivity OLcdpm) So000
1
formed
moo 3owo
1 i 10
20
30
40 Time (mini
50
60
30
CO Time (mini
50
60
r_adioactivity ?H dpm) Star&h
0 10
Chromatographic system Reversed-phase HPLC of lipoxygenase products was carried out on a Nucleosil 5C,, column. The solvent system A was tetrahydrofuran/ methanol/O.l% (w/v) EDTA solution in water/ acetic acid (25/30/45/0.1), adjusted to pH 5.5 with ammonium hydroxide. Mobile phases were filtered by vacuum filtering through a Millipore filter and degas& with helium. The flow rate was 0.9 ml/mm and the absorption was measured at 280, 270 and 234 nm for the identification of the mono-HETE species. The column was equilibrated with the mobile phase (A) at an oven temperature of 37 o C. Fractions were collected for scintillation counting. After each run (80 min) the column was rinsed with methanol for at least -30 min, because of contamination with the calcium
Eicosanoids
20
Fig. 1. Chromatogram of tritiated standards and cyclooxygenase products (‘4C-labeled) formed by Kupffer cells. 100 pl of a concentrated sample (300 pl), containing the “C-labeled arachidonic acid metabolites formed by Kupffer cells (0.85 mg) as well as the 3H-labeled standards, was applied to a Zorbax C,, column to separate the prostaglandins. In each fraction sample (1 ml), 3H and 14C radioactivity was counted. TxB,, thromboxane b.
ionophore and [‘4C]arachidonic acid, which elute after 115 and 140 min, respectively, in system A. Reversed-phase HPLC of prostaglandins was per; formed on a Zorbax C,, column. The solvent system B comprised acetonitrile/ benzene/ water/ acetic acid (24/0.2/76/0.1). The flow rate of this eluent was 2 ml/mm. Fractions were collected for
146 TABLE I
Quantitative
FORMATION OF CYCLOOXYGENASE AND LIPOXYGENASE PRODUCTS BY ISOLATED PARENCHYMAL, KUPFFER AND ENDOTHELIAL LIVER CELLS
The settings for double-label scintillation counting were such that there was no spillover of radioactivity of ‘H into the 14C channel. dpm calculations were carried out using quenched standard sets. A plotting system was programmed in order to obtain data of total counts covering the peak areas. Amounts were calculated in dpm of each channel and were plotted as separate chromatograms.
The liver cells were incubated for 10 min with labeled arachidonic acid (2.5 PCi) and the products formed were determined as indicated in the Materials and Methods section. cell protein& The results are expressed as r4C dpm.10-3/mg S.E. (n = 3). TxBa, thromboxane &. Type of eicosanoid
Cell type endothelial
Kupffer
6-Keto-PGF,,
6.4* 3.4 41.4* 9.2 42.4* 9.0 32.4* 5.3 230.2f15.2
10.3f 2.7 0.4kO.2 198.7* 28.8 0.6kO.3 95.0f 48.9 2.2k1.3 46.0f 23.9 0.4kO.l 1095.4f104.6 1.6k0.6 199.8f 40.4 2.8f0.4 106.0* 17.8 0.7kO.2 198.0* 24.2 l.OkO.3 43.lf 22.1 1.0*0.3
TxB, PGF,, PGE, PGD, Di-HETE/HHT IS-HETE 12-HETE 5-HETE
55.4 f 24.8* 65.5 f 16.1+
8.8 5.7 6.5 6.7
evaluation
parenchymal
scintillation counting. The column was rinsed with acetonitrile for 30 min after each sample to elute the lipoxygenase products [22]. Fig. 1 shows a representative reversed-phase HPLC chromatogram of the cyclooxygenase products from Kupffer cells. The tritiated standards, which were used to identify the lipoxygenase and cyclooxygenase products from the liver cells, had a somewhat shorter retention time (Fig. 1) due to a slightly higher polarity. Identification of the various prostaglandins was also performed using specific antibodies, as described extensively before by Zijlstra et al. [22,23]. The 3H-labeled standards were also used to measure the recovery of the various eicosanoids throughout the extraction and purification procedure. The recoveries f S.E. (mean of nine experiments) of the various eicosanoids were as follows: 6-keto-PGF,, (89% + 2.8); thromboxane B2 (75% f 2.4); PGF,, (25% + 1.0); PGE, (50% + 2.0); PGD, (58% f 2.8); leukotriene B4 (77% f 6.3); 15-HETE (37% f 1.2); 1ZHETE (35% f 0.9) and 5-HETE (25% f 0.9). These recoveries were used to quantify the production of the individual eicosanoids by endothelial, Kupffer and parenchymal cells (Table I).
Glucose production by parenchymal
cells
Freshly isolated parenchymal cells were incubated in the presence or absence of PGD, (lop6 M) in glucose-free Krebs-Ringer bicarbonate buffer (pH 7.4) saturated with O,/CO, (95%/5%). After a 10 min incubation, the cell suspension was rapidly cooled and centrifuged (500 X g, 5 min) and subsequently glucose was determined in the supematant by the glucose oxidase-ABTS method ~41.
The incorporation of 14C-labeled arachidonic acid into cyclooxygenase and lipoxygenase products by parenchymal, Kupffer and endothelial cells (per mg cell protein) is shown in Table I. From these data, it is clear that the two non-parenchymal cell types, Kupffer and endothelial cells, showed a much higher capacity to produce eicosanoids than parenchymal cells. To exclude the possibility that a difference in substrate availability between the various liver cell types would influence these differences in the apparent eicosanoid production rate, we also measured the percentage of added arachidonic acid incorporated into triacylglycerols, phospholipids and free fatty acids in the three cell types (Table II). No significant differences were found between endothelial, Kupffer and parenchymal cells, indicating that the observed differences in eicosanoid production between the various cell types (Table I) arose due to a difference in the activity of the specific enzymes involved in this pathway. The quantitatively most important prostaglandin synthesized by Kupffer cells was PGD,, while relatively high amounts of thromboxane 4, diHETE/HHT and 1ZHETE were also produced
147 TABLE II DISTRIBUTION OF 14C FROM [t4C]ARACHIDONIC IN VARIOUS LIVER CELL TYPES
ACID AMONG EICOSANOIDS, LIPIDS AND FREE FATTY ACIDS
The distribution of i4C from arachidonic acid (2.5 aCi) after a 10 min incubation with endothelial, Kupffer and parenchymal cells (0.1 to 0.5 mg cell protein/ml), among formed eicosanoids, lipids, free fatty acids and arachidonic acid was measured. Cell type
Eicosanoids
Triacylglycerol
Phospholipid
Free fatty acid
Arachidonic acid
Endothelial Kupffer Parenchymal
18.6k2.2 33.1 f 5.0 1.1*0.3
3.5kl.O * 3.9*1.2 * 5.s*1.4 *
4.8kl.O * 5.2k1.4 * 6.6k1.3 *
0.6kO.l * 0.7 *0.1 * 0.9kO.2 *
74.0 f 2.6 55.1 f4.4 83.9 f4.0
* No significant difference observed between the various cell types.
TABLE III EFFECT OF [i4C]ARACHIDONIC ACID CONCENTRATION KUPFFER AND ENDOTHELIAL CELLS
ON EICOSANOID
PRODUCTION
BY PARENCHYMAL,
The liver cells were incubated for 10 min with 2.5 or 5 gCi [‘4C]arachidonic acid and the total production of eicosanoids as well as the relative formation of prostaglandin D, was measured, as described in the Materials and Methods section. Data represent the mean f S.E. of three experiments. Cell type
endothelial Amount of added arachidonic acid Total eicosanoid production (t4C dpm. lo- ‘/mg cell protein Relative formation of PGDr (W of total)
Kupffer
2.5
5
521.2 f 36.9 44.2*
5
2.5
592.7 f 49.3 *
2.8
parenchymal
40.4*
3.1 *
1992.3 f 151.6 55.0f
5.3
2.5
1893.5 f 136.4 * 52.4f
4.3 *
5
10.7 f 1.9
11.7k2.1
15.0f4.3
17.1 f3.1 *
l
* Not significantly different from values found with 2.5 pCi arachidonic acid.
TABLE IV THE INFLUENCE
OF THEINCUBATION
TIME ON THE EICOSANOID PRODUCTION
BY LIVER CELLS
The various liver cell types were incubated with 2.5 pCi [‘4C)arachidonate for 10 or 20 min. Total eicosanoid formation (sum of individual eicosanoids) and relative percentage of PGD, was measured as described in the Materials and Methods section. Data represent the mean f. S.E. of three experiments. Ceil type endothelial Time @in Total eicosanoid formation (14C dpm.10-3/mg cell protein) Relative formation of PGD, (W of total)
10
521.2 f 36.9 44.2*
Kupffer 20
2.8
739.0* 88.6 39.4* 4.1 *
* Not significantly different from the 10 min level.
parenchymal
10
20
1992.3 f 151.6 55.0f
5.3
2 368.8 f 210.5 51.1*
4.6 *
10
20
10.7 f 1.9
15.6k1.9
15.0* 5.6
17.3 * 3.4 +
148
70 Endothelial
cells
60 I
F ;
50 40
s k a
30 20 10
70 Kupffer
cells
60 -
Parenchymal
cells
60
10
Fig. 2. Patterns of eicosanoid production by endothelial, Kupffer and parenchymal cells. The relative contribution of the various cyclooxygenase and lipoxygenase products to total eicosanoid production by endothelial, Kupffer and parenchymal cells was calculated from the data in Table I. The data are presented as the mean (f S.E.) of three experiments. Txb , thromboxane &.
(Fig. 2). Endothelial cells also formed PGD, as the main product, although the level of PGD, formation (230. lo3 dpm/mg cell protein) was about 4-fold lower than in Kupffer cells (Table I). Other relatively important endothelial cell products were thromboxane I$,, di-HETE/HHT and 5-HETE (Fig. 2). Parenchymal cells showed a completely different pattern of eicosanoid production, with PGF,, and di-HETE/HHT as the main products. The two eicosanoids, di-HETE and HHT, could not be fully separated by reversedphase HPLC, but on the basis of the elution profile of unlabeled HHT and di-HETE, it is concluded that about 80% of this peak is formed by HHT for all three cell types. The amount of 14C incorporated into the major eicosanoids produced by parenchymal cells was relatively small (2000-3000 dpm/mg cell protein, Table I), as compared to the non-parenchymal cell types. When the production of eicosanoids in the various liver cells was measured with 5 pCi arachidonate instead of 2.5 PCi, the absolute amounts of eicosanoid formed were comparable, indicating that after the addition of 2.5 PCi arachidonate, the maximal eicosanoid production had already been reached in the various liver cell types (Table III). The relative eicosanoid production was found to be identical under both conditions and PGD, was still the major eicosanoid formed by the non-parenchymal liver cell types. Analysis of the eicosanoid production by Kupffer, endothelial and parenchymal cells after a 20 min incubation revealed that the relative patterns were unchanged, as is illustrated by the unchanged contribution of PGD, to total eicosanoid production. The absolute amount of [‘4C]arachidonic acid incorporated into the eicosanoids after a 20 min incubation was 1.2-1.5-fold higher. However, the relative importance of endothelial and Kupffer cells in the production of eicosanoids as compared to parenchymal cells was virtually unchanged (Table IV). When the eicosanoid production by Kupffer and endothelial cells was measured in the absence of the calcium ionophore, no lipoxygenase products could be detected, whereas the level of total prostanoid production appeared to be lower (40%) in the absence of the calcium ionophore. The relative amounts of prostanoids produced by
149 TABLE
V
PRODUCTION OF PROSTANOIDS KUPFFER CELLS
IN THE PRESENCE
AND ABSENCE
OF Ca*+ IONOPHORE
BY ENDOTHELIAL
AND
The prostanoid production of endothelial and Kupffer cells was measure during a 10 min incubation in the absence and presence of Ca*+ ionophore A23187 (10 pm) as indicated in the Materials and Methods section. The results are expressed as the mean f S.E. of three experiments (14C dpm.10W3/mg cell protein). TxBa, thromboxane Ba. Endothelial
6-Keto-GPF,, TxB, PGF,, PGE, PGD,
Kupffer
cells
- A23187
+A23187
- A23187
+ A23187
2.6kO.l 39.9*7.3 33.1 f 2.4 26.5 f 4.4 150.5*1.3
6.4f 4.3 47.4* 9.2 42.4* 9.0 32.4* 5.3 230.2 f 15.2
5.3* 0.6 91.7* 9.3 56.1 f 11.0 35.7f 3.8 673.0 f 79.1
10.3+ 2.7 198.7 f 28.8 95.0* 48.9 46.0+ 23.9 1095.4 f 104.6
cell types to total liver [18,19], the contribution of each liver cell type to the production of the different eicosanoids by total liver was calculated (Fig. 3). Kupffer cells were the main source of PGD,, thromboxane Bz, di-HETE/HHT and 12-HETE, which were also the main products of the total liver. Endothelial cells and Kupffer cells were equally active in the production of PGE,, each producing about 40% of the PGE, synthesized by
Kupffer and endothelial cells were comparable, however, both in the presence and in the absence of the Ca2+ ionophore (Table V). Interestingly, the level of prostanoid production by Kupffer cells was, both in the presence and the absence of the Ca2+ ionophore, about 4-times higher than prostanoid production by edothelial cells. From the data on the eicosanoid production (Table I) and the relative contribution of the various liver 100 80
H & 2 g
6KPCFla
cells
TxB2
1
60
40 20
EC
KC
EC
KC
PC
PC
EC
EC
KC
PC
EC
KC
PC
EC
KC
PC
KC
PC
EC
KC
PC
EC
KC
PC
EC
KC
PC
Fig. 3. Contribution of endothelial (EC), Kupffer (KC) and parenchymal (PC) cells to the production of the different eicosanoids by total liver. The contribution (mean& S.E., n = 3) of endothelial, Kupffer and parenchymal cells to the total liver production of the different eicosanoids was determined as described in the Materials and Methods section.
150 TABLE VI COMPARISON OF PGDs FORMATION BY ENDOTHELIAL, KUPFFER AND PARENCHYMAL CELLS FROM ADDED ARACHIDONIC ACID (14C-LABELED) OR ENDOGENOUS ARACHIDONIC ACID The PGD, formation from t4C-labeled arachidonate in the presence of Ca*+ ionophore (A23187, 10 FM) was calculated using the data in Table I. The PGD, formation from endogenous arachidonic acid was measured after a 10 min incubation of freshly isolated liver cells, using a radioimmunoassay. The results are expressed as the mean of three experiments (pg PGD, /mg cell protein per min) f SE. Cell type
PGD, formation + [ l4Qrachidonic + A23187
Endothelial Kupffer
0.0617 f 0.0041 0.2941 f 0.0281
Parenchymal
0.004
acid
+ A23187 0.020 f 0.002 0.094 *0.011 O.oool *o.oooo1
kO.0002
the liver. Parenchymal cells appeared to be the main source of 6-keto-PGF,, and SHETE (Fig. 3), but these two eicosanoids are only minor liver eicosanoid products. In order to quantify the amount of PGD, formed by the various cell types from endogenous arachidonic acid, we applied a recently developed radioimmunoassay for PGD,. It appears that the Kupffer cells produce 3.9-times the amount of PGD, (per mg cell protein) produced by the endothelial cells, and 940~times that produced by parenchymal cells. These data indicate that the relative contribution of the various cell types to PGD, formation from endogenous substrate reflects the maximal capacities of this pathway, as
TABLE VII EFFECT OF PGDr ON THE AMOUNT OF GLUCOSE PRODUCED BY ISOLATED PARENCHYMAL CELLS The glucose output of freshly isolated parenchymal liver cells was measured in the absence and presence of PGD, (10m6 M). Data are meanf S.E. of four experiments. * Significant difference from control (P < 0.002). Addition
Glucose output (nmol glucose/ 10 min per mg cell protein)
None PGD, (1O-6 M)
31.7*1.7 53.3*3.3
*
measured by adding excess [ “C]arachidonic acid (Table VI). Preliminary results show that PGD,, the main eicosanoid produced by Kupffer and endothelial cells, is able to exert a metabolic effect on parenchymal cells. When PGD, (10e6 M) was added to freshly isolated parenchymal cells, the glucose output by these cells was found to be significantly enhanced (Table VII). Discussion
The data presented in this article show that the three liver cell types used, parenchymal, endothelial and Kupffer cells, all have the capacity to produce eicosanoids from exogenous arachidonic acid. Parenchymal cells, however, had a very low capacity to produce these arachidonate metabolites and it can be calculated, using the relative contribution of the various liver cells to total liver [19,20], that these cells can only be responsible for 12% of the total liver eicosanoid production. Both types of non-parenchymal cell had a much higher capacity to produce eicosanoids than parenchymal cells: the capacity (per mg cellular protein) was higher by a factor of 48 and 183 in endothelial and Kupffer cells, respectively, than in parenchymal cells. The main product of both liver endothelial and Kupffer cells was PGD,, representing 44.2 and 55.48, respectively, of the eicosanoids formed by these cell types. This large PGD, production is surprising, especially in liver endothelial cells, since endothelial cells from other sites in the body were shown to produce PGI, as the main eicosanoid [11,12]. In liver endothelial cells, PGI, (measured as 6-keto-PGF,,) comprises only 2.5% of the eicosanoids produced. The fact that PGD2 was found as the main product in Kupffer cells is less surprising [lo], although the relative amount of PGD, is much higher (55%) for freshly isolated Kupffer cells than has been described for cultured Kupffer cells (30%) [lo]. The results obtained with these rat liver macrophages are comparable with those obtained with murine tissue-macrophage cell lines, which also convert labeled arachidonate preferentially to PGD, [25]. Human or rat peritoneal macrophages, however, convert 30% of the labeled arachidonate to 5-HETE and 40% to di-
151
HETE/HHT [lo]. The finding that PGD, is the most prominent prostaglandin in the liver is in full agreement with results obtained by measuring the capacity of a whole liver homogenate to form prostaglandins, which showed that PGD, was the main prostaglandin formed [26]. We applied a system with added [‘4C]arachidonic acid and added the Ca*+ ionophore in order to assess the maximal capacities of the relative cell types to form both cyclooxygenase and lipoxygenase products. In the absence of the Ca*+ ionophore, no lipoxygenase products were formed (Table V), while the amount of cyclooxygenase products is lowered to about 40%. In order to obtain results on the relative contribution of the various cell types to total liver eicosanoid production from endogenous arachidonic acid, we carried out an immunoassay for PGD,. Although the absolute amounts of PGD, formed were lower, it was found that the relative contribution of the various cell types to total liver PGD, formation was similar to that found under maximally stimulating conditions (Table VI). This indicates that the maximal capacity of the various cell types to form PGD, is related to their production from endogenous arachidonic acid. There is great similarity between the liver endothelial cells and the Kupffer cells with respect to their ability to form the various eicosanoids. The main differences are a much higher capacity in Kupffer cells for eicosanoid formation than in endothelial cells (3.8~times higher per mg cell protein) and a relatively high capacity of endothelial cells for the formation of PGE,. The resemblance between these cell types is certainly not due to a contamination of the endothelial cells with Kupffer cells, since the purity was checked microscopically after a Kupffer-cell-specific peroxidase staining (see Materials and Methods). Furthermore, earlier data from our laboratory show that the liver endothelial cells, prepared by procedures similar to those used in this study, perform specific functions in relation to receptor-ligand interactions [17]. In addition, it must be remembered that liver endothelial cells can certainly not be compared with other endothelial cells in the body, as they contain fenestrae [27], numerous lysosomes [28] and also multiple receptors which make then sometimes more comparable to macrophages than
to endothelial cells [29]. Although the protein contribution of Kupffer and endothelial cells to total liver protein is only 2.5% and 3.3%, respectively, the high specific activity of these cell types to form eicosanoids leads to a calculation which indicates that Kupffer cells may contribute 65% to total liver eicosanoid production. This calculation assumes that the production of eicosanoids by the freshly isolated cells reflects their in vivo capacity to produce these products. Because, during culture, parenchymal cells change their pattern of protein synthesis [30], liver endothelial cells change their morphology [16], and Kupffer cells lose their peroxidase activity [16], we tend to conclude that the production of eicosanoids by freshly isolated cells, as reported here, may represent a production pattern which is the closest reflection of the physiological situation. The physiological function of the eicosanoids produced by non-parenchymal cells is unknown; however, in preliminary experiments we were able to show that PGD, is able to enhance the glucose output by parenchymal cells, suggesting that it may mediate cellular communication between the various liver cell types. Furthermore, it may be speculated that the PGD, produced (a vasodilator) performs an important function in the control of the sinusoidal liver blood flow. Since Kupffer cells, the main source of liver eicosanoids, produce, besides PGD,, significant amounts of the vasoconstrictive prostanoids thromboxane b and PGF,,, changes in the relative formation of these three compounds could regulate the sinusoidal liver blood flow. Another possible function of PGD, may be the inhibition of platelet aggregation in the liver: during phagocytosis, enzymes are released into the medium surrounding the Kupffer cells and one of the enzymes, phospholipase A,, induces the aggregation of blood platelets. It is possible that Kupffer cells release PGD, during phagocytosis in order to prevent formation of platelet aggregates, which could lead to obstruction of the sinusoidal liver blood flow. A change in sinusoidal blood flow may also have a profound effect on the parenchymal cell metabolism. Furthermore, the rapid conversion of PGD, by the intact liver to 9,,11,-PGF, (a potent vasoconstrictor) is also interesting with respect to the control of liver blood
1.52
flow. The identification of PGD, as the major product from liver sinusoidal cells will therefore stimulate further studies on the role of this prostaglandin in the communication between the various cell types inside the liver.
Acknowledgements
This research was supported by grant No. 523-066 from the Foundation for Medical Health Research (Medigon). We thank Dr. Vincent for critical reading of the manuscript Martha Wieriks for it’s preparation.
900and J.E. and
References 1 Bakhle, Y.S. (1983) Br. Med. Bulletin 39, 214-218. 2 Uehare, N., Ormstad, K. Oring, L. and Hammerstrom, S. (1983) B&him. Biophys. Acta 732, 69-74. 3 Wong, P.Y.-K., Lee, W.H., Quilley, C.P. and McGiff, J.C. (1981) Fed. Proc. 40,2001-2004. 4 Reingold, D.F., Kawasaki, A. and Neddleman, P. (1981) Biochim. Biophys. Acta 659, 179-188. 5 Pughese, G., Spokas, E.G., Marcinkiewicz, E. and Wong, P.Y.-K. (1985) J. Biol. Chem. 260, 14621-14625. 6 Liston, T.E. and Roberts, L.J., II (1985) Proc. Natl. Acad. Sci. USA 82, 6030-6034. 7 Chong, K.P., Burch, R.M., Black, M., Maloney, E., Jollow, D.J. and Hahtshka, P.V. (1983) Prostaglandins 26,397-408. 8 Birmelin, M. and Decker, K. (1984) Eur. J. B&hem. 142, 219-225. 9 Decker, K. and Birmehn, M. (1984) in Prostaglandins and Membrane Iron Transport (Braquet, P, ed.), pp. 113-118. 10 Ouwendijk, J., Zijlstra, F.J., Van den Broe.k, A.M.W.C., Brouwer, A., Wilson, J.H.P. and Vincent, J.E. (1986) Thesis, Erasmus University, Rotterdam. 11 Tremoli, E., jaffe, E.A., Goldman, K.T. and Weksler, B.B. (1985) Arteriosclerosis 5, 178-185.
12 Spector, A.A., Scanu, A.M., Kaduce, T.L., Figard, P.H., Fless, G.M. and Czervionke, R.L. (1985) J. Lipid Res. 26, 288-297. 13 Gorman, R.R., Oglesby, T.D., Bundy, G.L. and Hopkins, N.K. (1985) Circulation 72, 700-712. 14 Johnson, A.R., Revtyak, G.E., Ibe, B.O. and Campbell, W.B. (1985) Prog. Clin. Biol. Res. 199, 185-196. 15 S&or, K. (1985) Basic Res. Cardiol. 80, 502-514. 16 De Leeuw, A.M., Martindale, J.E. and Knook, D.L. (1982) in Sinusoidal Liver Cells (Knook, D.L. and Wisse, E., eds.), pp. 139-146, Elsevier, Amsterdam. 17 Nagelkerke, J.F., Barto, K.P. and Van Berkel, Th.J.C. (1983) J. Biol. Chem. 258, 12221-12227. 18 Blouin, A., Bolender, R.P. and Weibel, E.R. (1977) J. Cell. Biol. 72, 229-232. 19 Munthe-Kaas, AC., Berg, T. and Sjeljelid, R. (1976) Exp. Cell Res. 99, 146-154. 20 Knook, D.L. (1983) in The Handbook Series in Aging, Section D., Biological Sciences, CelI Biology (Christagolo, V.J., ed.), Vol. 2, CRC Press, Boca Raton. 21 Van Berkel, Th.J.C. (1982) in Metabolic Compartmentation (Sies, H., ed.), pp. 437-482, Academic Press, New York. 22 Zijlstra, J.F. and Vincent, J.E. (1984) J. Chromatogr. 311, 39-50. 23 Verhaagen, J., Walstra, P., Veldink, G.A. and Vhegenthart, J.F.G. (1984) Prostaglandin Leukot. Med. 13, 15-20. 24 Werner, W., Rey, H.G. and Wielingen, H. (1970) Zeitschr. Anal. Chem. 252, 224-228. 25 McGuire, J.C., Richard, K.A., Sun, F.F. and Tracey, D.E. (1985) Prostaglandins 30, 949-967. 26 Chiabrando, C., Naseda, A., Castagnoli, M.N., Salmona, M. and Farrelli, R. (1984) Biochim. Biophys. Acta 794, 292-297. 27 Wisse, E. (1970) J. Ultrastruct. Res. 31, 125. 28 Wisse, E. (1972) J. Ultrastruct. Res. 31, 528. 29 Hubbard, A.L., Wilson, G., Ashwell, G. and Stukenbrok, H. (1979) J. Cell Biol. 83, 47-64. 30 Auberger, P. and Le Cam, A. (1982) Biochim. Biophys. Acta 718. 92-108.
Biochimica et Biophysics Acta 959 (1988) 143-152 Elsevier
BBA 52114
Identification of prostaglandin D, as the major eicosanoid from liver endothelial and Kupffer cells
Johan Kuiper a, Freek J. Zijlstra b, Jan A.A.M. Kamps a and Theo J.C. van Berkel a a Division of Biopharmaceutics, and b Department
Center for Bio-Pharmaceutical Sciences, University of kiden, Leiden of Pharmacology, Erasmus University, Rotterdam (The Netherlands)
(Received 17 August 1987) (Revised manuscript received 7 December 1987)
Key words: Icosanoid release; Prostaglandin D,; Endothelial cell; Kupffer cell; Parenchymal cell; (Rat liver)
The capacity of freshly isolated endothelial, Kupffer and parenchymal rat liver cells to produce eicosanoids from [l-‘4C]arachidonic acid was investigated in order to determine the relative importance of these cells to total liver eicosanoid production. Based upon the total formation of [l-‘4C]arachidonate metabolites in the liver, it can be calculated that Kupffer and endothelial cells are responsible for 65 and 234, respectively, of the total amount of eicosanoids produced by the liver. Consequently, parenchymal liver cells, representing 92.5% of the total liver mass, contribute only 12% to the total liver production of eicosanoids. The main product of Kupffer cells was prostaglandin D, (PGD,), representing 55% of the total amount of eicosanoids produced. Liver endothelial cells produced about 44imes less eicosanoids (per mg cell protein) than Kupffer cells, and PGD, was also the main product of these cells (44%). The production of eicosanoids by parenchymal cells was lower by a factor of 180 (per mg cell protein) than that in Kupffer cells. Besides the ability to form eicosanoids from added “C-labeled arachidonic acid, Kupffer and endothelial liver cells were also able to produce significant amounts of PGD, (the main liver prostaglandin) from endogenous arachidonic acid, as determined by a radioimmunoassay. It is concluded that inside the liver, Kupffer cells together with endothelial cells are of major importance in the production of eicosanoids, while the parenchymal cells may be considered metabolic target cells for these products, as indicated by the finding that the major liver prostalgandin, PGD,, could stimulate the glucose output in isolated parenchymal cells.
Abbreviations: PG, prostaglandin; di-HETE, 5,12dihydroxy5,8,10,14-eicosatetraenoic acid; HHT, 12-hydroxy-5,8,10heptadecatetraenoic acid; 15-HETE, 15-hydroxy-5,8,11,13eicosatetraenoic acid; HPLC, high-pressure liquid chromatography. Correspondence: J. Kuiper, Division of Biopharmaceutics, Center for Bio-Pharmaceutical Sciences, University of Leiden, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The Netherlands. 0005-2760/88/$03.50
Introduction The liver is an important organ in the metabolism of prostaglandins [1,2]. For example, the liver is the main organ for the processing of PGI,, which is converted to 6-keto-PGE,,, a vasodilat& and potent inhibitor of platelet aggregation [3]. Also, PGDz can be converted by a microsomal enzyme from liver, 11-keto-reductase, to PGF,, [4]
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
144
and more recently, PGD, has been described to be transformed in the liver to 9,,lls-PGF,, a newly discovered prostanoid which is a very potent vasoconstrictor and an inhibitor of platelet aggregation [5,6]. Besides the processing of prostaglandins, the liver is able to synthesize several prostaglandins and leukotrienes. Hepatocytes, the main cell type in liver, can produce thromboxane Bz when stimulated with vasopressin [7]. Kupffer cells, which form the tissue macrophages of the liver, have been described to produce, in culture, PGE,, PGD,, PGF,,, PGI, and leukotrienes [8-lo]. In addition to parenchymal and Kupffer cells, the liver also contains endothelial cells, and although extrahepatic endothelial cells have been described to produce a variety of eicosanoids, such as PGI, [11,12], 15-HETE [13,14], PGEi and PGF,, [15], the capacity of the liver endothelial cells to produce eicosanoids is unknown, probably because adequate culture conditions for liver endothelial cells were unavailable until recently [16]. In our laboratory, we developed a cell isolation and purification procedure using collagenase, which allows a direct comparison of the ability of parenchymal, Kupffer and endothelial liver cells to perform receptor-dependent uptake of ligands [17]. In the present experiments, we have compared quantitatively and qualitatively the capacity of the various liver cell types to synthesize both cyclooxygenase and lipoxygenase products from arachidonic acid. Materials and Methods Materials Collagenase type I was obtained from Sigma (St. Louis, MO); Ca2+ ionophore A23187 was from Hoechst (Calbiochem-Behring, U.S.A.); ail other radiolabeled compounds, [l- l4 Clarachidonic acid, 5-D-[5,6,8,9,11,12,14,15(n)-3H]-HETE, 12-L[5,6,8,9,11,12,14,15(n)-3H]-HETE, 15-L-[5,6,8,9, 11,12,14,15(n)-3H]-HETE, [5,6,8,9,11,12,14,15(n)3H]leukotriene B4, 6-keto[5,8,9,11,12,14,15(n)-3H]prostaglandin Fi,, [5,6,8,9,11,12,14,15(n)-3H]thromboxane &, [5,6,8,11,12,14,15(n)-3H]prostaglandin E,, [5,6,8,9,11,12,14,15(n)-3H]prostaglandin Fza and [5,6,8,9,12,14,15(n)-3H]prostaglandin D, and a radioimmunoassay kit for specific PGD,
determination were from Amersham International (U.K.). Sep Pak C,,, silica cartridges and HPLC filters HA (0.45 PM), FH (0.5 PM) and Millex (0.45 pm) were obtained from Waters/ Millipore (The Netherlands). Prepacked HPLC columns, Nucleosil5C,, and Zorbax C,, were obtained from Chrompack Middelburg (The Netherlands). Liver cell isolation The three types of liver cell (i.e., parenchymal, endothelial and Kupffer cells) were isolated and purified from livers of male Wistar rats (3 months old, weighing about 200 g) by in situ perfusion of the rat liver with 0.05% collagenase, as described previously [ 171, with one modification, whereby hepatocytes were removed by centrifugation (2 min, 75 x g), and not by elutriation. Cell viability was checked during the incubations by measuring the ATP content in the three cell types. The ATP content appeared to be constant, indicating no loss of cell viability. Purity of the cell fractions was examined by peroxidase staining with diaminobenzine. The parenchymal and endothelial cell fractions were pure and contained no other type of liver cell. The Kupffer cell fraction contained 91 k 5% (S.E.) (n = 3) peroxidase-positive cells, the remainder being (large) endothelial cells. The calculation of the relative contribution of the various liver cells to total liver eicosanoid production was performed as follows. According to Blouin et al. [18], hepatocytes contribute 92.5% to the total liver cell volume, the endothelial 3.3%, and the Kupffer cells 2.5%. The remaining 1.7% is contributed by fat-storing cells and pit cells. Since the protein concentration (in mg per ml of cell volume) in the parenchymal, Kupffer and endothelial cells is identical [19,20], these percentages can be directly related to protein. The validity of this assumption was demonstrated earlier by enzyme distribution studies with peroxidase (a specific marker for Kupffer cells), lysosomal enzymes and pyruvate kinase [21]. Cell incubations Liver cells were incubated for 10 min in 10 ml Krebs-Henseleit buffer (cell concentrations: Kupffer and endothelial cells, 0.1-0.2 mg protein/ml and parenchymal cells, 0.5-0.8 mg pro-
145
tein/ml). Tubes were placed in a water-bath (37 o C) and the cell suspensions were continuously gassed through a thin pipette with 95% 0,/5% CO,. The addition of 2.5 or 5 PCi [l“C]arachidonic acid (59.6 mCi/mmol) was followed by the addition of glutathione (final concentration, 2 mM) and the calcium ionophore A23187 (10 PM). At the end of a 10 or 20 mm 3H-labeled leukotrienes and 3Hincubation, labeled prostaglandins were added, and the liver cells were centrifuged (10 min, 1400 x g, 4O C). The supernatants of each cell type were then applied to two Sep Pak C,, and silica cartridges (the cartridges were prewashed with 10 ml ethanol and 10 ml distilled water). The samples were eluted with 5 ml ethanol and the eluates were evaporated to dryness with a Speed Vat concentrator (Savant) at 4O’C. Thereafter, the dried samples were dissolved in 0.3 ml solvent A, centrifuged and purified by a Millex filter, and kept in an HPLC microvial (Weichmann, Switzerland). Volumes of 100 ~1 were injected onto the columns and chromatographed using a 1082B HPL chromatograph (Hewlett Packard, Brussels, Belgium). Fractions were collected with a Superrac fraction collector (LKB, Sweden) and counted in a 3255 Tricarb liquid scintillation counter (Hewlett Packard). The percentage of arachidonic acid incorporated in the lipid fraction was assessed by a Folch extraction. Lipids were separated by thin-layer chromatography.
radioactivity OLcdpm) So000
1
formed
moo 3owo
1 i 10
20
30
40 Time (mini
50
60
30
CO Time (mini
50
60
r_adioactivity ?H dpm) Star&h
0 10
Chromatographic system Reversed-phase HPLC of lipoxygenase products was carried out on a Nucleosil 5C,, column. The solvent system A was tetrahydrofuran/ methanol/O.l% (w/v) EDTA solution in water/ acetic acid (25/30/45/0.1), adjusted to pH 5.5 with ammonium hydroxide. Mobile phases were filtered by vacuum filtering through a Millipore filter and degas& with helium. The flow rate was 0.9 ml/mm and the absorption was measured at 280, 270 and 234 nm for the identification of the mono-HETE species. The column was equilibrated with the mobile phase (A) at an oven temperature of 37 o C. Fractions were collected for scintillation counting. After each run (80 min) the column was rinsed with methanol for at least -30 min, because of contamination with the calcium
Eicosanoids
20
Fig. 1. Chromatogram of tritiated standards and cyclooxygenase products (‘4C-labeled) formed by Kupffer cells. 100 pl of a concentrated sample (300 pl), containing the “C-labeled arachidonic acid metabolites formed by Kupffer cells (0.85 mg) as well as the 3H-labeled standards, was applied to a Zorbax C,, column to separate the prostaglandins. In each fraction sample (1 ml), 3H and 14C radioactivity was counted. TxB,, thromboxane b.
ionophore and [‘4C]arachidonic acid, which elute after 115 and 140 min, respectively, in system A. Reversed-phase HPLC of prostaglandins was per; formed on a Zorbax C,, column. The solvent system B comprised acetonitrile/ benzene/ water/ acetic acid (24/0.2/76/0.1). The flow rate of this eluent was 2 ml/mm. Fractions were collected for
146 TABLE I
Quantitative
FORMATION OF CYCLOOXYGENASE AND LIPOXYGENASE PRODUCTS BY ISOLATED PARENCHYMAL, KUPFFER AND ENDOTHELIAL LIVER CELLS
The settings for double-label scintillation counting were such that there was no spillover of radioactivity of ‘H into the 14C channel. dpm calculations were carried out using quenched standard sets. A plotting system was programmed in order to obtain data of total counts covering the peak areas. Amounts were calculated in dpm of each channel and were plotted as separate chromatograms.
The liver cells were incubated for 10 min with labeled arachidonic acid (2.5 PCi) and the products formed were determined as indicated in the Materials and Methods section. cell protein& The results are expressed as r4C dpm.10-3/mg S.E. (n = 3). TxBa, thromboxane &. Type of eicosanoid
Cell type endothelial
Kupffer
6-Keto-PGF,,
6.4* 3.4 41.4* 9.2 42.4* 9.0 32.4* 5.3 230.2f15.2
10.3f 2.7 0.4kO.2 198.7* 28.8 0.6kO.3 95.0f 48.9 2.2k1.3 46.0f 23.9 0.4kO.l 1095.4f104.6 1.6k0.6 199.8f 40.4 2.8f0.4 106.0* 17.8 0.7kO.2 198.0* 24.2 l.OkO.3 43.lf 22.1 1.0*0.3
TxB, PGF,, PGE, PGD, Di-HETE/HHT IS-HETE 12-HETE 5-HETE
55.4 f 24.8* 65.5 f 16.1+
8.8 5.7 6.5 6.7
evaluation
parenchymal
scintillation counting. The column was rinsed with acetonitrile for 30 min after each sample to elute the lipoxygenase products [22]. Fig. 1 shows a representative reversed-phase HPLC chromatogram of the cyclooxygenase products from Kupffer cells. The tritiated standards, which were used to identify the lipoxygenase and cyclooxygenase products from the liver cells, had a somewhat shorter retention time (Fig. 1) due to a slightly higher polarity. Identification of the various prostaglandins was also performed using specific antibodies, as described extensively before by Zijlstra et al. [22,23]. The 3H-labeled standards were also used to measure the recovery of the various eicosanoids throughout the extraction and purification procedure. The recoveries f S.E. (mean of nine experiments) of the various eicosanoids were as follows: 6-keto-PGF,, (89% + 2.8); thromboxane B2 (75% f 2.4); PGF,, (25% + 1.0); PGE, (50% + 2.0); PGD, (58% f 2.8); leukotriene B4 (77% f 6.3); 15-HETE (37% f 1.2); 1ZHETE (35% f 0.9) and 5-HETE (25% f 0.9). These recoveries were used to quantify the production of the individual eicosanoids by endothelial, Kupffer and parenchymal cells (Table I).
Glucose production by parenchymal
cells
Freshly isolated parenchymal cells were incubated in the presence or absence of PGD, (lop6 M) in glucose-free Krebs-Ringer bicarbonate buffer (pH 7.4) saturated with O,/CO, (95%/5%). After a 10 min incubation, the cell suspension was rapidly cooled and centrifuged (500 X g, 5 min) and subsequently glucose was determined in the supematant by the glucose oxidase-ABTS method ~41.
The incorporation of 14C-labeled arachidonic acid into cyclooxygenase and lipoxygenase products by parenchymal, Kupffer and endothelial cells (per mg cell protein) is shown in Table I. From these data, it is clear that the two non-parenchymal cell types, Kupffer and endothelial cells, showed a much higher capacity to produce eicosanoids than parenchymal cells. To exclude the possibility that a difference in substrate availability between the various liver cell types would influence these differences in the apparent eicosanoid production rate, we also measured the percentage of added arachidonic acid incorporated into triacylglycerols, phospholipids and free fatty acids in the three cell types (Table II). No significant differences were found between endothelial, Kupffer and parenchymal cells, indicating that the observed differences in eicosanoid production between the various cell types (Table I) arose due to a difference in the activity of the specific enzymes involved in this pathway. The quantitatively most important prostaglandin synthesized by Kupffer cells was PGD,, while relatively high amounts of thromboxane 4, diHETE/HHT and 1ZHETE were also produced
147 TABLE II DISTRIBUTION OF 14C FROM [t4C]ARACHIDONIC IN VARIOUS LIVER CELL TYPES
ACID AMONG EICOSANOIDS, LIPIDS AND FREE FATTY ACIDS
The distribution of i4C from arachidonic acid (2.5 aCi) after a 10 min incubation with endothelial, Kupffer and parenchymal cells (0.1 to 0.5 mg cell protein/ml), among formed eicosanoids, lipids, free fatty acids and arachidonic acid was measured. Cell type
Eicosanoids
Triacylglycerol
Phospholipid
Free fatty acid
Arachidonic acid
Endothelial Kupffer Parenchymal
18.6k2.2 33.1 f 5.0 1.1*0.3
3.5kl.O * 3.9*1.2 * 5.s*1.4 *
4.8kl.O * 5.2k1.4 * 6.6k1.3 *
0.6kO.l * 0.7 *0.1 * 0.9kO.2 *
74.0 f 2.6 55.1 f4.4 83.9 f4.0
* No significant difference observed between the various cell types.
TABLE III EFFECT OF [i4C]ARACHIDONIC ACID CONCENTRATION KUPFFER AND ENDOTHELIAL CELLS
ON EICOSANOID
PRODUCTION
BY PARENCHYMAL,
The liver cells were incubated for 10 min with 2.5 or 5 gCi [‘4C]arachidonic acid and the total production of eicosanoids as well as the relative formation of prostaglandin D, was measured, as described in the Materials and Methods section. Data represent the mean f S.E. of three experiments. Cell type
endothelial Amount of added arachidonic acid Total eicosanoid production (t4C dpm. lo- ‘/mg cell protein Relative formation of PGDr (W of total)
Kupffer
2.5
5
521.2 f 36.9 44.2*
5
2.5
592.7 f 49.3 *
2.8
parenchymal
40.4*
3.1 *
1992.3 f 151.6 55.0f
5.3
2.5
1893.5 f 136.4 * 52.4f
4.3 *
5
10.7 f 1.9
11.7k2.1
15.0f4.3
17.1 f3.1 *
l
* Not significantly different from values found with 2.5 pCi arachidonic acid.
TABLE IV THE INFLUENCE
OF THEINCUBATION
TIME ON THE EICOSANOID PRODUCTION
BY LIVER CELLS
The various liver cell types were incubated with 2.5 pCi [‘4C)arachidonate for 10 or 20 min. Total eicosanoid formation (sum of individual eicosanoids) and relative percentage of PGD, was measured as described in the Materials and Methods section. Data represent the mean f. S.E. of three experiments. Ceil type endothelial Time @in Total eicosanoid formation (14C dpm.10-3/mg cell protein) Relative formation of PGD, (W of total)
10
521.2 f 36.9 44.2*
Kupffer 20
2.8
739.0* 88.6 39.4* 4.1 *
* Not significantly different from the 10 min level.
parenchymal
10
20
1992.3 f 151.6 55.0f
5.3
2 368.8 f 210.5 51.1*
4.6 *
10
20
10.7 f 1.9
15.6k1.9
15.0* 5.6
17.3 * 3.4 +
148
70 Endothelial
cells
60 I
F ;
50 40
s k a
30 20 10
70 Kupffer
cells
60 -
Parenchymal
cells
60
10
Fig. 2. Patterns of eicosanoid production by endothelial, Kupffer and parenchymal cells. The relative contribution of the various cyclooxygenase and lipoxygenase products to total eicosanoid production by endothelial, Kupffer and parenchymal cells was calculated from the data in Table I. The data are presented as the mean (f S.E.) of three experiments. Txb , thromboxane &.
(Fig. 2). Endothelial cells also formed PGD, as the main product, although the level of PGD, formation (230. lo3 dpm/mg cell protein) was about 4-fold lower than in Kupffer cells (Table I). Other relatively important endothelial cell products were thromboxane I$,, di-HETE/HHT and 5-HETE (Fig. 2). Parenchymal cells showed a completely different pattern of eicosanoid production, with PGF,, and di-HETE/HHT as the main products. The two eicosanoids, di-HETE and HHT, could not be fully separated by reversedphase HPLC, but on the basis of the elution profile of unlabeled HHT and di-HETE, it is concluded that about 80% of this peak is formed by HHT for all three cell types. The amount of 14C incorporated into the major eicosanoids produced by parenchymal cells was relatively small (2000-3000 dpm/mg cell protein, Table I), as compared to the non-parenchymal cell types. When the production of eicosanoids in the various liver cells was measured with 5 pCi arachidonate instead of 2.5 PCi, the absolute amounts of eicosanoid formed were comparable, indicating that after the addition of 2.5 PCi arachidonate, the maximal eicosanoid production had already been reached in the various liver cell types (Table III). The relative eicosanoid production was found to be identical under both conditions and PGD, was still the major eicosanoid formed by the non-parenchymal liver cell types. Analysis of the eicosanoid production by Kupffer, endothelial and parenchymal cells after a 20 min incubation revealed that the relative patterns were unchanged, as is illustrated by the unchanged contribution of PGD, to total eicosanoid production. The absolute amount of [‘4C]arachidonic acid incorporated into the eicosanoids after a 20 min incubation was 1.2-1.5-fold higher. However, the relative importance of endothelial and Kupffer cells in the production of eicosanoids as compared to parenchymal cells was virtually unchanged (Table IV). When the eicosanoid production by Kupffer and endothelial cells was measured in the absence of the calcium ionophore, no lipoxygenase products could be detected, whereas the level of total prostanoid production appeared to be lower (40%) in the absence of the calcium ionophore. The relative amounts of prostanoids produced by
149 TABLE
V
PRODUCTION OF PROSTANOIDS KUPFFER CELLS
IN THE PRESENCE
AND ABSENCE
OF Ca*+ IONOPHORE
BY ENDOTHELIAL
AND
The prostanoid production of endothelial and Kupffer cells was measure during a 10 min incubation in the absence and presence of Ca*+ ionophore A23187 (10 pm) as indicated in the Materials and Methods section. The results are expressed as the mean f S.E. of three experiments (14C dpm.10W3/mg cell protein). TxBa, thromboxane Ba. Endothelial
6-Keto-GPF,, TxB, PGF,, PGE, PGD,
Kupffer
cells
- A23187
+A23187
- A23187
+ A23187
2.6kO.l 39.9*7.3 33.1 f 2.4 26.5 f 4.4 150.5*1.3
6.4f 4.3 47.4* 9.2 42.4* 9.0 32.4* 5.3 230.2 f 15.2
5.3* 0.6 91.7* 9.3 56.1 f 11.0 35.7f 3.8 673.0 f 79.1
10.3+ 2.7 198.7 f 28.8 95.0* 48.9 46.0+ 23.9 1095.4 f 104.6
cell types to total liver [18,19], the contribution of each liver cell type to the production of the different eicosanoids by total liver was calculated (Fig. 3). Kupffer cells were the main source of PGD,, thromboxane Bz, di-HETE/HHT and 12-HETE, which were also the main products of the total liver. Endothelial cells and Kupffer cells were equally active in the production of PGE,, each producing about 40% of the PGE, synthesized by
Kupffer and endothelial cells were comparable, however, both in the presence and in the absence of the Ca2+ ionophore (Table V). Interestingly, the level of prostanoid production by Kupffer cells was, both in the presence and the absence of the Ca2+ ionophore, about 4-times higher than prostanoid production by edothelial cells. From the data on the eicosanoid production (Table I) and the relative contribution of the various liver 100 80
H & 2 g
6KPCFla
cells
TxB2
1
60
40 20
EC
KC
EC
KC
PC
PC
EC
EC
KC
PC
EC
KC
PC
EC
KC
PC
KC
PC
EC
KC
PC
EC
KC
PC
EC
KC
PC
Fig. 3. Contribution of endothelial (EC), Kupffer (KC) and parenchymal (PC) cells to the production of the different eicosanoids by total liver. The contribution (mean& S.E., n = 3) of endothelial, Kupffer and parenchymal cells to the total liver production of the different eicosanoids was determined as described in the Materials and Methods section.
150 TABLE VI COMPARISON OF PGDs FORMATION BY ENDOTHELIAL, KUPFFER AND PARENCHYMAL CELLS FROM ADDED ARACHIDONIC ACID (14C-LABELED) OR ENDOGENOUS ARACHIDONIC ACID The PGD, formation from t4C-labeled arachidonate in the presence of Ca*+ ionophore (A23187, 10 FM) was calculated using the data in Table I. The PGD, formation from endogenous arachidonic acid was measured after a 10 min incubation of freshly isolated liver cells, using a radioimmunoassay. The results are expressed as the mean of three experiments (pg PGD, /mg cell protein per min) f SE. Cell type
PGD, formation + [ l4Qrachidonic + A23187
Endothelial Kupffer
0.0617 f 0.0041 0.2941 f 0.0281
Parenchymal
0.004
acid
+ A23187 0.020 f 0.002 0.094 *0.011 O.oool *o.oooo1
kO.0002
the liver. Parenchymal cells appeared to be the main source of 6-keto-PGF,, and SHETE (Fig. 3), but these two eicosanoids are only minor liver eicosanoid products. In order to quantify the amount of PGD, formed by the various cell types from endogenous arachidonic acid, we applied a recently developed radioimmunoassay for PGD,. It appears that the Kupffer cells produce 3.9-times the amount of PGD, (per mg cell protein) produced by the endothelial cells, and 940~times that produced by parenchymal cells. These data indicate that the relative contribution of the various cell types to PGD, formation from endogenous substrate reflects the maximal capacities of this pathway, as
TABLE VII EFFECT OF PGDr ON THE AMOUNT OF GLUCOSE PRODUCED BY ISOLATED PARENCHYMAL CELLS The glucose output of freshly isolated parenchymal liver cells was measured in the absence and presence of PGD, (10m6 M). Data are meanf S.E. of four experiments. * Significant difference from control (P < 0.002). Addition
Glucose output (nmol glucose/ 10 min per mg cell protein)
None PGD, (1O-6 M)
31.7*1.7 53.3*3.3
*
measured by adding excess [ “C]arachidonic acid (Table VI). Preliminary results show that PGD,, the main eicosanoid produced by Kupffer and endothelial cells, is able to exert a metabolic effect on parenchymal cells. When PGD, (10e6 M) was added to freshly isolated parenchymal cells, the glucose output by these cells was found to be significantly enhanced (Table VII). Discussion
The data presented in this article show that the three liver cell types used, parenchymal, endothelial and Kupffer cells, all have the capacity to produce eicosanoids from exogenous arachidonic acid. Parenchymal cells, however, had a very low capacity to produce these arachidonate metabolites and it can be calculated, using the relative contribution of the various liver cells to total liver [19,20], that these cells can only be responsible for 12% of the total liver eicosanoid production. Both types of non-parenchymal cell had a much higher capacity to produce eicosanoids than parenchymal cells: the capacity (per mg cellular protein) was higher by a factor of 48 and 183 in endothelial and Kupffer cells, respectively, than in parenchymal cells. The main product of both liver endothelial and Kupffer cells was PGD,, representing 44.2 and 55.48, respectively, of the eicosanoids formed by these cell types. This large PGD, production is surprising, especially in liver endothelial cells, since endothelial cells from other sites in the body were shown to produce PGI, as the main eicosanoid [11,12]. In liver endothelial cells, PGI, (measured as 6-keto-PGF,,) comprises only 2.5% of the eicosanoids produced. The fact that PGD2 was found as the main product in Kupffer cells is less surprising [lo], although the relative amount of PGD, is much higher (55%) for freshly isolated Kupffer cells than has been described for cultured Kupffer cells (30%) [lo]. The results obtained with these rat liver macrophages are comparable with those obtained with murine tissue-macrophage cell lines, which also convert labeled arachidonate preferentially to PGD, [25]. Human or rat peritoneal macrophages, however, convert 30% of the labeled arachidonate to 5-HETE and 40% to di-
151
HETE/HHT [lo]. The finding that PGD, is the most prominent prostaglandin in the liver is in full agreement with results obtained by measuring the capacity of a whole liver homogenate to form prostaglandins, which showed that PGD, was the main prostaglandin formed [26]. We applied a system with added [‘4C]arachidonic acid and added the Ca*+ ionophore in order to assess the maximal capacities of the relative cell types to form both cyclooxygenase and lipoxygenase products. In the absence of the Ca*+ ionophore, no lipoxygenase products were formed (Table V), while the amount of cyclooxygenase products is lowered to about 40%. In order to obtain results on the relative contribution of the various cell types to total liver eicosanoid production from endogenous arachidonic acid, we carried out an immunoassay for PGD,. Although the absolute amounts of PGD, formed were lower, it was found that the relative contribution of the various cell types to total liver PGD, formation was similar to that found under maximally stimulating conditions (Table VI). This indicates that the maximal capacity of the various cell types to form PGD, is related to their production from endogenous arachidonic acid. There is great similarity between the liver endothelial cells and the Kupffer cells with respect to their ability to form the various eicosanoids. The main differences are a much higher capacity in Kupffer cells for eicosanoid formation than in endothelial cells (3.8~times higher per mg cell protein) and a relatively high capacity of endothelial cells for the formation of PGE,. The resemblance between these cell types is certainly not due to a contamination of the endothelial cells with Kupffer cells, since the purity was checked microscopically after a Kupffer-cell-specific peroxidase staining (see Materials and Methods). Furthermore, earlier data from our laboratory show that the liver endothelial cells, prepared by procedures similar to those used in this study, perform specific functions in relation to receptor-ligand interactions [17]. In addition, it must be remembered that liver endothelial cells can certainly not be compared with other endothelial cells in the body, as they contain fenestrae [27], numerous lysosomes [28] and also multiple receptors which make then sometimes more comparable to macrophages than
to endothelial cells [29]. Although the protein contribution of Kupffer and endothelial cells to total liver protein is only 2.5% and 3.3%, respectively, the high specific activity of these cell types to form eicosanoids leads to a calculation which indicates that Kupffer cells may contribute 65% to total liver eicosanoid production. This calculation assumes that the production of eicosanoids by the freshly isolated cells reflects their in vivo capacity to produce these products. Because, during culture, parenchymal cells change their pattern of protein synthesis [30], liver endothelial cells change their morphology [16], and Kupffer cells lose their peroxidase activity [16], we tend to conclude that the production of eicosanoids by freshly isolated cells, as reported here, may represent a production pattern which is the closest reflection of the physiological situation. The physiological function of the eicosanoids produced by non-parenchymal cells is unknown; however, in preliminary experiments we were able to show that PGD, is able to enhance the glucose output by parenchymal cells, suggesting that it may mediate cellular communication between the various liver cell types. Furthermore, it may be speculated that the PGD, produced (a vasodilator) performs an important function in the control of the sinusoidal liver blood flow. Since Kupffer cells, the main source of liver eicosanoids, produce, besides PGD,, significant amounts of the vasoconstrictive prostanoids thromboxane b and PGF,,, changes in the relative formation of these three compounds could regulate the sinusoidal liver blood flow. Another possible function of PGD, may be the inhibition of platelet aggregation in the liver: during phagocytosis, enzymes are released into the medium surrounding the Kupffer cells and one of the enzymes, phospholipase A,, induces the aggregation of blood platelets. It is possible that Kupffer cells release PGD, during phagocytosis in order to prevent formation of platelet aggregates, which could lead to obstruction of the sinusoidal liver blood flow. A change in sinusoidal blood flow may also have a profound effect on the parenchymal cell metabolism. Furthermore, the rapid conversion of PGD, by the intact liver to 9,,11,-PGF, (a potent vasoconstrictor) is also interesting with respect to the control of liver blood
1.52
flow. The identification of PGD, as the major product from liver sinusoidal cells will therefore stimulate further studies on the role of this prostaglandin in the communication between the various cell types inside the liver.
Acknowledgements
This research was supported by grant No. 523-066 from the Foundation for Medical Health Research (Medigon). We thank Dr. Vincent for critical reading of the manuscript Martha Wieriks for it’s preparation.
900and J.E. and
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