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Pages 1049-1055

1991

EFFECT OF INTERLEUKIN 6 ON PHENOBARBITAL INDUCTION CYTOCHROME P-450IIB IN CULTURED RAT HEPATOCYTES Joseph F. Williams ‘+ , William

J. Bement’,

OF

Jacqueline F. Sinclair”**

and Peter R. Sinclair’** ‘VA Hospital,

White River Junction, VT 05009

**Department

of Biochemistry,

Dartmouth

+Department

of Pharmacology

and Therapeutics, University of South Florida, Tampa, FL 33612

Received

June

14,

Medical School, Hanover, NH 03756

1991

Human recombinant interleukin 6 (rhIL-6) caused a dose dependent decrease in the phenobarbital induction of benzyloxyresorufin 0-deethylase activity in cultured rat hepatocytes. Decreased enzymatic activity was associated with a decrease in the amount of immunoreactive P-450IIB1/2. rhIL-6 also prevented the PB-induced increase in the steady state level of P-45011B mRNA. These results suggest that altered P-450 levels observed in vivo during the acute phase reaction may be due to interleukin 6. 0 1991 Academic Pres*, Inc. Interleukin

6 (IL-6) is the major cytokine that alters the hepatic production

plasma proteins and intracellular inflammation,

enzymes during the acute phase response to

infection and other noxious stimuli (1). Compared to IL-6, other cytokines

released during the acute phase response, such as interleukin

I (IL-l),

tumor necrosis

factor (TNF), and gamma interferon, alter the synthesis of only a limited acute phase proteins.

alpha, macroglobulin,

In contrast, IL-6 decreases the synthesis of albumin, Endotoxin

spectrum of the

In rodents and in rat hepatocyte cultures, IL-6 increases the

synthesis of alpha, acid glycoprotein,

administered

of

fibrinogen,

and hemopexin.

and transferrin (see 1 for review).

and other agents that evoke the acute phase reaction when

to rodents also cause marked decreases in the level of total hepatic

+To whom reprints should be addressed at Department of Pharmacology and Therapeutics, College of Medicine, University of South Florida, Tampa, Fl. 33612. Abbreviations: rh, recombinant human; IL-l and IL6, interleukin 1 and 6; TNF, tumor necrosis factor; P-450, cytochrome P-450; P-45OIIBl (also known as P-450b) and P45OIIB2 (also known as P-450e); PB, phenobarbital, BZROD, benzylo~esorufin Odeethylase; 3MC, 3-methylcholanthrene.

1049

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cytochrome P-450 (P-450) and the associated monooxygenase activities (2-4). Administration

of the cytokines IL-l, TNF, and interferon to rodents also decreases total

P-450 (2-11). Recent studies indicate that endotoxin, IL-l,

and interferon

most likely

decrease the synthesis of P-450 by decreasing the level of P-450 mRNA (12-15). Although some P-450 forms present in control, phenobarbital cholanthrene

(PB) or 3-methyl-

(3-MC) induced animals are decreased, other forms are increased or

unaffected (16-18). These results suggest that P-450, like the acute phase proteins, may be transcriptionally

regulated by the mediators of the acute phase response. However, in

whole animal studies hepatic effects may result from several cytokines and/or other hormones released during the acute phase reaction or by the administered cytokines. Rat hepatocyte cultures offer a more defined system than whole animals to study the direct effect of agents to regulate hepatocyte P-450 expression and activity. only recently have rat hepatocyte cultures been shown to be inducible P-450IIBl

and P-450IIB2

However,

by PB for

to levels equivalent to those induced by PB in vivo (19-21).

The present study utilized such rat hepatocyte culture system to examine the effect of IL-6 on the PB induction of P-450IIB. MATERIALS

AND METHODS

Preoaration of Heuatocvtes: Hepatocytes were prepared from male Fischer 344 rats and plated on Matrigel-coated culture plates as previously described (20). Cell yield was typically in excess of 2.5 x 10’ cells/liver, and viability above 85%, as determined by trypan blue exclusion. Cultures were maintained at 37” in an atmosphere of 5% CO, in air, and the culture medium was changed every 24 hr. Chemicals were added to cultures at 72 hr or 96 hr after inoculation. PB, sodium salt (Baker Chemical Co, Phillipsburg, NJ) was dissolved in water. Recombinant human IL-6 (1 x lo6 U/mg), kindly provided by Dr. G. Wong, Genetics Institute, Cambridge, MA., was diluted with Williams E medium. Assays: Benzyloxyresorufin 0-deethylase (BZROD) activity was determined in sonicate of cells harvested in 20 mM Tris buffer, pH 7.8 essentially as described (20). Hemopexin in the culture medium was determined by titration of its heme-binding capacity with a heme-albumin solution, and the concentration of hemopexin calculated from the increased absorption at 414 nm, using an E,, of 19.2 (22). Protein was determined by the method of Lowry ti. (23) using bovine serum albumin as standard. To correct for Matrigel protein, Matrigel-coated plates not inoculated with hepatocytes were processed identically as the cultured cells. Immunoblots: Sonicate proteins were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted as previously described (20). A monoclonal antibody reacting with both P-45OIIBl and P-450IIB2 (P-450IIB1/2) was a gift from Dr. M. Adesnick, NYU Medical Center, New York, NY. Solution Hvbridization: Total RNA was extracted from hepatocytes as previously described (24), and purified by CsCl, gradient centrifugation. Solution hybridization was performed (24) using a [“PI-labeled synthetic oligonucleotide DNA complementary to a DNA sequence (ACTGCGGTCATCAAGGGTTGGTAG) in exon seven of P-45OIIBl. This sequence differs from the sequence in the corresponding region of the closely related P-450IIB2 (25). 1050

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RESULTS Fig. 1 shows the effect of rhIL-6 on the PB-mediated activity of cultured rat hepatocytes.

induction

PB (2 mM) caused a marked increase in BZROD

activity in agreement with previous reports (20). rhIL-6 (50 U/ml) suppressed the PB-induced

increase in BZROD

induction of BZROD

activity.

by 0.05 U/ml

and essentially complete inhibition

shows an immunoblot microsomal

completely

activity.

Fig. 2A shows the effects of increasing concentrations rhIG6,

of BZROD

of rhIL-6 on the PB

The PB induction of BZROD

activity was decreased 42%

was seen at 50 U/ml.

of the hepatocyte sonicates and, for comparison, a hepatic

sample from a PB-treated rat. The microsomal

sample from the PB-treated

rat (lane 9) shows three bands. The upper two correspond to P-450IIBl The lower molecular

Fig. 2B

weight protein is an unidentified

and P-450IIB2.

protein (possibly P-45Of) with

which the antibody also cross-reacts (26); this protein was not affected by either PB or rhIL-6.

Compared to untreated hepatocytes (lane l), incubation

increased the amount of both P-450IIBl

and IIB2 (lane 2). The presence of rhIL-6

(lanes 3-8) caused a decrease in the PB induction

of both P-450IIBl

essentially complete suppression observed at 50 U IG6/ml U/ml)

caused no decrease in a 90 min incorporation

precipitable

of hepatocytes with PB and P-450IIB2,

(lanes 3 and 8).

rhIL-6 (50

of [14C]-leucine into TCA-

cellular proteins in PB-treated cells (results not shown), indicating

rhIL-6 does not cause a general inhibition

that

of protein synthesis. Fig. 3 shows the effect of

I-

1

CNTRL

with

PB

1

IL-6

PB

+

IL-6

Fig. 1. Effect of rhIM on PB induction of BZROD activity in cultured rat hepatocytes. PB (2mM) and rhIG6 (50 U/ml) were added to hepatocytes (two plates/treatment) at 72 hr after inoculation, and cells were harvested 24 hr later. BZROD activity was determined in duplicate in cell sonicate from individual plates. Values are mean and S.D. of determinations from three experiments. 1051

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A loo c 5 F Y

80

IL-6

1

(UNITS/ML)

2

3

4

5

6

7

8

Fig. 2. Effects of varying concentrations of IL-6 on PB induction of P-450IIB1/2. A. BZROD activity. At 72 hr after inoculation, hepatocytes were treated (two plates/treatment) with PB (2mM) and rhIL-6 (0.0550 U/ml), and cells were harvested 24 hr later. BZROD activity was determined as indicated in Fig. 1. Values are expressed as percent of PB induced BZROD activity and are the mean and range of the duplicate treatments. Control and PB-induced BZROD activity were 3.5 and 109.2 pmoles/mg protein/min, respectively. B. Western blot of P-450IIB1/2. Lanes l-8 contained 20 pg of sonicate protein/lane (Lane 1, control; lane 2, PB (2mM); lanes 3-8: PB and 50, 2.5, 0.5, 0.25, 0.05, and 50 U IG6/ml, respectively). Lane 9 contained 0.33 pg hepatic microsomal protein from a PB-induced rat.

rhIG6

on the PB-induced

expression of P-450IIBl

7-fold increase in the steady-state level of P-450IIBl

mRNA.

Within 5 hr, PB caused a

mRNA,

and rhIL-6 (50 U/ml)

essentially blocked this effect. 60

1

CNTRL

PB

ElPB+IL-6

Fig. 3. Effect of rhIL-6 on the PB induced expression of P-450IIBl mRNA. PB (2mM) and rhIL6 (50 U/ml) were added to hepatocytes (2 plates/ treatment) at 96 hr after inoculation, and cells were harvested 5 hr later. Total RNA was extracted and mRNA for P-450IIBl in the duplicate samples was determined by solution hybridization. Values are mean and range of duplicate samples. 1052

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The synthesis of the acute phase protein hemopexin in rat hepatocyte cultures has been reported to be increased by rhIL-6 (27). Therefore, the effect of rhIL6 hemopexin was determined rhIL-6.

as an independent

assay of the hepatocellular

In two separate experiments, rhIL-6 (50 U/ml)

hemopexin

accumulation

in the medium

in medium

from untreated cells (4.2 and 5.9 pg/ml).

to increase

effect of

increased the amount of

(10.5 and 10.6 pg/ml)

compared to the amount

DISCUSSION In this study with cultured rat hepatocytes we have shown that IG6, the major cytokine responsible for inducing hepatic acute phase proteins, prevents the PB induction of P-45011B as indicated by decreased BZROD proteins, and P-450IIBl

mRNA.

dependent on the concentration

activity, immunoreactive

The decrease in PB-induced BZROD of rhIL-6.

of P-450IIBl

activity was

The decreased enzymatic activity most likely

reflects the effect of rhIL-6 to prevent induction by altering the accumulation

P-450IIB1/2

of immunoreactive

mRNA.

Preliminary

that rhIL-6 did not affect the stability of the mRNA

P-450IIB1/2

experiments indicated

(unpublished

observations),

suggesting that the observed decrease is probably an effect on transcription. concentrations

of rhIL-6 needed to alter P-450IIB1/2

proteins

The

expression are similar to those

shown to affect acute phase protein synthesis in cultured rat or mouse hepatocytes (2830). A concentration and immunoreactive

of 50 U IL6/ml

the PB-induction

of P-450IIBl

mRNA

protein by > 80% yet increased hemopexin concentration.

increase in hemopexin production, incorporation

inhibited

The

and the lack of effect of rhIL-6 on [‘4C]-leucine

into cellular protein indicate that the effect on P-450 induction was not a

toxic response. It is suggested that IL6

may negatively regulate these P-450 forms by a

mechanism similar to that involved in the regulation

of the acute phase proteins.

This

mechanism may involve the same IL-6 responsive element in the regulatory region of the P-450 genes that is present in the genes of the acute phase proteins (1). The results suggest that the decrease in P-450 and the associated monooxygenase activity seen in vivo during the acute phase reaction may be caused by released IL-6. Whether other cytokines may have a similar direct effect on P-450 regulation

is not

known. The results also indicate that cultured rat hepatocytes can be used to directly study the mechanism of the effects of IL-6 and other cytokines to regulate different forms of P-450. Preliminary

results (unpublished)

ethoxyresorufin

activity in cultured rat hepatocytes is not inhibited

concentrations

0-deethylase

of IL-6 that inhibited

indicate that the 3-MC induction of

the PB-induction

of BZROD.

by

Thus, IL-6 may

regulate the expression of some, but not all forms of P-450. These results are consistent 1053

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effects of endotoxin administration

on

of P-450s.

Cytokines interact in an additive, synergistic, or antagonistic manner to regulate acute phase protein synthesis (31). The hepatocyte culture system will be useful to examine whether similar interactions between cytokines exist in the regulation The regulation

of P-450 by the various cytokines may be clinically important

understanding

the altered pharmacokinetics

of P-450s. in

of drugs during infection, inflammation,

other conditions of altered immune function as well as when the recombinant

and

cytokines

are used as therapeutic modalities. ACKNOWLEDGMENTS: This work was supported by funds from the Dept of Veterans Affairs, NIH Grant CA 25012, and the R. J. Reynolds Tobacco Co. We thank H. Walton, and N. Gorman for assistance in preparation of the Western blots, and Dr. S. Shedlofsky, V. A. Hospital, Lexington, KY for reading the manuscript. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Heimich, P.C., C&tell, J.V. and T. Andus. (1990) Biochem. J. 262,621-636. Renton, K. W. (1983) In Biological Basis of Detoxification. (J. Caldwell and W. B. Jakoby, Eds.) pp. 307-324. Academic Press, New York, NY. Williams, J.F. and A. Szentivanyi. (1985) In The Reticuloendothelial System (J.W. Hadden and A. Szentivanyi, Eds.). Vol. VIII, pp. l-25. Plenum Press, New York, N.Y. Williams, J.F. (1990) In Immunopharmacological Reviews (J.W. Hadden and A. Szentivanyi, Eds.). Vol. I, pp. 65-87. Plenum Press, New York, NY. Ghezzi, P., Saccardo, B., Villa, P., Rossi, V., Bianchi, M. and C.A. Dinarello. (1986) Infect. Immunol. 54,837-840. Peterson, T.C. and K.W. Renton. (1986) Immunopharmacol. 11,21-28. Shedlofsky, S.I., Swim, A.T., Robinson, J.M., Gallichio, V.S., Cohen, D.A. and C.J. McClain. (1987) Life Sci. 402331-2336. Bertini, R., Bianchi, M., Villa, P. and P. Ghezzi. (1988) Int. J. Immunopharmacol. 10,525-530. Craig, PI., Williams, S.J., Cantrill, E. and G. C. Farrell. (1989) Gastroenterology 97,999-1004. Moochhala, S.M., Renton, KW. and N. Stebbing. (1989) Biochem. Pharmacol. 38,439-447. Pous, C., Giroud, J.-P., Damais, C., Raichvarg, D. and L. Chauvelot-Moachon. (1990) Drug Metab. Disp. 18,467-470. Morgan, E.T. (1989) Molec. Pharmacol. 36,699-707. Morgan, E.T. and C.A. Norman. (1990) Drug Metab. Disp. 18649-653. Renton, K.W. and L.C. Knickle. (1990) Can. J. Physiol. Pharmacol. 68, 777-781. Wright, K. and E.T. Morgan. (1990) FEBS Letters 271,59-61. Stanley, L.A., Adams, D.J., Lindsay, R., Meehan, R.R., Liao, W. and C.R. Wolf. (1988) Eur. J. Biochem. 174:31-36. Coto, J.A. and J.F. Williams. (1989) Int. J. Immunopharmacol. 11,623-628. Craig, PI., Mehta, I., Murray, M., McDonald, D., Astrom, S., van der Meide, P.H. and G.C. Farrell. (1990)‘MoIec. Pharmacol. 38,313-318. Schuetz, E.G., Li, D., Omiecinski, C.J., Muller-Eberhard, U., Kleinman, H.K., Elswick, B. and P.S. Guzelian. (1988) J. Cell. Physiol. 134:309-323. 1054

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1055

Effect of interleukin 6 on phenobarbital induction of cytochrome P-450IIB in cultured rat hepatocytes.

Human recombinant interleukin 6 (rhIL-6) caused a dose dependent decrease in the phenobarbital induction of benzyloxyresorufin O-deethylase activity i...
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