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Cell population kinetics of Kupffer cells during the onset of fibrosis in rat liver by chronic carbon tetrachloride administration
50
Journal of Hepatology, 1988; 6:50-56
Elsevier HEP 00368
Cell population kinetics of Kupffer cells during the onset of fibrosis in rat liver by chronic carbon tetrachloride administration
Albert Geerts, Paul Schellinck, Luc Bouwens and Eddie Wisse Laborato~3'Jbr Cell Biology and Htstology. Free Umversttv Brussels, V. U B., Brussels (Belgium)
(Received 28 April 1987) (Accepted 14 September 1987)
Summary The cell population kinetics of Kupffer cells (KCs) were investigated during the induction of fibrosis in rat liver bv carbon tetrachloride. KCs, characterized microscopically by their specific peroxidase pattern, increased in number during the first 24 h after CC14 injection. After repeated injections of the toxin the number of KCs increased logarithmically. After 9 weeks of CCI~ treatment, the KC population reached a new steady state with a 2.6-times-higher cell number than in control animals. Local KC proliferation was demonstrated by the metaphase arrest technique using vinblastine. It was calculated that at 24 h and at 72 h after one injection of CC14, local proliferation did not account for the entire growth of the population. This means that, in addition to local proliferation of KCs, an influx of mononuclear phagocytes into the fiver sinusoids occurred.
Introduction The liver parenchyma is composed of several cell types including parenchymal cells, sinusoidal endothelial cells, Kupffer cells (KCs), fat-storing cells and pit cells [1]. These cells are in permanent contact with extracellular matrix (ECM) components such as collagens, non-collagenous glycoproteins, glycosaminoglycans [2] and elastin [3]. The different cell types and their surrounding ECM are in dynamic equilibrium. In healthy liver, the relative proportions
of liver cells and matrix remain constant. The mechanisms which control this complex equilibrium are unknown. As a result of chronic liver damage, this balance is often lost and hepatic fibrosis develops [4]. Liver fibrosis is characterized by a 5-10-fold increase in the amount of collagen per g of liver [5,6]. Concomitantly, the cellular mass of the liver decreases and its composition changes. Fat-storing cells increase in number [7-9] and differentiate into myofibroblast-like cells [10,11]. The number of macrophages also increases [12,13]. However, quantitative
This work was financially supported by NFWO grants No. 30.0040.80 and No. 30.0028.86. Correspondence: A. Geerts, Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel (V.U.B.), Laarbeeklaan 103, 1090 Brussels-Jene, Belgium. Tel: (32-2)478 48 90, ext. 1406/1407. 0168-8278/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
KUPFFER CELLS DURING CCI4-INDUCED FIBROSIS
51
data are lacking. Furthermore, it is not clear whether such an increase of liver macrophages is the result of KC proliferation or is due to infiltrating mononuclear phagocytes. In the present study, we have examined the population kinetics of KCs during the onset of liver fibrosis, using chronic carbon tetrachloride (CC14) intoxication of the male Wistar rat as a model system [14].
in the cytoplasm. The number of KCs per microscopic field (NKC/f:f = 0.077 mm 2) was counted according to Bouwens et al. [16], using a Zeiss Photomicroscope III. Only KCs in which the nucleus was visible were counted. Ten microscopic fields per section were taken at random; at least three sections per rat were examined. The livers of six animals were studied for each time point. The statistical significance between two groups of rats was tested by applying Student's t-test [18].
Materials and Methods
Metaphase index (/met) of KCs
Experimental animals Sixty-six male Wistar rats, weighing 225-250 g, were given subcutaneous injections of 0.1 ml of CCi 4 per 100 g body weight twice a week. The CCI 4 was dissolved in an equal volume of paraffin oil [6]. Six animals were sacrificed at each of the following time points: 1 day, 3 days, 1, 2, 3, 4, 5, 7, 9 and 13 weeks. Six animals (controls) received paraffin oil alone.
Fixation Low-pressure (12 cm HEO ) perfusion fixation of the liver through the portal vein was performed according to De Zanger and Wisse [15]. The fixation was preceded by a brief rinse of the liver with isotonic buffer. The fixative contained 1.5% glutaraldehyde (Merck), 0.1 M cacodylate buffer pH 7.4, 0.01% CaCI2.2H20 and 2.5% sucrose, and was applied for 3 min.
The metaphase index or fraction of KCs arrested in metaphase was determined by injecting vinblastine sulphate (Velbe-Lilly; 2 mg/kg body weight) 6 h before perfusion of the liver. The metaphase-arrested cells were easily recognized by their distinct chromosomes and the diffuse brown staining in the cytoplasm. Chromosomes were visualized by briefly counterstaining the Epon sections with 1% toluidine blue dissolved in 1% borax [16]. From the metaphase indices we have calculated the birth rate (kB) of the KCs, i.e., the number of cells generated per unit of time and per cell. k B has been calculated for two extreme cell population growth models [19]. First, a cell population in steady state was considered. For such a population, k B can be easily found as follows:
kB.s -
met
(1)
tA
Peroxidase cytochemistry and KC counts KCs were characterized by a modification [16] of the original staining method for endogenous peroxidase [17]. 50-70tLm Vibratome sections of glutaraldehyde-perfused liver tissue were incubated for 1 h at 22 °C in a peroxidase medium containing 0.1% diaminobenzidine.4HCl (Fluka), 0.02% H202 and 0.14 M cacodylate buffer. The final pH of this medium was 6.9. Subsequently, the sections were post-fixed for 1 h in 1% Millonig's OsO4, and embedded in Epon 812. KCs were recognized by light microscopy in semithin Epon sections by the presence of a brown rim around the nucleus and a diffuse brown staining
(/met: metaphase index, tA: arrest time (6 h)) Second, an exponentially growing cell population from which no cells leave the cell compartment was assumed. Then k B equals:
kB.c = 2.303 log(l + Imct) _ In(1 +/met) tA
(2)
tA
Expansion of equation 2 in an infinite series [20] results in the following equation:
52
A GEERTS et al.
1 imct 3 /mct kB,c = ~ ( / m e t - - + tA 2 3 IfflCl - -
NKC/f
a /tact --'") 4
t
20-
t
I (if/me t ~< 0.01)
tA Thus if lmct ~ 0.01, then kB.c = Imct/tA for an exponentially growing population as well.
Cell population kinetics The following equation was used to describe an exponentially expanding population [18]: 10 ¸
N, = N,_te kB.~(q-q-i)
(3)
N, and N,-i represent the mean number of cells per field at time t, and t,_t after injecting CC14. kB., is the growth rate constant at time t,.
Results
i
0
-1
5
10
time
( weeks
)
Fig. I. Average number of Kupffer cells per microscopical field (NKC/f; f = 0.077 mm 2) during the reduction of fibrosis by CC14 Kupffer cells prohferate from the very beginning of the CCI~ administration. After 9 weeks of CCI a treatment a new steady state is reached.
b~duction of fibrosis In keeping with the findings of previous studies [14], a single or a few injections of a sublethal dose of CCI4 produced steatosis and parenchymal cell necrosis in the centrilobular areas of the liver lobules in all animals. This was followed by a progressive increase in the amount of connective tissue in this zone; there was individual variation in the degree of fibrosis between animals which had received an equal number of injections. Further CC14 intoxication induced the formation of fibrotic septa. From 9 weeks onwards, all animals showed liver fibrosis.
KC population kinetics KCs increased in number from the first day of the CCla administration (+17.4%, P < 0.01; Fig. 1). Repeated injections of CCI4 provoked further growth of the KC population (Fig. 1); after 2 weeks the population had doubled. Microscopical examination of necrotic pericentral areas showed KCs infiltrating these regions prior to the deposition of excess ECM. Prolonged administration of CC14 led to a steadily decreasing growth of the population. After 9 weeks the KC population reached a new steady state in which the average number of cells per microscopic field was
2.6-times higher than in control animals. By applying the metaphase arrest technique, KCs were shown to divide at all stages of chronic CC14 intoxication (Fig. 2). We have found KCs arrested in metaphase in all zones of the liver lobule. The meta/phase index of the KC population remained fairly Constant at a level of about 1% throughout the experiment, except for animals treated for t week (2 injections). In such animals the metaphase index was about 2% (Table 1). The cell birth rate (kB), calculated from the observed metaphase indices, seemed to be almost completely independent of the assumed population growth model (Table 1). At 24 h and 72 h after one injection of C C I 4, the maximal increase of the number of KCs due to autoproliferation of the population was calculated by substituting the obtained values for k B in equation 3 (see Materials and Methods). In Table 2, the results of these calculations are summarized. By comparing the maximal number of KCs that could have been obtained by autoproliferation of the population with the number of cells that was observed microscopically, it is concluded that local proliferation alone did not account for the total growth of the population.
KUPFFER CELLS DURING CCI4-INDUCEDFIBROSIS
53
Fig. 2. Kupffercell arrested in metaphase. Distinct chromosomes(C) are visible. The rough endoplasmicreticulum showsendogenous peroxidase activity (arrow). Lead citrate counterstaining (x 10 800). P: parenchymalcell.
Discussion
The present experiments were undertaken to study the population kinetics of KCs during the onset of liver fibrosis by chronic administration of CC14 in rats. KCs respond to CC14 within 24 h by expanding their population. This finding is in accordance with previous observations in mice, which were acutely intoxicated with CCI4 [12]. After 9 weeks of treatment, the KC population reached a new steady state at a
level 2.6-times higher than the control level. Earlier studies on isolated sinusoidal liver cells from CCl4-intoxicated rats [13,21,22] indicated an increase in the number of KCs following CC14 administration. However, it was not clear from these studies whether the yields of purified KCs from normal and CCl4-treated rats were equal. Furthermore, it was not established in these studies whether the increased population of macrophages was exclusively composed of cells showing the typical histochemical characteristics of KCs.
54
A GEERTS et al.
TABLE 1 METAPHASE INDICES (`/met) OF KCs DURING THE INDUCTION OF FIBROSIS BY CCI~ From `/m~tthe birth rate of KCs. i.e., the number of new cells generated per ceil and per day. has been calculated assuming both a steady-state KC population (k a ,) and an exponentially growing populauon (kB.¢). Treatment time 0 1 day
3 days l week 2 weeks 3 weeks 4 weeks 5 weeks 7 weeks 9 weeks 11 weeks 13 weeks
In( 1 + `/me0 (day-I x 10 -4)
'/met (x 10-41
kB, = Imct/t A (day-ix 10-4)
k u ~ = t,-x I
0 121 + 23"
0 485
0 480
90 + 10 185 + 31 70 4- 14 90 4- 6 92 + 19 121 4- 23 104 +_ 17 71 + 5 126 + 14 50 + 6
360 739 281 360 367 485 415 283 504 199
358 734 278 358 365 480 413 283 504 199
" Standard error.
In earlier papers [2,22], it was suggested that K C s
that liver m a c r o p h a g e s are n o n - d i v i d i n g cells, de-
may m o d u l a t e the p r o l i f e r a t i o n of o t h e r liver cells
rived f r o m m o n o c y t e s [24], the p r e s e n t findings indi-
and thetr capacity to synthesize c o n n e c t i v e tissue
cate that, f o l l o w i n g c h r o n i c CCI 4 i n t o x i c a t i o n , t h e r e
c o m p o n e n t s . In a recent study, in which isolated K C s
is local p r o l i f e r a t i o n of KCs. Such a u t o p r o l i f e r a t i o n
were c o - c u l t u r e d with fat-storing cells, it was d e m o n -
of KCs has also b e e n f o u n d after s t i m u l a t i o n of the
strated that KCs isolated f r o m livers of CC14-treated
reticulo-endothelial
rats stimulate fat-storing cells to divide [23]. O u r ob-
[16,25], R E S b l o c k a d e [25] and partial h e p a t e c t o m y
servation that the K C p o p u l a t i o n e x p a n d s signifi-
[16,25].
system
(RES)
with
zymosan
cantly during the i n d u c t i o n of fibrosis s u p p o r t s the
A t 24 h and 72 h after o n e i n j e c t i o n of CC14, we cal-
hypothesis that KCs m a y play a crucial role during the onset of fibrosis.
/culated f r o m the cell birth c o n s t a n t (kB) the m a x i m a l
By injecting vinblastine 6 h b e f o r e fixation of the
KCs by assuming (i) an e x p o n e n t i a l cell p r o l i f e r a t i o n
liver, we have b e e n able to d e m o n s t r a t e a r r e s t e d mi-
m o d e l and (ii) that no KCs left the liver. In short-
toses of KCs t h r o u g h o u t the p e r i o d of CCI~ treat-
t e r m e x p e r i m e n t s , the latter a s s u m p t i o n is r e a s o n -
ment. Thus, in contrast to the widely held o p i n i o n
able [24,26,27]. U s i n g this analysis, we h a v e d e m o n -
p o p u l a t i o n g r o w t h due to a u t o p r o l i f e r a t i o n of the
TABLE 2 OVERALL GROWTH VS. GROWTH DUE TO MITOSIS OF THE KC POPULATION AT 24 h AND 72 h AFTER ONE INJECTION OF CCI 4 The maximal increase of the number of KCs per microscopical field at 24 h and 72 h after one injection of CCI 4 which could be accounted for by mitosis of the resident KC populauon was calculated by applying formula 3. Since the observed numbers of KCs (column 4) exceeds these calculated numbers (column 3), it is concluded that extrahepatic recrmtment of KCs must take place. Treatment ume (h) 0
24 72
Observed k B (day -I × 10 -4)
Calculated number of cells tf only KC mitosis occurred
Observed number of KCs
--
--
--
482 359
7.78 9.36
8.71 9.54
KUPFFER CELLS DURING CCI4-INDUCED FIBROSIS
55
strated that 1 day after CCI 4 i n j e c t i o n , a r o u n d 3 0 % of
of the e x p e r i m e n t , a simple q u a n t i t a t i v e r e p l a c e m e n t
the growth of the p o p u l a t i o n can be e x p l a i n e d by lo-
of resident liver m a c r o p h a g e s by m o n o c y t e s or o t h e r
cal p r o l i f e r a t i o n . T h u s , at least 7 0 % of the n e w cells would a p p e a r to h a v e b e e n r e c r u i t e d f r o m e x t r a h e p -
e x t r a h e p a t i c m a c r o p h a g e s is not likely to take place
atic
origin,
although
they
have
cytochemical
during the induction of fibrosis by CC14 in the rat liver.
characteristics in c o m m o n with KCs. T h r e e days after one CCIz i n j e c t i o n , local p r o l i f e r a t i o n was s h o w n to account for 8 0 % of the p o p u l a t i o n growth. In ex-
Acknowledgements
p e r i m e n t s in which C f l 4 was a d m i n i s t e r e d for long periods of time, it c a n n o t be a s s u m e d that all KCs
T h e authors wish to t h a n k M r Paul Lens and M r
stay in the liver. A m e t h o d to e v a l u a t e the cell loss
Ronald
from the K C p o p u l a t i o n is at p r e s e n t not available,
t h r o u g h o u t the e x p e r i m e n t s . W e t h a n k Dr. A . D .
De
Zanger
for their helpful suggestions
and c o n s e q u e n t l y q u a n t i t a t i v e e s t i m a t e s of the im-
Burt ( P a t h o l o g y D e p a r t m e n t ,
p o r t a n c e of K C mitoses during l o n g - t e r m CCI4 ad-
gow, U . K . ) for c o r r e c t i n g the m a n u s c r i p t , Mrs Mar-
ministration are unreliable. N e v e r t h e l e s s , since we
leen D e P a u w for typing it, and Mrs Chris D e r o m for the p h o t o g r a p h i c work.
have o b s e r v e d K C mitoses during the entire d u r a t i o n
References 1 Wisse E. Ultrastructure and function of Kupffer cells and other smusoldal cells in the liver. In: Wlsse E, Knook DL, eds. Kupffer Cells and Other Liver Sinusoldal Cells. Amsterdam: Elsevier/North-Holland Biomedical Press, 1977; 33-60. 2 Rojkind M, Ponce-Noyola P. The extracellular matrix of the liver. Coil Relat Rcs 1982; 2: 151-175. 3 Popper H. Summary of the conference. In: Gerlach U, Pon G, Rauterberg ,l, Voss B, eds. Connective Tissue of the Normal and Fibrouc Human Liver. Stuttgart, New York: Georg Thieme Verlag, 1982; 246-258. 4 Rojkind M. Kershenobish D. Extracellular matrix, fibrosis and cirrhosis. In: Arias IM, Frcnkel M, Wilson ,IHP, eds. The Liver Annual, Vol. 5. Amsterdam, New York, Oxford: Elsevier, 1982; 180-210. 5 Rojkind M. Giambrone MA, Biempica L. Collagen types in normal and cirrhotic liver. Gastroenterology 1979; 76: 710-719. 6 Seyer ,lM. lntcrstmal collagen polymorphism in rat liver with CCI.cinduced cirrhosis. Biochim Biophys Acta 1980; 629: 49(I-498. 7 Kent G, Inouye T, Minick OT, Bahu RM Role oflipocytes (pensmusoidal cells) in fibrogenesis. In: Wisse E, Knook DL. eds. Kupffer Cells and Other Lwer Sinusoidal Cells. Amsterdam. Elsevier/North-Holland Biomedical Press, 1977; 73-82. S Leo MA, Lieber CS. Hepauc fibrosis after long-term administration of ethanol and moderate vitamin A supplementation in the rat. Hepatology 1983; 3:1-11. 9 Ballardini G, Degh Esposti S, Bianchi FB, et al Correlation betwcen Ito cclls and fibrogenesis in an experimental model of hepatic fibrosis. A sequential stereological study. Liver 1983; 3: 58-63. 10 Mak KM, Leo MA, Lleber CS. Alcoholic liver injury in ba-
U n i v e r s i t y of Glas-
boons: transformation of hpocytes to transitional cells Gastroenterology 1984; 87: 188-200. 11 Callea F, Desmet V,l Transformation of fat storing cells (Ito cells) to myofibroblasts. J Hepatol 1985; 2 (suppl): $206. 12 Thompson WD, ,lack AS. Patrick RS. The possible role of macrophages in transient hepatic fibrogenesis induced by acute carbon tetrachlonde injury..l Pathol 1980; 13(1: 65-73. 13 Kulonen E, Karl K, Fransilla K. Effects of long-term treatment of rats with ethanol, carbon tetrachloride and high fatlow protein diet on the Kupffer cell distribution w~th reference to chemical composition of the hver. Acta Pathol MIcrobiol Immunol Scand [C] 1983; 91. 221-225 14 Tamayo RP. Is cirrhosis of the hver experimentally produced by CCI4 an adequate model of human cirrhosis? Hepatology 1983: 3:112-120. 15 De Zanger R, Wisse E. The filtration effect of rat l.ver rehesitated sinusoidal endothelium on the passage of (remnant) chylomicrons to the space of Disse. In' Knook DL, Wisse E, eds. Sinusoldal Liver Cells. Amsterdam: Elsevier Biomedical Press, 1982; 69-76. 16 Bouwens L, Baekeland M, Wisse E. Importance of local proliferation in the expanding Kupffcr cell population of rat liver after Zymosan stimulation and partml hepatectomy. Hepatology 1984, 4:213-219 17 Widmann J-,l, Cotran RS, Fahimi HD. Mononuclear phagocytcs (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver smusoids by endogenous peroxidase activity. J Cell Biol 1972; 52: 159-170 18 Snedecor GW. The comparison of two groups. In: Snedecor GW, ed. Statistical Methods. Ames, Iowa: The Iowa State College Press, 1950; 75-88. 19 Aherne WA, Camplejohn RS, Wright NA. Cell cycle events. In: Aherne WA, Camplejohn RS, Wright NA, eds. An Introduction to Cell Population Kmeucs. London: E
56 Arnold, 1977; 8-16. 20 Salas SL, Hdle E. Infinite series In: Salas SL, Hdle E, eds Calculus. One and Several Variables with Analytical Geometry. Part 1 New York: John Wiley and Sons, 1982; 533-598. 21 Rojkmd M, Reyes-Romero M, Cerbon-Ambnz J, Rosas M Cell-cell and cell-matrix interactions in hver cirrhosis. In Van Bezooljen CFA, ed. Proceedings of the Eurage Workshop on Pharmacological and Physiological Aspects of Liver Aging. Rijswijk. Eurage, 1983, 59-70. 22 Rojkmd M, Valadez G. Regulation of fibroblast proliferation by Kupffer cells and monocytcs. In. Badey AJ. ed. Fibrosis, Ctba Foundation Symposmm 114. London: Pitman, 1985: 208-221. 23 Shlratori Y. Geerts A, Ichida T, Kawase T, Wtsse E. Kupffer cells from CCl:induced fibrotic livers stimulate prohferat,on of fat-storing cells. J Hepatol 1986; 3:
A GEERTS et al. 294-303. 24 Van Furth R, Crofton RW, Dlesselhoff-den Dulk MMC. The bone marrow origin of Kupffer cells. In: W,sse E, Knook DL, eds. Kupffer Cells and Other Liver S,nusoidal Cells. Amsterdam: Elsevier/North-Holland Biomedical Press, 1977; 471-480. 25 Wlsse E. Kupffer cell reactions in rat liver under various conditions as observed in the electron microscope. J Ultrastruct Res 1974; 46:499-520 26 Volkman A. The unsteady state of the Kupffer cell. In: Wisse E, Knook DL, eds. Kupffer Cells and Other Liver Sinusoidal Cells. Amsterdam: Elsevier/North-Holland Biomedical Press, 1977; 459-469. 27 Bouwens L, Baekeland M, De Zanger R, Wisse E. Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver. Hepatology 1986: 6: 718-722.
Journal of Hepatology, 1988; 6:50-56
Elsevier HEP 00368
Cell population kinetics of Kupffer cells during the onset of fibrosis in rat liver by chronic carbon tetrachloride administration
Albert Geerts, Paul Schellinck, Luc Bouwens and Eddie Wisse Laborato~3'Jbr Cell Biology and Htstology. Free Umversttv Brussels, V. U B., Brussels (Belgium)
(Received 28 April 1987) (Accepted 14 September 1987)
Summary The cell population kinetics of Kupffer cells (KCs) were investigated during the induction of fibrosis in rat liver bv carbon tetrachloride. KCs, characterized microscopically by their specific peroxidase pattern, increased in number during the first 24 h after CC14 injection. After repeated injections of the toxin the number of KCs increased logarithmically. After 9 weeks of CCI~ treatment, the KC population reached a new steady state with a 2.6-times-higher cell number than in control animals. Local KC proliferation was demonstrated by the metaphase arrest technique using vinblastine. It was calculated that at 24 h and at 72 h after one injection of CC14, local proliferation did not account for the entire growth of the population. This means that, in addition to local proliferation of KCs, an influx of mononuclear phagocytes into the fiver sinusoids occurred.
Introduction The liver parenchyma is composed of several cell types including parenchymal cells, sinusoidal endothelial cells, Kupffer cells (KCs), fat-storing cells and pit cells [1]. These cells are in permanent contact with extracellular matrix (ECM) components such as collagens, non-collagenous glycoproteins, glycosaminoglycans [2] and elastin [3]. The different cell types and their surrounding ECM are in dynamic equilibrium. In healthy liver, the relative proportions
of liver cells and matrix remain constant. The mechanisms which control this complex equilibrium are unknown. As a result of chronic liver damage, this balance is often lost and hepatic fibrosis develops [4]. Liver fibrosis is characterized by a 5-10-fold increase in the amount of collagen per g of liver [5,6]. Concomitantly, the cellular mass of the liver decreases and its composition changes. Fat-storing cells increase in number [7-9] and differentiate into myofibroblast-like cells [10,11]. The number of macrophages also increases [12,13]. However, quantitative
This work was financially supported by NFWO grants No. 30.0040.80 and No. 30.0028.86. Correspondence: A. Geerts, Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel (V.U.B.), Laarbeeklaan 103, 1090 Brussels-Jene, Belgium. Tel: (32-2)478 48 90, ext. 1406/1407. 0168-8278/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
KUPFFER CELLS DURING CCI4-INDUCED FIBROSIS
51
data are lacking. Furthermore, it is not clear whether such an increase of liver macrophages is the result of KC proliferation or is due to infiltrating mononuclear phagocytes. In the present study, we have examined the population kinetics of KCs during the onset of liver fibrosis, using chronic carbon tetrachloride (CC14) intoxication of the male Wistar rat as a model system [14].
in the cytoplasm. The number of KCs per microscopic field (NKC/f:f = 0.077 mm 2) was counted according to Bouwens et al. [16], using a Zeiss Photomicroscope III. Only KCs in which the nucleus was visible were counted. Ten microscopic fields per section were taken at random; at least three sections per rat were examined. The livers of six animals were studied for each time point. The statistical significance between two groups of rats was tested by applying Student's t-test [18].
Materials and Methods
Metaphase index (/met) of KCs
Experimental animals Sixty-six male Wistar rats, weighing 225-250 g, were given subcutaneous injections of 0.1 ml of CCi 4 per 100 g body weight twice a week. The CCI 4 was dissolved in an equal volume of paraffin oil [6]. Six animals were sacrificed at each of the following time points: 1 day, 3 days, 1, 2, 3, 4, 5, 7, 9 and 13 weeks. Six animals (controls) received paraffin oil alone.
Fixation Low-pressure (12 cm HEO ) perfusion fixation of the liver through the portal vein was performed according to De Zanger and Wisse [15]. The fixation was preceded by a brief rinse of the liver with isotonic buffer. The fixative contained 1.5% glutaraldehyde (Merck), 0.1 M cacodylate buffer pH 7.4, 0.01% CaCI2.2H20 and 2.5% sucrose, and was applied for 3 min.
The metaphase index or fraction of KCs arrested in metaphase was determined by injecting vinblastine sulphate (Velbe-Lilly; 2 mg/kg body weight) 6 h before perfusion of the liver. The metaphase-arrested cells were easily recognized by their distinct chromosomes and the diffuse brown staining in the cytoplasm. Chromosomes were visualized by briefly counterstaining the Epon sections with 1% toluidine blue dissolved in 1% borax [16]. From the metaphase indices we have calculated the birth rate (kB) of the KCs, i.e., the number of cells generated per unit of time and per cell. k B has been calculated for two extreme cell population growth models [19]. First, a cell population in steady state was considered. For such a population, k B can be easily found as follows:
kB.s -
met
(1)
tA
Peroxidase cytochemistry and KC counts KCs were characterized by a modification [16] of the original staining method for endogenous peroxidase [17]. 50-70tLm Vibratome sections of glutaraldehyde-perfused liver tissue were incubated for 1 h at 22 °C in a peroxidase medium containing 0.1% diaminobenzidine.4HCl (Fluka), 0.02% H202 and 0.14 M cacodylate buffer. The final pH of this medium was 6.9. Subsequently, the sections were post-fixed for 1 h in 1% Millonig's OsO4, and embedded in Epon 812. KCs were recognized by light microscopy in semithin Epon sections by the presence of a brown rim around the nucleus and a diffuse brown staining
(/met: metaphase index, tA: arrest time (6 h)) Second, an exponentially growing cell population from which no cells leave the cell compartment was assumed. Then k B equals:
kB.c = 2.303 log(l + Imct) _ In(1 +/met) tA
(2)
tA
Expansion of equation 2 in an infinite series [20] results in the following equation:
52
A GEERTS et al.
1 imct 3 /mct kB,c = ~ ( / m e t - - + tA 2 3 IfflCl - -
NKC/f
a /tact --'") 4
t
20-
t
I (if/me t ~< 0.01)
tA Thus if lmct ~ 0.01, then kB.c = Imct/tA for an exponentially growing population as well.
Cell population kinetics The following equation was used to describe an exponentially expanding population [18]: 10 ¸
N, = N,_te kB.~(q-q-i)
(3)
N, and N,-i represent the mean number of cells per field at time t, and t,_t after injecting CC14. kB., is the growth rate constant at time t,.
Results
i
0
-1
5
10
time
( weeks
)
Fig. I. Average number of Kupffer cells per microscopical field (NKC/f; f = 0.077 mm 2) during the reduction of fibrosis by CC14 Kupffer cells prohferate from the very beginning of the CCI~ administration. After 9 weeks of CCI a treatment a new steady state is reached.
b~duction of fibrosis In keeping with the findings of previous studies [14], a single or a few injections of a sublethal dose of CCI4 produced steatosis and parenchymal cell necrosis in the centrilobular areas of the liver lobules in all animals. This was followed by a progressive increase in the amount of connective tissue in this zone; there was individual variation in the degree of fibrosis between animals which had received an equal number of injections. Further CC14 intoxication induced the formation of fibrotic septa. From 9 weeks onwards, all animals showed liver fibrosis.
KC population kinetics KCs increased in number from the first day of the CCla administration (+17.4%, P < 0.01; Fig. 1). Repeated injections of CCI4 provoked further growth of the KC population (Fig. 1); after 2 weeks the population had doubled. Microscopical examination of necrotic pericentral areas showed KCs infiltrating these regions prior to the deposition of excess ECM. Prolonged administration of CC14 led to a steadily decreasing growth of the population. After 9 weeks the KC population reached a new steady state in which the average number of cells per microscopic field was
2.6-times higher than in control animals. By applying the metaphase arrest technique, KCs were shown to divide at all stages of chronic CC14 intoxication (Fig. 2). We have found KCs arrested in metaphase in all zones of the liver lobule. The meta/phase index of the KC population remained fairly Constant at a level of about 1% throughout the experiment, except for animals treated for t week (2 injections). In such animals the metaphase index was about 2% (Table 1). The cell birth rate (kB), calculated from the observed metaphase indices, seemed to be almost completely independent of the assumed population growth model (Table 1). At 24 h and 72 h after one injection of C C I 4, the maximal increase of the number of KCs due to autoproliferation of the population was calculated by substituting the obtained values for k B in equation 3 (see Materials and Methods). In Table 2, the results of these calculations are summarized. By comparing the maximal number of KCs that could have been obtained by autoproliferation of the population with the number of cells that was observed microscopically, it is concluded that local proliferation alone did not account for the total growth of the population.
KUPFFER CELLS DURING CCI4-INDUCEDFIBROSIS
53
Fig. 2. Kupffercell arrested in metaphase. Distinct chromosomes(C) are visible. The rough endoplasmicreticulum showsendogenous peroxidase activity (arrow). Lead citrate counterstaining (x 10 800). P: parenchymalcell.
Discussion
The present experiments were undertaken to study the population kinetics of KCs during the onset of liver fibrosis by chronic administration of CC14 in rats. KCs respond to CC14 within 24 h by expanding their population. This finding is in accordance with previous observations in mice, which were acutely intoxicated with CCI4 [12]. After 9 weeks of treatment, the KC population reached a new steady state at a
level 2.6-times higher than the control level. Earlier studies on isolated sinusoidal liver cells from CCl4-intoxicated rats [13,21,22] indicated an increase in the number of KCs following CC14 administration. However, it was not clear from these studies whether the yields of purified KCs from normal and CCl4-treated rats were equal. Furthermore, it was not established in these studies whether the increased population of macrophages was exclusively composed of cells showing the typical histochemical characteristics of KCs.
54
A GEERTS et al.
TABLE 1 METAPHASE INDICES (`/met) OF KCs DURING THE INDUCTION OF FIBROSIS BY CCI~ From `/m~tthe birth rate of KCs. i.e., the number of new cells generated per ceil and per day. has been calculated assuming both a steady-state KC population (k a ,) and an exponentially growing populauon (kB.¢). Treatment time 0 1 day
3 days l week 2 weeks 3 weeks 4 weeks 5 weeks 7 weeks 9 weeks 11 weeks 13 weeks
In( 1 + `/me0 (day-I x 10 -4)
'/met (x 10-41
kB, = Imct/t A (day-ix 10-4)
k u ~ = t,-x I
0 121 + 23"
0 485
0 480
90 + 10 185 + 31 70 4- 14 90 4- 6 92 + 19 121 4- 23 104 +_ 17 71 + 5 126 + 14 50 + 6
360 739 281 360 367 485 415 283 504 199
358 734 278 358 365 480 413 283 504 199
" Standard error.
In earlier papers [2,22], it was suggested that K C s
that liver m a c r o p h a g e s are n o n - d i v i d i n g cells, de-
may m o d u l a t e the p r o l i f e r a t i o n of o t h e r liver cells
rived f r o m m o n o c y t e s [24], the p r e s e n t findings indi-
and thetr capacity to synthesize c o n n e c t i v e tissue
cate that, f o l l o w i n g c h r o n i c CCI 4 i n t o x i c a t i o n , t h e r e
c o m p o n e n t s . In a recent study, in which isolated K C s
is local p r o l i f e r a t i o n of KCs. Such a u t o p r o l i f e r a t i o n
were c o - c u l t u r e d with fat-storing cells, it was d e m o n -
of KCs has also b e e n f o u n d after s t i m u l a t i o n of the
strated that KCs isolated f r o m livers of CC14-treated
reticulo-endothelial
rats stimulate fat-storing cells to divide [23]. O u r ob-
[16,25], R E S b l o c k a d e [25] and partial h e p a t e c t o m y
servation that the K C p o p u l a t i o n e x p a n d s signifi-
[16,25].
system
(RES)
with
zymosan
cantly during the i n d u c t i o n of fibrosis s u p p o r t s the
A t 24 h and 72 h after o n e i n j e c t i o n of CC14, we cal-
hypothesis that KCs m a y play a crucial role during the onset of fibrosis.
/culated f r o m the cell birth c o n s t a n t (kB) the m a x i m a l
By injecting vinblastine 6 h b e f o r e fixation of the
KCs by assuming (i) an e x p o n e n t i a l cell p r o l i f e r a t i o n
liver, we have b e e n able to d e m o n s t r a t e a r r e s t e d mi-
m o d e l and (ii) that no KCs left the liver. In short-
toses of KCs t h r o u g h o u t the p e r i o d of CCI~ treat-
t e r m e x p e r i m e n t s , the latter a s s u m p t i o n is r e a s o n -
ment. Thus, in contrast to the widely held o p i n i o n
able [24,26,27]. U s i n g this analysis, we h a v e d e m o n -
p o p u l a t i o n g r o w t h due to a u t o p r o l i f e r a t i o n of the
TABLE 2 OVERALL GROWTH VS. GROWTH DUE TO MITOSIS OF THE KC POPULATION AT 24 h AND 72 h AFTER ONE INJECTION OF CCI 4 The maximal increase of the number of KCs per microscopical field at 24 h and 72 h after one injection of CCI 4 which could be accounted for by mitosis of the resident KC populauon was calculated by applying formula 3. Since the observed numbers of KCs (column 4) exceeds these calculated numbers (column 3), it is concluded that extrahepatic recrmtment of KCs must take place. Treatment ume (h) 0
24 72
Observed k B (day -I × 10 -4)
Calculated number of cells tf only KC mitosis occurred
Observed number of KCs
--
--
--
482 359
7.78 9.36
8.71 9.54
KUPFFER CELLS DURING CCI4-INDUCED FIBROSIS
55
strated that 1 day after CCI 4 i n j e c t i o n , a r o u n d 3 0 % of
of the e x p e r i m e n t , a simple q u a n t i t a t i v e r e p l a c e m e n t
the growth of the p o p u l a t i o n can be e x p l a i n e d by lo-
of resident liver m a c r o p h a g e s by m o n o c y t e s or o t h e r
cal p r o l i f e r a t i o n . T h u s , at least 7 0 % of the n e w cells would a p p e a r to h a v e b e e n r e c r u i t e d f r o m e x t r a h e p -
e x t r a h e p a t i c m a c r o p h a g e s is not likely to take place
atic
origin,
although
they
have
cytochemical
during the induction of fibrosis by CC14 in the rat liver.
characteristics in c o m m o n with KCs. T h r e e days after one CCIz i n j e c t i o n , local p r o l i f e r a t i o n was s h o w n to account for 8 0 % of the p o p u l a t i o n growth. In ex-
Acknowledgements
p e r i m e n t s in which C f l 4 was a d m i n i s t e r e d for long periods of time, it c a n n o t be a s s u m e d that all KCs
T h e authors wish to t h a n k M r Paul Lens and M r
stay in the liver. A m e t h o d to e v a l u a t e the cell loss
Ronald
from the K C p o p u l a t i o n is at p r e s e n t not available,
t h r o u g h o u t the e x p e r i m e n t s . W e t h a n k Dr. A . D .
De
Zanger
for their helpful suggestions
and c o n s e q u e n t l y q u a n t i t a t i v e e s t i m a t e s of the im-
Burt ( P a t h o l o g y D e p a r t m e n t ,
p o r t a n c e of K C mitoses during l o n g - t e r m CCI4 ad-
gow, U . K . ) for c o r r e c t i n g the m a n u s c r i p t , Mrs Mar-
ministration are unreliable. N e v e r t h e l e s s , since we
leen D e P a u w for typing it, and Mrs Chris D e r o m for the p h o t o g r a p h i c work.
have o b s e r v e d K C mitoses during the entire d u r a t i o n
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