Induction of hepatocyte mitosis in intact adult rat by interleukin-1 α and interleukin-6

Life Sciences, Vol. 49, pp. 1263-1270 Printed in the U.S.A.

Pergamon Press

INDUCTION OF HEPATOCYTE MITOSIS IN INTACT ADULT RAT BY I N T E R L E U K I N - I U AND INTERLEUKIN-6 1 Mutuyosi

Koga 2 and Hisatake Ogasawara 3

2Department of Physiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, and 3Department of Internal Medicine, Daisan Hospital, The Jikei University School of Medicine, Izumi Hon-machi, Komae City 201, Japan (Received in final form August 19, 1991)

Summary In the adult rat the liver is normally quiescent, but it proliferates rapidly in response to partial hepatectomy (PH). A hepatectomized rat is subjected to stress by the operation. We have examined the effects of acute phase cytokines. To investigate the mediation of hepatocyte growth, recombinant human i n t e r l e u k i n - 1 ~ (IL-I) and interleukin-6 (IL-6) were injected into male rats. A d m i n i s t r a t i o n of IL-I or IL-6 followed by NH C1 and glucagon could induce hepatocyte mitosis 30 h after the first injection. This activity was lost when interleukins were exposed to 90°C for 30 minutes. NH4CI augmented the effects of IL-I and IL-6. These results suggest that the IL-I and IL-6 are important mediators of liver regeneration after PH. We present a hypothesis for the triggering mechanism of hepatocyte proliferation. Although most hepatocytes are quiescent in the adult rat liver, as is well known, the cells respond to PH by replicating their DNA (peak at 24 h) and undergoing mitosis (peak at 30h) (I). Many growth factors have been proposed as regulators for hepatic regeneration, with much supporting evidence (I-9). Knowledge of precise trigger(s) that induce hepatocytes to enter the cell cycle from the quiescent state is lacking. To learn about the stimuli that are evoked by PH, the conditions have been sought for inducing hepatocyte proliferation in unoperated rats. We previously found that the combinations of various amines followed by glucagon induced DNA synthesis and mitosis in hepatocytes of intact rats (10). Studies of the primary culture of adult rat hepatocytes have shown that the hepatocytes were induced into DNA synthesis and cell division by insulin and IEssentials of this paper were presented at the Fourth Canda, in International Congress of Cell Biology, Montreal, August, 1988. 2To whom reprint requests

should be addressed.

0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc

IL-I, IL-6 & Hepatocyte Proliferation

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g l u c a g o n in s e r u m - f r e e K o g a ' s L m e d i u m (11, 12). We t u r n e d a g a i n to in v i v o e x p e r i m e n t s b e c a u s e we c o n s i d e r e d that the isolated hepatocytes were already triggered during the isolating procedure. P r o l i f e r a t i o n of these h e p a t o c y t e s can be i n d u c e d by i n s u l i n and g l u c a g o n , w h i c h are not t r i g g e r s (I). P a r t i a l l y h e p a t e c t o m i z e d rats are s u b j e c t e d to the stress of operation. Stress induces events that lead to a complex homeostatic r e a c t i o n k n o w n as the a c u t e p h a s e responses (i.e., increase of h e p a t i c a m i n o a c i d flux, i n c r e a s e of acute phase protein synthesis by hepatocytes, increase of hepatic zinc concentration, a n d d e c r e a s e of s e r u m a l b u m i n ) (13-15). Similar r e s p o n s e s w e r e o b s e r v e d in the rats a f t e r PH (I, 16, 17). In prestressed rats, DNA s y n t h e s i s f o l l o w i n g PH was a c c e l e r a t e d (I, 18). In the p r e s e n t paper, we examined whether hepatocyte p r o l i f e r a t i o n m a y be i n d u c e d by c y t o k i n e s (IL-I and IL-6), which are produced and r e l e a s e d by a c t i v a t e d m a c r o p h a g e s and which e l i c i t acute phase p r o t e i n s y n t h e s i s by h e p a t o c y t e s (19-21).

Materials

and M e t h o d s

Chemicals: G l u c a g o n was p u r c h a s e d from C a l b i o c h e m (30 ug/ml, San Diego, CA, USA). R e c o m b i n a n t h u m a n IL-le (172 ~g/200 ~i, lot No. 6703) was k i n d l y s u p p l i e d by Dr. Y a m a d a ( D a i n i p p o n P h a r m a c e u t i c a l Co., Osaka, Japan). R e c o m b i n a n t h u m a n IL-6 (4 u n i t s / n g , Lot No. 685-3 and 200 ng/~l, Lot No. 703) was kindly provided by Ajinomoto Co., Central Research Institute (Kawasaki, Japan). E n d o t o x i n (5 ~g/ml, E. coli 055: B5) was from D i f c o L a b o r a t o r i e s (Detroit, MI, USA). The saline s o l u t i o n was a p y r o g e n i c 0.9% NaCI solution. Each c o m p o u n d was d i s s o l v e d in the s a l i n e solution. All s o l u t i o n s w e r e s t e r i l i z e d by f i l t r a t i o n t h r o u g h a M i l l i p o r e f i l t e r (0.2 ~m, M i l l i p o r e Corp., Bedford, MA, USA.). Animals: W i s t a r m a l e rats, w e i g h i n g 150-160 g, were used. The animals were k e p t in a t e m p e r a t u r e c o n t r o l l e d r o o m with 12 h alternating light and dark cycles. They were given laboratory food and w a t e r for I w e e k p r i o r to the e x p e r i m e n t s . T r e a t m e n t of animals: The rats in e a c h g r o u p were i n j e c t e d w i t h I ml per 100 g B.W. at the i n d i c a t e d time w i t h o u t e t h e r a n e s t h e s i a by a m o d i f i e d p r o c e d u r e as p r e v i o u s l y d e s c r i b e d (10). At 30 h a f t e r the first i n j e c t i o n or PH, the rats w e r e k i l l e d u n d e r d e e p ether-induced anesthesia and the m e d i a n lobe or the remaining right lobe was removed. A PH was p e r f o r m e d at 9:00 a.m. by the method of H i g g i n s and A n d e r s o n (22), the left l a t e r a l lobe (34% PH) was removed by the m e t h o d of B u c h e r and Swaffield (23). Hepatocyte p r o l i f e r a t i o n a c t i v i t y was e x p r e s s e d as the rate of mitoses. Examination of the m i t o t i c rate was p r e p a r e d by the s q u a s h m e t h o d of M a k i n o and T a n a k a (24) (FIG. I, No. 2, 4). The number of m i t o s e s per 1,000 h e p a t o c y t e n u c l e i was u s e d as the index of the m i t o t i c ratio. P a r a f f i n s e c t i o n s w e r e cut in 5 ~m t h i c k n e s s and s t a i n e d w i t h h a e m a t o x y l i n and e o s i n (FIG. I, No. I, 3).

Results Table

I shows

that

hepatocyte

mitosis

in i n t a c t

adult

rats

Vol. 49, No. 17, 1991

IL-I, IL-6 & Hepatocyte Proliferation

TABLE Effect Time 0 h

of I L - l e and

I

IL-6 on Rat H e p a t o c y t e

of A d m i n i s t r a t i o n 2 h 4, 6, 8 h

Saline Saline Saline Saline

1265

Mitosis

Mitosis

per 1,000 30 h

Saline Saline NH4CI NH4CI

Saline G Saline G

I L - I ~ (ng/ml) I0 100 200 500 I ,000 200 (heated) 200 200

IL-le + NH4Cl NH4CI NH4CI NH4CI NH4CI NH4CI Saline Saline

G G G G G G G Saline

0 5.0 ± I .3, 11.6 ± 2.8 8.2 ± I .9 7.5 ± 2.2 0 I .5 ± 0.7 0

IL-6 (ng/ml) 20 50 100 50 (heated) 50 50 50

IL-6 + NH4CI NH4CI NH4CI NH4CI NH4CI Saline Saline

G G G G Saline G Saline

4.2 13.2 8.7 0 0 2.4 0

Endotoxin Endotoxin

Saline NH4CI

Saline G

0 14.0

Sham Sham

Saline NH4CI

Saline G

0 0

34% PH 34% PH PH

Saline NH4CI -

Saline G -

2.7 33.0 39.7

nuclei

0 0 0 0

± I .7, ± 3.2 ± 2.0

± 0.6

, ± 2.3

± 1.1 ± 3.1,, ± 2.6

Treatment of the a n i m a l s is d e s c r i b e d in "Materials and Methods". One ml per 100 g B. W. was i n j e c t e d at the i n d i c a t e d time. Saline: 0.9% NaCI solution, sc; G: glucagon, 30 ~g/ml, sc; NH4CI: 8 mg/ml, sc; IL-I~: r e c o m b i n a n t h u m a n IL-le, ip; IL-6: - r e c o m b i n a n t h u m a n IL-6, ip. H e a t e d IL-I and IL-6 w e r e i n c u b a t e d at 90°C for 30 min. Endotoxin: 5 Hg/ml, sc; Sham: s h a m - o p e r a t e ; 34% PH: r e m o v e d left l a t e r a l lobe; 68% PH: r e m o v e d left lateral plus median. V a l u e s are mean ± S. E. for four or five experiments. *: P>0.05 c o m p a r e d to IL-6; **: P<0.05.

significantly i n c r e a s e d 30 h a f t e r the first i n j e c t i o n s of IL-I (200 n g / 1 0 0 g rat) or IL-6 (50 n g / 1 0 0 g rat), w h i c h w e r e f o l l o w e d by N H 4 C I a n d g l u c a g o n . A d m i n i s t r a t i o n of e a c h s u b s t a n c e had no effect on h e p a t o c y t e m i t o s i s Although investigators that approximately one mitosis is seen in 10,000 t o r e ~ h e p a t o c y t e s in a d u l t a n i m a l s (1,7), the m i t o t i c rate per 1,000-

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IL-I, IL-6 & Hepatocyte Proliferation

FIG.

Vol. 49, No. 17, 1991

I

Mitotic appearance of liver 30 h after partial hepatectomy (I) (2) a n d a d m i n i s t r a t i o n fo IL-6 (50 n g / 1 0 0 g B.W.), N H 4 C I a n d g l u c a g o n d e s c r i b e d in T a b l e I (3) (4). Arrows indicate hepatocyte mitoses. There are f o u n d all p h a s e s of m i t o s i s as s h o w n in (2) (4) by the s q u a s h method. P: prophase; M: metaphase; A: anaphase; T: telophase. N o t m a n y fat d r o p l e t s in the f i e l d of the r e g e n e r a t i n g l i v e r (2).

2,000 nuclei was practically zero in the saline-injected controls. W h e n IL-I a n d IL-6 w e r e i n c u b a t e d at 90°C for 30 m i n p r i o r to t h e i r i n j e c t i o n , t h e i r s t i m u l a t o r y e f f e c t in h e p a t o c y t e proliferation was lost. T h e i n d u c t i o n of m i t o s i s was a u g m e n t e d by injection of NH4CI. Sham and sham plus NH.Cl and glucagon i n j e c t i o n y i e l d e d no i n c r e a s e in r a t e of m l t o s l s . B u t the n u m b e r of m i t o s e s r o s e r e m a r k a b l y w h e n the 34% h e p a t e c t o m i z e d rat was injected with NH4Cl and glucagon, t h o u g h few m i t o s e s w e r e f o u n d a f t e r 34% PH. E x p e r i m e n t s of e n d o t o x i n as s t r e s s o r s h o w t h a t the m a x i m u m i n c r e a s e in h e p a t o c y t e m i t o s i s , w i t h a d m i n i s t r a t i o n of 5 ug/100 g B. W. in t e s t e d d o s e s b e t w e e n 0.1 a n d 50 u g / 1 0 0 g B. W. (not s h o w n in T a b l e I), was 14.0 ± 2.3. T h e m e a n of m i t o s e s 24 h a f t e r PH was 16 ± 5 . 8 / I , 0 0 0 nuclei. In the injected r a t s (IL-6, 50 n g / 1 0 0 g B. W., NH4CI and glucagon), this was 1.2 ± 0 . 8 / 1 , 0 0 0 n u c l e i . T h e PH and the injected r a t s a c h i e v e d the h i g h e s t level at 30 h 39.7 ± 2.6 and 13.2 ± 3 . 2 / 1 , 0 0 0 nuclei, r e s p e c t i v e l y . T h e m i t o s i s 40 h a f t e r PH was 23.1 ± 4 . 3 / 1 , 0 0 0 n u c l e i a n d in the i n j e c t e d r a t s it r e t u r n e d to the s a l i n e c o n t r o l l e v e l at 40 h. E a c h r e s u l t r e p r e s e n t s the m e a n ± SE of 3-4 rats.

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IL-1, IL-6 & Hepatocyte Proliferation

1267

All phases of mitotic changes were found (FIG. I, No. 2, 4). Necrotic changes of hepatic tissues were not found in any instance (FIG. I, No. I, 3). Mitotic figures in epithelial cells of kidney tubules in the rats were induced with IL-I (200 ng/100 g B.W.), NH4CI and glucagon. The number of mitoses increased in number (4/1,000 nuclei in the mean of 4 rats, P<0.05 compared to normal rats), but in the case of injection of IL-6, increase of mitoses was not observed.

Discussion IL-I and IL-6 treatment at physiological doses (25-27) markedly induced hepatocyte mitosis in nearly the same length of period as that in the regenerating liver. Though the influence on the liver regeneration has not been addressed by the studies reported here, these interleukines may be important mediators of the liver regeneration. The results of sham and 34% PH experiments (Table I) seem to be concerned with the reports that smaller incisions (I-1.5 cm, sham) yielded no response of acute stress (28) and 34% PH resulted in small increases of DNA synthesis (23). Mitotic changes are also found in kidney tubules in the case of IL-I, but the occurrence with IL-I is apparently less marked. It seems likely that different conditions may elicit different "triggering" mechanisms (I-8). Bucher et al. (9) reported that regulation of hepatic growth appears to depend on the synergistic of combinations of many substances. Extreme hormone imbalances can stimulate liver growth without any surgical intervention under various experimental or stressful conditions (10, 29). In FIG. 2, we have attempted to summarize what we currently know about the hepatocyte proliferation trigger mechanism. (I) The partially hepatectomized rats suffer from surgical stress. The stress hormones including adrenalin (4, 10), vasopressin (30), serotonin and histamine (10), caused by the surgical trauma and bleeding, promote fibrinolysis (31). Fibrinolytic activity of euglobulin fraction of rat serum significantly increased at an early stage (I-10 h) after PH (our unpublished data). The plasmin-derived fibrinogen stimulates the production of macrophage hepatocyte-stimulating factor (HSF)(14). Recently, this molecule was shown to be identical to IL-6 (20, 21). (2) Gut-derived endotoxins are continuously present in the portal vein. The loss of the phagocytic capacity of Kupffer cells by PH increases the portal and systemic concentration of endotoxins compared to the amount in normal rats (32). It is known that macrophages (Kupffer cells) are activated by endotoxins which then cause secretion of cytokines such as IL-I and IL-6 (19-21). Cornell (32) reported that treatments to restrict endogenous endotoxin impaired DNA synthesis in the regenerating liver. In the present study, the combination of endotoxin, NH4CI and glucagon induced mitosis in hepatocytes (Table I). (3) Castell et al.

(33) reported that about 80% of the 125I-

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IL-I, IL-6 & Hepatocyte Proliferation

Vol. 49, No. 17, 1991

Hypothetical Schema for Initiating Rat Liver Regeneration (1) Stressors

(2) Endotoxin

Fibrinolysis ~Plasminogen

activator~

Fibrinogen Degradation Products

I MONOCYTES ,. MACROPHAGES, KUPFFER CELLS

IL-1 (4) Ammonia overload

---

(3) IL-1

FIBROBLASTS ENDOTHELIAL CELLS KERATINOCYTES

IL-6 -

HEPATOCYTES

l

(5)

Proliferation FIG.

Hepatotrophic growth factors

2

(I) B l e e d i n g and stress from a partial hepatectomy s t i m u l a t e s the r e l e a s e of a d r e n a l i n e , vasopressin, s e r o t o n i n and h i s t a m i n e . These, in turn, p r o m o t e fibrinolytic activity, r e s u l t i n g in the p r o d u c t i o n of f i b r i n o g e n d e g r a d a t i o n p r o d u c t s (FDP). The FDP a c t i v a t e s m a c r o p h a g e s to p r o d u c e IL-6. (2) The r e l a t i v e a m o u n t s of g u t - d e r i v e d e n d o t o x i n and lipopolysaccharide i n c r e a s e w i t h the r e m o v a l of 68% of the liver. K u p f f e r cells (macrophages) are a c t i v a t e d by the i n c r e a s e d a m o u n t of e n d o t o x i n , a n d p r o d u c e IL-I and IL-6. (3) IL-I a c t i v a t e s f i b r o b l a s t s , e n d o t h e l i a l cells and keratinocytes. T h e y p r o d u c e and r e l e a s e IL-6. (4) The g u t - d e r i v e d a m m o n i a c a n n o t be fully d e t o x i f i e d by the r e m a i n i n g liver. (5) H e p a t o t r o p h i c g r o w t h factors; insulin, glucagon, EGF, TGF- e , h e p a t o p o i e t i n - A and B, and h e p a t o c y t e g r o w t h factor, etc. labelled IL-6 that had d i s a p p e a r e d from c i r c u l a t i o n was found the liver and that the l a b e l l e d IL-6 was e x c l u s i v e l y l o c a l i z e d the s u r f a c e of the h e p a t o c y t e s . In vivo, the h e p a t o c y t e s s e e m be the m a j o r t a r g e t cells of the IL-6.

mass, tasks that from

in on to

(4) The r e m a i n i n g liver w i t h o n l y a t h i r d of the original must efficiently and i m m e d i a t e l y p e r f o r m the metabolic of an intact liver a f t e r PH. F a u s t o et al. (33) r e p o r t e d in the r e g e n e r a t i n g liver, the a b i l i t y to remove ammonia the b l o o d is close to its m a x i m u n limit and that ammonia

Vol. 49, No. 17, 1991

o v e r l o a d leads then causing decarboxylase (35).

IL-I, IL-6 & Hepatocyte Proliferation

1269

to the p r o d u c t i o n of large a m o u n t s of o r o t i c acid, marked elevation of the hepatic ornithine a c t i v i t y w h i c h is n e c e s s a r y for liver restoration

We previously proposed a two step hypothesis for the i n d u c t i o n of h e p a t o c y t e p r o l i f e r a t i o n in the liver of intact rats (10). The first step factors (triggers) a f t e r PH m a y be the release of stress h o r m o n e s by the o p e r a t i o n (I in Fig. 2), and the i n c r e a s e of r e l a t i v e a m o u n t s of e n d o t o x i n (2 in Fig. 2) and a m m o n i a in the portal v e i n (4 in Fig. 2). The s e c o n d step m a y be combination of h e p a t o t r o p h i c h o r m o n e s (I-8)(5 in Fig. 2). It will be necessary in f u r t h e r s t u d i e s to experiment with the quiescent, d i f f e r e n t i a t e d h e p a t o c y t e s in s e r u m - f r e e cell c u l t u r e systems, because in v i v o e x p e r i m e n t s can i n v o l v e m a n y s e c o n d a r y effects.

Acknowlegment We

thank

Ms.

K. T a b e r

for r e a d i n g

the m a n u s c r i p t .

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