Isolation and culture of Kupffer cells from human liver

Isolation and culture of Kupffer cells from human liver

JournalofHepatology, 1988; 6:36-49 Elsevier 36 HEP 00369 Isolation and culture of Kupffer cells from human liver Ultrastructure, endocytosis and pro...

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JournalofHepatology, 1988; 6:36-49 Elsevier

36 HEP 00369

Isolation and culture of Kupffer cells from human liver Ultrastructure, endocytosis and prostaglandin synthesis A . B r o u w e r I , R . J . B a r e l d s I , A . M . d e L e e u w 1, E . B l a u w I , A . P l a s 2, S . H . Y a p 3, A.M.W.C.

van den Broek 4 and D.L. Knook l

/ TNO Institute]'or Experimental Gerontology, Rtjswqk, 2Diviston of Hematology, and ~Dtvtsion of Gastrointestinal and Liver Diseases', Department of Medicine, Umversity Hospital of Nijmegen, Ni]megen, and 4Department of Pharmacology, Erasmus Umverstty, Rotterdam (The Netherlands) (Received 23 June 1987) (Accepted 14 September 1987)

Summary Kupffer cells and other sinusoidal cells were isolated after perfusion and incubation with pronase and collagenase of pieces of liver tissue obtained from organ donors. The resulting cell preparations contained endothelial cells, Kupffer cells and fat-storing cells as well as considerable numbers of leucocytes. Attempts to purify the different sinusoidal cell types by density centrifugation and centrifugal elutriation were successful only for Kupffer cells. Kupffer cells, in contrast to endothelial cells and fat-storing cells, could be kept in maintenance culture for at least 5 days. Cultured Kupffer cells were active in the/ endocytosis of foreign substances, such as colloidal carbon, latex beads, horseradish peroxidase and bacterial endotoxin. The cultured Kupffer cells synthesized and secreted considerable amounts of prostaglandins PGE 2, PGF2., 6-keto-PGFla and thromboxane B 2. The production of prostaglandins was influenced by the presence of Escherichia coli endotoxin.

Introduction The Kupffer, endothelial and fat-storing cells of the mammalian liver play an essential role in a great number of functions that are of vital importance to the organism as a whole. This is extensively documented in a number of symposium proceedings and review articles [1-4]. Most of what is known about the specific characteristics of sinusoidal liver cells has been derived from studies with experimental animals

such as rat or guinea pig. Only a few studies on the isolation or culture of sinusoidal liver cells of human origin have been reported [5-9]. These studies did not provide information on cell yield, and only one report included data on the viability and purity of isolated and cultured Kupffer cells [9]. In this paper, we report a reliable and reproducible method for the isolation and culture of human Kupffer cells. In addition, studies were conducted wi:h cultured Kupffer cells to characterize their ca-

Correspondence: A Brouwer. TNO Institute for Experimental Gerontology, P.O. Box 5815, 2280 HV Rijswqk, The Netherlands. 0168-8278/88/$03.50 © 1988Elsevier Sctence Publishers B.V. (Biomedical Division)

ISOLATION AND CULTURE OF HUMAN KUPFFER CELLS pacity for the uptake of a number of different foreign compounds and the synthesis of prostaglandins. The results show that Kupffer cells of the human liver are closely similar to those from rat liver in terms of ultrastructure and function.

Materials and Methods

Materials Horseradish peroxidase (HRPO) was obtained from Sigma Chemical Co. (Saint Louis, MO). Higgins indian ink obtained from Faber Castell was used as a colloidal carbon preparation. Bacterial lipopolysaccharide (endotoxin) from Escherichia coli (026B6; phenol extract) was from Sigma Chemical Co., and latex particles (0.65 /~m) were obtained from Polysciences Inc. (Warrington, PA). Isolation and purification of sinusoidal liver cells from human liver Sinusoidal liver cells were isolated from samples of post-mortem human livers obtained from organ donors who died after severe traumatic brain injury. After the informed consent of relatives was obtained, the liver was taken out during routine human donor nephrectomy, immediately after heparinization and kidney removal [10]. Liver tissue was kept immersed in sterile saline at 4 °C until use. Within 3-5 h after excision, cell isolation was commenced. For sinusoidal cell isolation, 30-80 g of liver tissue were used. The piece of tissue had a single sectioned surface area which was kept as small as possible; the other surfaces were surrounded by the liver capsule. The tissue was first washed by gentle massage in several changes of sterile saline to remove most of the blood. The tissue was then perfused with sterile Gey's balanced salt solution (GBSS) [11] (310 mosmol) through four individual cannulae which were connected with the main portal vessels that were visible on the sectioned surface. Cannulae were secured into the vessels by gluing with cyanoacrylate adhesive. The tissue was perfused with GBSS at 37 °C for 30 min to remove blood, followed by a linear 10 min perfusion at 37 °C with 250 ml GBSS containing 0.2%

37

pronase E (Merck, Darmstadt, F.R.G.) and a circular perfusion at 37 °C with GBSS containing 0.2% pronase and 0.2% collagenase type I (Sigma) for 45 min. The flow rate of the perfusion was 15-20 ml.min -1 per cannula. The tissue was then cut into smaller pieces and well-perfused areas (about half of the total tissue) were selected for further processing. These were then minced carefully and incubated for 45 min at 37 °C under magnetic stirring in 300 ml of GBSS containing 0.2% pronase and 0.1% collagenase. After filtration through nylon gauze, the cells present in the liver digest were pelleted by centrifugation (10 rain; 300 x g), resuspended in GBSS and then purified by centrifugation in a single-layer discontinuous 16.7% Nycodenz (Nyegaard and Co., Oslo, Norway) density gradient as described for rat liver cells [11]. The sinusoidal cells were collected from the top of the gradient and washed with GBSS. For one donor (donox E a, Table 1), sinusoidal liver cells were also isolated from a total liver cell preparation isolated from liver tissue perfused with collagenase only (as described in Ref. 10). The supernatant fraction obtained after centrifugation of the parenchymal cells at 50 x g for 3 rain was used. Purification of this sinusoidal cell preparation by Nycodenz density gradient centrifugation was done exactly as for pronase-collagenase isolated cells. Sinusoidal liver cells were further separated into various fractions using centrifugal elutriation [11,12]. Unless stated otherwise, successive fractions were collected at flow rates of 15, 19, 32, 45 and 100 ml per min at a rotor speed of 3250 rpm using a Sanderson separation chamber.

Culture of Kupffer cells and other sinusoidal liver cells Isolated cells were generally cultured in Dulbecco's modified Eagles medium (DMEM) (310 mosmoo with t_-glutamine (Gibco) containing 20 mM Hepes and 10 mM HCO.~ (pH 7.4). This medium was supplemented with penicillin (100 units.ml-l), streptomycin (100 ug.ml -I) and 20% (v/v) newborn calf serum (NBCS). The endocytic capacities of the cells in culture were studied by incubation with various ligands as specified in the Results section. For phase contrast microscopy, cells were cultured on

38

A. BROUWER et al.

glass using a Bachofer test chamber [13].

Assessment of purity and ultrastructural properties of cells Isolated and cultured cells were prepared for electron microscopy as described earlier [14]. The purity of all fractions was assessed electron-microscopically. Incubation for the demonstration of peroxidase activity was done as described before [15]. For endogenous peroxidase activity, unfixed cells or cells that were fixed in 1% glutaraidehyde for 1, 5 or 10 min were used. Cells incubated with H R P O were fixed in glutaraldehyde solution for at least 60 min to selectively inactivate endogenous peroxidase activity.

Determination of prostaglandins The prostaglandins PGF2a, PGE2 and 6-ketoPGF~a and thromboxane (Tx) B 2 w e r e determined in culture supernatants by radioimmunoassay as described elsewhere [16]. Culture supernatants were stored frozen at - 7 0 °C. They were applied to a couple of Sep-Pak CIs and silica cartridges prior to the assay.

Results

Isolation and identification of sinusoidal liver cells Between 6 and 20 x 108 nucleated cells were isolated from 30-80 g of tissue. The compositions of the

different sinusoidal cell preparations obtained during this study varied significantly between donors (Table 1). All three major sinusoidal cell types, as well as considerable numbers of white blood cells, were observed in these preparations. Both Kupffer and endothelial cells were consistently present, at percentages of 7 - 3 6 % and 5 - 2 3 % , respectively. Fat-storing cells were generally lower in number ( 2 - 8 % ) . Pit cells were rarely observed. The remainder of the cells ( 4 4 - 8 3 % ) were mostly white blood cells, such as monocytes, granulocytes and lymphocytes. The average yield in the sinusoidal cell preparations was about 5, 6 and 1.2 x 106 ceils per g liver tissue for endothelial, Kupffer and fat-storing ceils, respectively. The composition of the sinusoidal cell preparation obtained after collagenase perfusion was qualitatively similar, but the numbers of Kupffer and fat-storing cells were lower than after pronase-collagenase treatment (donor E a in Table 1). Fig. la gives an overview of the ultrastructural appearance of the cells present in the sinusoidal cell preparations. In endothelial cells (Fig. lb), the sponge-like structures representing the retracted cell processes containing the sieve plates were prominent features. Often, the endothelial cells were not fully rounded up and displayed a highly irregular outline. Fat-storing cells (Fig. lc), which could be identified "by their lipid droplets, also had a very irregular outline. The ultrastructure of isolated Kupffer cells (Fig. la) was very reminiscent of that of macrophages from other sources. The cells contained numerous,

TABLE 1 COMPOSITION OF SINUSOIDAL LIVER CELL PREPARATIONS OBTAINED FROM VARIOUS HUMAN DONORS Donor

Composition of cell preparation (% of total)

Age (year)

Sex

A

51

M

23

11

B

47

M

13

36

C D E E" F

20 46 15

M " M M

31

F

21 5 8 12 8

12 13 15 8 7

3 4 2 0.5 1

EC

KC

FC

PC

Blood cells

Other cells

8

1

48

10

4

-

44

3

4 -

62 75 70 73 83

2 3 5 2 1

a Supernatant fraction of collagenase perfusion after centrifugation of parenchymal cells (see Materials and Methods). EC = endothelial cells; KC = Kupffer cells; FC = fat-storing cells; PC = parenchymal cells.

ISOLATION A N D C U L T U R E O F H U M A N K U P F F E R CELLS

39

C

Fig. 1. Transmission electron micrographs of freshly isolated human sinusoidal liver cells. (a) Overview of a sinusoidal liver cell fraction showing the different cell types: K = Kupffer cell; E = endothelial cell; F = fat-storing cell; L = lymphocyte; G = granulocyte. (x 3600). (b,c) Higher magnification of (b) an endothelial cell with sieve plates (x6800) and (c) a fat-storing cell ( x 8800) displaying the most prominent cellular characteristics. S = sieve plates; F = fenestrations; N = nucleus; L = lipid droplets; G = Golgi complex; M = mitochondria.

40

A. B R O U W E R et al.

TABLE 2 YIELD AND COMPOSITION OF KUPFFER CELL FRACTIONS OBTAINED BY CENTRIFUGAL ELUTRIATION Donor

A B C D E F

Number of cells elutriated ( x 106)

Yield ( x 106)

497 610 640 1005 980 1270

17.1 38.5 30.3 27.8 19.2 41.8

Composition (%) KC

EC

FC

BC

Other

55 66 68 26 69 76

19 4 10 5 7 -

6 1 2

21 28 9 63 13 15

1 13 5 11 7

EC = endothelial cells; KC = Kupffer cells; FC = fat-storing cells; BC = blood cells.

often e l e c t r o n - d e n s e , lysosomal structures.

e n r i c h e d in K u p f f e r cells. T h e yield in t h e s e fractions

Purification of sinusoidal cells

5 - 1 2 x 108 n u c l e a t e d cells ( T a b l e 2). T h e purity was

v a r i e d b e t w e e n 17 and 42 x 106 cells, o b t a i n e d f r o m T h e application of centrifugal e l u t r i a t i o n to the dif-

b e t w e e n 55 and 7 6 % with o n e e x c e p t i o n ( d o n o r D ) .

ferent h u m a n sinusoidal cell p r e p a r a t i o n s has y i e l d e d

T h e ultrastructural a p p e a r a n c e o f the cells i n d i c a t e d

variable results. F o r K u p f f e r cells, the purification

a g o o d viability (Fig. 2). Small c l u m p s o f bile duct

was g e n e r a l l y successful. T h e fraction eluting be-

epithelial cells w e r e o f t e n e n c o u n t e r e d in the n o n eluted pellet fraction (not shown).

t w e e n 45 and 100 ml/min was n e a r l y always highly

T h e results with e n d o t h e l i a l and fat-storing cells have, up to n o w , not b e e n such that a r e p r o d u c i b l e m e t h o d for purification of these cells could be estab-

Fig. 2. Transmission electron micrograph of a purified Kupffer cell fraction obtained after centrifugal elutriation of sinusoidal liver cells: K = Kupffer cell; L = lymphocyte. Note the abundance of lysosomal vacuoles (V) that are occasionally filled with electron-dense material. (× 1700).

Fig. 3. Transmission electron microscopy of a human Kupffer cell after 64 h in culture. Note the extensive areas with lysosomal structures (L), often filled with lipofuscin-like electrondense material and the very pronounced cytoplasmic extensions (lamellipodia: arrows). N = nucleus; M = mitochondria. ( x 3200).

ISOLATION AND CULTURE OF HUMAN KUPFFER CELLS lished. Likewise, separation of sinusoidal cells on the basis of density in various types of discontinuous density gradients was unsuccessful in separating these cells from the majority of contaminating blood cells. Occasionally, a relatively pure preparation containing 50-75% of either endothelial or fat-storing cells was obtained with a low yield and this was used for studying the behaviour of these cells in culture.

Culture of sinusoidal liver cells Kupffer cell cultures were successfully established by using the same conditions that are used in our laboratory for rat Kupffer cells [13] except for the osmolality of the DMEM (310 mosmol). Human Kupffer cells could be kept in maintenance culture for at least

.

41

4-5 days without obvious changes in their uitrastructural appearance or viability. In culture, the cells attached to the substratum (both on plastic and on glass coverslips) but the degree of spreading was generally lower than displayed by rat Kupffer cells. The cell membrane was extremely ruffled with numerous lamellipodia (Fig. 3), also visible by light microscopy (Figs. 4 and 5). The purity of the Kupffer cells in culture was always higher than that of the original suspension as assessed by both electron (Fig. 3) and light microscopy (Figs. 4 and 5). Most blood cells and endothelial cells did not attach and were removed with the change in culture medium after 24 h. In view of the difficulties in consistently producing purified populations of endothelial and fat-storing

,j

2"

(

.i "f,i , #v

Fig. 4. Phase contrast micrograph of unfixed human Kupffer cells cultured on glass coverslips. Overview of cells after 55 h of culture ( x 1440).

m 9

Fig. 5. Phase contrast micrograph of unfixed human Kupffer cells cultured on glass coverslips. Selection of cells displaying cytoplasmicextensions (arrows). (x 1440).

42 cells, only limited attempts were made at their cultivation. With semi-purified endothelial and fat-storing cells obtained by pronase-collagenase treatment and centrifugal separation the survival in maintenance culture was poor. Survival of these populations was tested in different media containing either newborn calf, rat or heterologous human serum. Coating of the plastic culture dishes with human fibronectin did not improve the survival of cells.

Endogenous peroxidase activity in isolated Kupffer cells When freshly isolated or cultured Kupffer cells were tested for the presence of endogenous peroxidase activity, no reaction product was observed electron-microscopically in the rough endoplasmic reticulum and nuclear envelope but some was present in lysosomal structures (results not shown). This positively staining material was also found in cells that were not fixed prior to incubation. Light-microscopically, a smaller percentage of the isolated Kupffer cells did appear to contain the brown reaction prod-

A. BROUWER et al. uct, but this was probably due to nonspecific reactions such as those displayed by haemoglobin from endocytosed erythrocytes.

Endocytosis by human Kupffer cells in culture The functional properties of the cultured human Kupffer cells were tested by incubation of the cells with various endocytosable substances, which are detectable by electron microscopy. These included colloidal carbon, latex particles, bacterial endotoxin from E, coli and H R P O . Firstly, it was shown that the cells were capable of endocytosing two colloidal substances generally used for the assessment of macrophage phagocytic activity in vivo and in vitro, viz. colloidal carbon and 0.65 k~m latex particles. Fig. 6a reveals the presence of colloidal carbon inside cellular vacuoles after a 30 min incubation, The amount of colloidal carbon found intracellularly varied considerably between individual cells. The colloid was only occasionally observed at the cell membrane (Fig. 6b) and rarely inside primary endocytic structures, such as bristle-coated mi-

Fig. 6. Transmission electron microscopy of cultured human Kupffer cells after incubation with colloidal carbon. After 64 (a) or 55 h (b) of culture, the cells were incubated with colloidal carbon (india ink, diluted 1:1000in incubation medium) for 30 mm. Colloidal carbon (arrows) is observed at the cell membrane (b), attached to the wall of translucent vacuoles (b) and in clusters inside larger vacuoles (a). (a: x8500; b: × 17 000).

ISOLATION AND CULTURE OF HUMAN KUPFFER CELLS

43

cropinocytotic vesicles. Latex particles were ob-

Bacterial endotoxins are probably among the most

served attached to the cell m e m b r a n e and at different intracellular sites (Fig.7a-c). Latex was found in association with iamellipodia in various stages of engulfment (Fig. 7a,c).

important substances that are taken up by Kupffer ceils in vivo under both normal and pathophysiological conditions [17]. In vitro incubation of h u m a n Kupffer cells with high concentrations of E. coli

Fig. 7. Transmission electron microscopy of cultured human Kupffer cells after incubation with latex particles (0.65 pm). Cells were incubated for 5 (b) or 30 (a,c) min with a 1:5 dilution of the original latex particle suspension in culture medium after 55 (a) or 64 (b,c) h. Latex particles are observed at the cell membrane (arrows), often surrounded by pseudopodia-like cytoplasmic extensions (barred arrows) and inside vacuoles (arrow heads) in the cell body. (a: × 13 250; b: x8270; c: x8050).

44 endotoxin revealed that these cells, indeed, are able to bind and endocytose endotoxin (Fig. 8a,b). Endotoxin 'particles' were not found in each Kupffer cell visible in the ultrathin sections. This may be the result of incidental selection of an area devoid of endotoxin within an actually endocytosing cell, but could also represent the existence of selective subpopulations of Kupffer cells that do not express the capacity to take up endotoxin. The Kupffer cells were also incubated with different concentrations of HRPO, which is taken up by interaction with the mannose/N-acetylglucosamine receptor. The cells displayed the peroxidase activity of this ligand at different intracellular sites (Fig. 9a-d). Initial uptake, visualized after incubation with a high HRPO concentration (500/xg.ml -l) for 5 min, occurred primarily by bristle-coated micropinocytosis (Fig. 9c). Some larger vacuoles containing HRPO were also observed (Fig. 9a,c). At first, HRPO was only present close to the vacuolar membrane, leaving the centre of the vacuole devoid of electron-dense reaction product. After a 30 min incubation larger vacuoles displaying HRPO activity throughout their

A. BROUWER et al. entire contents were observed (Fig. 9b,d).

Effects of long-term incubation with endotoxin on Kupffer cell morphology and prostaglandin production Bacterial endotoxins or lipopolysaccharides (LPS) are known to exert profound effects on organisms upon their appearance in the circulation [17]. These effects are strongly species- and dose-dependent and most probably involve Kupffer cells as well as products released by these cells in response to contact with endotoxins [17]. Therefore, cultures of human Kupffer cells were incubated with various concentrations of endotoxin for up to 24 h and cell survival, ultrastructure and prostaglandin release were assessed. The results showed that human Kupffer ceils were not appreciably affected in their ultrastructural appearance by concentrations of endotoxin up to 0.5 mg.ml -l. However, after 21-24 h of incubation with 2 mg.ml -t of endotoxin, all cells had detached from the plastic substratum. At concentrations of 0.1-0.5 mg.m1-1, light microscopy showed increased rounding up of the cells.

Fig. 8. Transmission electron microscopyof cultured human Kupffer cells after incubation with bacterial endotoxin. After 64 h of culture, the cells were incubated with 3 mg E. coli 026 B6 LPS per ml culture medium for 30 min. The typical two-layeredstructures representing the endotoxin particles (arrows) are found inside larger and smaller vacuoles. (a: x38 700; b: ×40 000).

ISOLATION AND CULTURE OF HUMAN KUPFFER CELLS

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TABLE 3 PROSTAGLANDINS PRESENT IN CULTURE MEDIA OF HUMAN KUPFFER CELLS CULTURED IN THE PRESENCE OR ABSENCE OF BACTERIAL LPS Purified Kupffer cells (of donors D, E and F) were cultured in normal culture medium for 22.5 (D), 55 (E) or 24 (F) h in 24-well tissue culture plates. Then, culture medium was removed and fresh medium with or without 026 B6 E. coli LPS was added. After incubation, the media were harvested and frozen at -70 °C until analysis. Each well contained 1 x 106 cells in 1 ml medium. LPSadded O~g.ml-t)

Incubation time(h)

(n)

0a 2 10 100 500 2000

24 24 24 24 24 24

2 2 2 2 2 2

0d 10 100 500 0" 10 100 500 0" 10 100 500 0" I0 100 500

2.5 2.5 2.5 2.5 21 21 21 21 2.5 2.5 2.5 2.5 24 24 24 24

Donor

Prostaglandin content (pg/well) TxB 2

PGF2a

PGE 2

D D D D D D

1340 1215 1190 1075 925 45

319 369 313 550 275 ~13

396 464 613 825 838 289

250 231 356 356 306 80

3 2 2 2

E E E E

79 75 60 30

1020 1240 495 200

597 679 535 186

101 141 85 ~25

3 2 3 2

E E E E

94 93 123 79

447 1120 1420 430

1073 1300 1422 183

247 275 226 41

3 3 3 3

F F F F

791 800 766 800

~160 ~160 ~160 ~125

18 33 30 26

40 49 45 47

3 3 3 3

F F F F

1016 1108 1250 1433

196 231 151 166

48 135 253 256

70 116 113 126

/

6-keto-PGFt, ,

When control culture media were analysed for the presence of endogenous LPS by the chromogenic Limulus amoebocyte assay [24], the equivalent of 40 ng LPS.ml -I was detected.

T a b l e 3 shows the a m o u n t s of v a r i o u s p r o s t a g l a n dins that could be d e t e c t e d

in culture m e d i a of

K u p f f e r cells after i n c u b a t i o n with v a r i o u s c o n c e n -

LPS ( 1 0 - 1 0 0 p g . m l - t ) , p r o d u c t i o n of p r o s t a g l a n d i n s o t h e r than T x B 2 was m o d e r a t e l y i n c r e a s e d (up to a b o u t 5-fold) as c o m p a r e d to controls.

trations of LPS. T h e levels of p r o s t a g l a n d i n s in m e d i a

T h e s e results s h o w that t h e r e is a d o s e - d e p e n d e n t

f r o m u n t r e a t e d K u p f f e r ceils w e r e d o n o r - d e p e n d e n t ,

influence of LPS on the p r o d u c t i o n of p r o s t a g l a n d i n s

especially for TxB2. This m a y reflect the d i f f e r e n c e s

by K u p f f e r cells in vitro which varies for the d i f f e r e n t

in the cellular c o m p o s i t i o n s of the cell p r e p a r a t i o n s

types of p r o s t a g l a n d i n s and m a y also d e p e n d on the donors.

(see T a b l e 2) but also actual d i f f e r e n c e s b e t w e e n K u p f f e r cells of different donors. T h e most p r o f o u n d effect of LPS was n o t e d at the highest c o n c e n t r a t i o n of 2 mg.m1-1. A t this c o n c e n t r a t i o n

there was a

Discussion

m a r k e d r e d u c t i o n in p r o s t a g l a n d i n c o n t e n t of the med i u m after 24 h. Usually at l o w e r c o n c e n t r a t i o n s of

This r e p o r t deals with the isolation, culture and

ISOLATION AND CULTURE OF HUMAN KUPFFER CELLS characterization of sinusoidal cells from human liver. The results show that, in spite of extensive perfusion, the sinusoidal cell preparations obtained were always much more contaminated by blood cells than those obtained from the rat liver [11,12]. The overall yield of sinusoidal cells per g liver was satisfactory. For Kupffer and endothelial cells, the yield was about 25-50% of that maximally obtained with rat liver [11,12]. Human endothelial and fat-storing cells appear to be more heterogeneous in size and physical properties than those isolated from rat liver. As a result, we have not yet succeeded in establishing a reproducible method for the purification of these two cell types. In contrast, Kupffer cells were generally obtained with a reasonably high purity and a yield sufficient for cell cultivation and biochemical analyses. These results compare favourably with the few earlier studies relating to the isolation of human Kupffer cells [5-9,18,19]. It has been suggested that the yield and purity of sinusoidal cells could be increased by perfusion of the whole liver in situ or directly after excision [9]. However, recent experiments that we performed showed no improvement in the degree of contamination by in situ perfusion (unpublished results). In situ perfusion will mostly be difficult to achieve, while the method presented here appears to be more generally applicable, since it can be easily carried out with smaller pieces of liver, that require no special treatment and can be much more easily obtained. When human liver sinusoidal cells or separated fractions thereof were kept in maintenance culture, the adherence and survival of both endothelial and fat-storing cells were very poor. This finding is in apparent contrast with those of the few studies on human hepatic endothelial cell culture reported by others [5-8,18]. However, these studies were concerned only with individual cells kept under different culture conditions, and information on tests of viability or survival of whole populations of cells was not given. Possibly, endothelial cells of human liver show improved survival in co-culture with other sinusoidal liver cells [18] or in the presence of autologous donor serum [8]. Alternatively, the time between excision of the liver and start of the isolation procedure could

47

be a determinant factor. Kupffer cell fractions obtained after centrifugal elutriation could be kept in culture for at least 4 days, using essentially the same conditions as for rat liver cells. Using this culture system, we have been able to study some of the major functional characteristics of human Kupffer cells. In contrast to rat Kupffer cells, the cells isolated from human liver appeared to lack the characteristic localization of endogenous peroxidase activity in rough endoplasmic reticulum and nuclear envelope. Since the functional significance of the presence of peroxidase in Kupffer cells is still obscure, it is not possible to infer the significance of its absence in the human cells [4]. The endocytic abilities of these cells were closely similar to those of rat Kupffer ceils. Foreign particles, such as colloidal carbon and latex beads, H R P O and bacterial endotoxin particles were all shown to be internalized by the cultured cells, by mechanisms that could not be distinguished ultrastructurally from those in rat liver macrophages [15]. These results clearly show that human Kupffer cells possess the same kinds of membrane receptors as do rat Kupffer cells, viz., the foreign body receptor, the mannose/N-acetylglucosamine glycoprotein receptor and the (poorly defined) recognition site(s) for endotoxins (cf. Ref. 20). Our results confirm and extend those of Kirn et al. [9], who reported on the endocytic capacities of human Kupffer cells for latex particles, opsonized sheep erythrocytes and FV3 virus. The production and excretion into the medium of various types of prostaglandins was also investigated. All four prostaglandins tested, viz., PGE 2, PGF2~,,6keto-PGFla, and TxB 2 were consistently detected. TxB, and 6-keto-PGFl(, are the stable metabolic products of TxA 2 and prostacyclin (PGI), respectively [20-22]. The prostanoid levels, especially those of TxB 2, varied greatly between the donors. This might be partly related to differences in the cellular composition of the Kupffer cell fractions. The synthesis of the different prostaglandins was sensitive to incubation of the cells with bacterial LPS, which generally resulted in a moderate increase at low concentrations of LPS for all prostaglandins, except TxB 2. The influence of the concentration of LPS varied considerably

48

A. BROUWER et al.

with the type of p r o s t a n o i d and with the d o n o r . This

total p r o s t a n o i d p r o d u c t i o n a p p e a r s to be h i g h e r in

was especially true for the inhibition of p r o s t a n o i d

h u m a n K u p f f e r cells than in t h o s e f r o m the rat.

synthesis at h i g h e r c o n c e n t r a t i o n s of L P S ( 5 0 0 - 1 0 0 0

In s u m m a r y , o u r results and those of o t h e r s [9] in-

/~g.ml-1). T h e range of c o n c e n t r a t i o n s of L P S that

dicate that h u m a n K u p f f e r cells are q u a l i t a t i v e l y v e r y

were effective in stimulating the p r o s t a n o i d r e s p o n s e

similar to rat K u p f f e r cells, in t e r m s of ultrastructural

of the cells was 10-100 ~ g . m l - l , which is c o m p a r a b l e

m o r p h o l o g y , b e h a v i o u r in c u l t u r e , m e t a b o l i s m and

to that f o u n d for rat K u p f f e r cells [20-26]. T h e types

e n d o c y t i c function. T h e s e findings imply that rat

and a m o u n t s of p r o s t a g l a n d i n s p r o d u c e d by h u m a n

K u p f f e r cells m a y be c o n s i d e r e d as a v a l u a b l e and

K u p f f e r cells in r e s p o n s e to LPS w e r e v a r i a b l e , and

relatively easily accessible e x p e r i m e n t a l m o d e l that

by rat

allows cautious e x t r a p o l a t i o n of findings o b t a i n e d

K u p f f e r cells [22-26]. PGE2, which a p p e a r s to be a

with these cells to the situation in h u m a n s . O u r study

somewhat

different f r o m

those

produced

K u p f f e r cells

also shows that it is also possible to isolate and culti-

[24-26] was p r o d u c e d in high a m o u n t s only by the

vate K u p f f e r cells f r o m h u m a n liver and that these

cells of two out of three h u m a n donors. T h e relative

cells can be used to verify the validity of certain ex-

contribution of T x B 2, PGF2, , and 6 - k e t o - P G F i , to the

trapolations f r o m rats to h u m a n s .

References

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