THE JOURNAL 0 1988 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Vol .263, NO. 15. Issue of May 25, pp. 7141-7146, 1988 Printed in U. S. A .

Induction of cul-Acid Glycoprotein by Recombinant Human Interleukin-1 in Rat Hepatoma Cells*

(Received for publication, August 26, 1987)

Thomas Geiger, Tilo Andus, Jan Klapproth, Hinnak Northoff$, and Peter C. HeinrichQ From the Bwchemisches Institut, Uniuersitat Freiburg, Hermann-Herder-Strasse 7, 0-7800 Freiburg and the SBlutspendezentrale des Deutschen Roten Kreuzes, Oberer Eselsberg, 0-7900 Ulm, Federal Republic of Germany

The induction of al-acid glycoprotein mRNA by re- combinant murine interleukin- 1, recombinant human interleukin- la , and recombinant human interleukin- 18 has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant hu- man interleukin- l a and recombinant murine interleu- kin-1 were almost identical, the concentrations of re- combinant human interleukin- 18 needed for half-max- imal induction of al-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in al-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-18. In parallel albumin mRNA levels decreased to about 30%. The al-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of >lo-’ M were needed, whereas concentrations of

The increase in al-acid glycoprotein mRNA after recombinant human interleukin-18 was followed by a 36-fold stimulation in al-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the in- duction of al-acid glycoprotein mRNA by recombinant human interleukin- 18 was impaired suggesting the in- volvement of a short-€ived protein in the induction of al-acid glycoprotein mRNA.

M were ineffective.

Tissue injury and inflammatory processes induce a host response commonly referred to as the acute-phase reaction (1). The acute-phase response is characterized by a series of complex local and systemic reactions. Among the latter, dras- tic changes in the serum levels of acute-phase proteins are observed. In the rat a,-macroglobulin, al-acid glycoprotein (aI-AGP),’ and cysteine proteinase inhibitor represent the major positive acute-phase reactants. Their serum levels are elevated between 10- and several hundred-fold during inflam- mation (reviewed in Refs. 1-3). The increase in serum levels is preceded by an increase in.the respective mRNA levels (4- 10). At the same time serum and mRNA levels of the negative

*This work was supported by grants from the Deutsche For- schungsgemeinschaft, Bonn, and the Fonds der Chemischen Indus- trie, Frankfurt. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 To whom reprint requests should be addressed Biochemisches Institut, Hermann-Herder-Str. 7, D-7800 Freiburg, F. R. G.

’ The abbreviations used are: a,-AGP, orl-acid glycoprotein; 11-1, interleukin-1; rh11-la, recombinant human interleukin-la; rh11-16,

terleukin-1. recombinant human interleukin-la; rm11-1, recombinant murine in-

acute-phase proteins albumin, transferrin, transthyretin, or al-inhibitor 3 decrease (11-14).

These changes are caused by the action of inflammatory mediators secreted by mononuclear phagocytes (15-19). Among the monokines 11-1 (for reviews see Refs. 20-22), tumor necrosis factor-a (23, 24) and hepatocyte-stimulating factor (18, 19, 25-29) have been found to be stimulants of acute-phase protein synthesis. For a long time 11-1 has been favored as the major mediator of acute-phase protein synthe- sis (20). Moreover, recombinant 11-1 has been available for a few years (30-34). Nevertheless, in only a few systems has acute-phase protein stimulation by 11-1 been demonstrated. Thus far however, recombinant 11-1 has not been shown to induce the full spectrum of acute-phase protein synthesis as found i n vivo during inflammation. Only a few of the acute- phase proteins investigated were inducible by 11-1 (23, 24,35, 36). Furthermore, the changes in acute-phase protein synthe- sis caused by 11-1 added to cell cultures were much weaker than those found i n vivo during the acute-phase response (23, 24, 35, 36). Hence, a suitable i n vitro system to study the action of 11-1 on the synthesis of acute-phase proteins has not been available.

In the present paper we describe that recombinant murine 11-1, and recombinant human 11-la and p change albumin, and al-AGP mRNA levels in a well-differentiated rat hepa- toma cell line to a similar extent as i n vivo during experimen- tal inflammation.

MATERIALS AND METHODS

Chemicals-~-[~~S]Methionine (>lo00 Ci/mmol) and deoxycyti- dine 5’-[a-32P]triphosphate (>3000 Ci/mmol) were purchased from Amersham Buchler (Braunschweig, Federal Republic of Germany (F. R. G.)). Cycloheximide, puromycin, and emetine were from Sigma (Munich, F. R. G.). RNase-free DNase I was from Pharmacia LKB Biotechnologies Inc. (Freiburg, F. R. G.). Recombinant murine inter- leukin-1 and recombinant human interleukin-la were generously supplied by Drs. P. Lomedico and A. Stern (Hoffmann-LaRoche, Nutley, NJ). Recombinant human interleukin-la was from Biogen (Geneva, Switzerland). Lipopolysaccharide from Salmonella abortus equi was a generous gift of Dr. C. Galanos, Max-Planck Institut fur Immunbiologie (Freiburg, F. R. G.). Albumin cDNA was kindly supplied by Dr. A. Alonso, DKFZ (Heidelberg, F. R. G.), a,-AGP cDNA by Dr. G. Schreiber (Melbourne, Australia), and the genomic clone of rRNA by Dr. I. Grummt (Wurzburg, F. R. G.). The Fao cell line established and characterized by Deschatrette and Weiss (37) was kindly supplied by Dr. F. Wiebel, Gesellschaft fur Strahlenfor- schung (Munich, F. R. G.).

Culture and Labeling of Fa0 Cells-Fao cells were grown in RPMI 1640 medium containing 5% fetal calf serum (Sigma), 100 mg/liter of streptomycin and 65 mg/liter of penicillin, and passaged twice a week by trypsinization. For most experiments the cells were plated on a 24-well dish (Falcon 3047) 2 days earlier. About 5 X lo6 cells/well were used for the cytoblots. For the radioactive labeling of a,-AGP 50 pCi of [35S]methionine were added to 2 X lo6 cells for 3 h.

Immunoprecipitation-Immunoprecipitations were carried out in

7141

7142 Induction of al-Acid Glycoprotein by Recombinant Interleukin-1

the presence of a monospecific anti-al-AGP as described in previous publications (10, 14, 29).

Isolation of RNA and Northern Analysis-Cells were lysed by guanidinium thiocyanate, and RNA was isolated according to the protocol of Chirgwin et al. (38). 15 pg of total RNA was separated on a 1% agarose, 6.6% formaldehyde gel prior to transfer to the gene screen filters (39).

Cytoblot and mRNA Hybridization-Cells (5 X 105/well) were lysed in 10 mM Tris-HC1 buffer, pH 7.0, containing 1 mM EDTA, and 1% Nonidet P-40, nuclei were removed by centrifugation, the cytoplasmic RNA was denatured in the presence of formaldehyde essentially as described by White and Bancroft (40), and RNA was blotted to a gene screen membrane using a Manifold dot-blot apparatus (Schleicher & Schull). After baking the filters at 80 "C for 2 h and prehybridization (39) for 6 h, the filters were hybridized (39) to 32P- labeled cDNA probes. The cDNAs were radioactively labeled using the random primer technique described by Feinberg and Vogelstein (41). Filters were washed 3 times in 0.1 X SSPE, 0.1% sodium dodecyl sulfate a t 55 "C for 30 min (20 X SSPE = 3.6 M sodium chloride, 0.2 M sodium phosphate, 0.02 M EDTA, pH 7.4) (39).

RESULTS

Recombinant 11-la and 11-1p and recombinant murine 11-1 were compared in respect to their capabilities to induce al- AGP mRNA in rat hepatoma cells (Fao). Prior to the addition to the hepatoma cell cultures the activities of the three 11-1 preparations had been determined in a thymocyte costimula- tory assay. It can be seen in Fig. lA that rhI1-la and P as well as rmI1-1 cause a dose-dependent stimulation of al-AGP mRNA. Whereas the stimulatory capacities of rhI1-la and rmI1-1 were almost identical, rhIl-lp was even more effective (filled triangles). Maximal induction of al-AGP mRNA by

alphal AG P

0 0.003 0.03 0.3 3 30 300 3000

rec IL- 1 (units /ml)

rhIl-lp was about 50% higher than the maximum achieved with rhI1-la or rm11-1. Furthermore, for half-maximal induc- tion rhII-lp is required at concentrations which were lower by three orders of magnitude than those of rhI1-la or rm11-1. For comparison we have analyzed the influence of the three interleukins on the negative acute-phase protein albumin mRNA levels (Fig. 1B). Dose-dependent decreases were found for rhI1-la or p as well as for rm11-1. Again, rhIl-lP had the strongest effect (filled triangles). Since the recombinant in- terleukins isolated from E. coli may be contaminated by lipopolysaccharide, it was necessary to exclude that lipopoly- saccharide exerts a stimulatory effect on the al-AGP mRNA induction. Lipopolysaccharide in concentrations ranging from 1 pg/ml to 10 pg/ml in the presence of M dexamethasone had no effect on al-AGP mRNA levels in Fao cells. Moreover, preincubation of 11-1 with the lipopolysaccharide inactivator polymyxin B (10 pg/ml) did not diminish the stimulatory effect of 11-1 (data not shown).

To study the al-AGP mRNA induction in greater detail, we selected rhI1-1P as inducing agent. Earlier studies revealed that acute-phase protein induction in the rat requires the permissive action of steroid hormones. We examined the effect of the synthetic glucocorticoid analog dexamethasone for the induction of al-AGP mRNA by rhI1-1P. Fig. 2 shows that the presence of dexamethasone is a prerequisite for al- AGP mRNA induction. A full stimulation was achieved with dexamethasone concentrations of M, whereas concen- trations of less than M were ineffective. The greatest influence on the stimulation of al-AGP mRNA synthesis was

ALBUMIN

B

la

- 1 ' " 0 0.003 0.03 0.3 3 30 300 3000

rec I L - 1 (units /ml)

FIG. 1. Induction of al-acid glycoprotein mRNA by recombinant human interleukin-la, recombinant human interleukin-la, and murine interleukin-1. Fao cells (5 X lo5 cells/well) were incubated with rhI1-la (O), rhI1-1B (A), and rmI1-1 (m) in concentrations indicated in the figure in the presence of M dexamethasone for 18 h. The various concentrations of 11-1 were obtained after dilution with RPMI 1640 medium containing 5% fetal calf serum. The cells were lysed, the cytoplasmic RNA extract blotted to a gene screen membrane, and hybridized with 32P-labeled a,-AGP cDNA (A) and albumin cDNA ( B ) . The radioactivity of each dot was determined in a liquid scintillation counter.

Induction of cr,-Acid Glycoprotein by Recombinant Interleukin-I 7143

1614 1012 lilo 0 lo6 10"

Der (M)

FIG. 2. Requirement of dexamethasone for the induction of al-acid glycoprotein mRNA by recombinant human interleu- kin-10. Fao cells (5 X IO5 cells/well) were incubated with 500 units/ ml of rhI1-10 in the presence of increasing concentrations of dexa- methasone for 18 h. The cells were lysed, the cytoplasmic RNA extract blotted to a gene screen membrane, and hybridized with :i2P- labeled al-AGP cDNA. The radioactivity of each dot was determined in a liquid scintillation counter.

0 5 10 15 20 25 30 3 5 time ( h )

FIG. 3. Time course of the induction of al-acid glycoprotein mRNA by recombinant human interleukin-10. Fao cells (5 X lo5 cells/well) were incubated in the presence of IO-' M dexametha- sone without (B) or with 500 units of rhII-l~/ml (0) for the times indicated in the figure. The cells were lysed, the cytoplasmic RNA extract blotted to a gene screen membrane, and hybridized with "P- labeled al-AGP cDNA. The radioactivity of each dot was determined in a liquid scintillation counter.

found between and 10"' M dexamethasone. To gain some information on the kinetics of al-AGP mRNA

induction by rh11-lB, Fao cells were exposed to rhI1-1B for varying lengths of time (Fig. 3). Although hardly visible in the figure, already 2 h after the addition of 500 units/ml an increase in al-AGP mRNA levels was observed when the radioactivity of the respective dot was quantitated. Maximal stimulation was found at 18 h followed by a rather steep decrease. By measuring the radioactivity of the dots, we found a 60-fold increase in al-AGP mRNA 18 h after rhIl-lB. Since it is known that aI-AGP is inducible by dexamethasone alone in rat hepatocytes (42), we measured, in a parallel experiment, the kinetics of al-AGP mRNA induction in the presence of lod6 M dexamethasone but without rhI1-18 (filled squares). I t

is evident that the sole addition of dexamethasone does not lead to the induction of al-AGP mRNA synthesis in Fao cells. Thus, Fao cells need the combined action of dexamethasone and rhIl-lB for al-AGP mRNA induction.

Since data obtained from dot-blots may sometimes contain unspecific hybridization signals, we studied the aI-AGP mRNA induction and the decrease in albumin mRNA at different times after the addition of rhIl-lB to Fao cells by Northern analysis. I t can be seen from Fig. 4 that aI-AGP mRNA with a size of about 850 bases increased and that albumin mRNA with a mobility corresponding to a length of about 2100 bases decreased in a time-dependent manner. There is a rather sharp decrease in albumin mRNA levels between 8 and 12 h. Since the kinetics of al-AGP mRNA induction does not show such a discontinuity and since the amounts of 28 S and 18 S rRNA were the same for the various time points, it is unlikely that the sharp decrease in albumin mRNA is due to a difference in RNA added to the gel. 20 h after rhIl-lB al-AGP mRNA levels were elevated about 56- fold over the basal level as determined by counting the radio- activity of the excised bands. This increase could either be the result of an increased synthesis, a decreased degradation of al-AGP mRNA or a combination of both processes.

T o examine whether the al-AGP mRNA induction requires ongoing protein synthesis, we have studied the effect of var- ious inhibitors of protein synthesis on their influence on al- AGP mRNA induction. When cycloheximide, puromycin, or emetine were added in increasing concentrations to the cul- ture media of Fao cells, total protein synthesis was blocked to about 95-98% (Fig. 5, A-C, filled circles). In parallel to the inhibition of protein synthesis, the induction of al-AGP mRNA was impaired (filled triangles). It is interesting that the al-AGP mRNA induction was more sensitive to low

28S-

3 albumin

. a,AGP

0 4 8 12 16

time [h] 20 24

FIG. 4. Northern analysis of mRNA levels for al-acid gly- coprotein and rat serum albumin at different times after addition of recombinant human interleukin-10. Fao cells (5 X 10" cells/dish) were stimulated with rhI1-16 (500 units/ml) in the presence of M dexamethasone. A t the times indicated total RNA was extracted as described under "Materials and Methods." For each time point 15 pg of total RNA were separated on a denaturing agarose gel and blotted to a gene screen transfer membrane. The RNA was hybridized to a mixture of "P-labeled rat serum albumin and a,-AGP cDNAs as detailed under "Materials and Methods."

7144 Induction of al-Acid Glycoprotein by Recombinant Interkukin-1

0 0 2 0 5 1 2 0 5 10 15 30 60 0 10 20 30

C H X (pg/ml) Pura ( p w n l ) Em (pg/ml I

FIG. 5. Effect of various inhibitors of protein synthesis on the induction of al-acid glycoprotein mRNA by recombinant human interleukin-18. Fao cells (5 X 10' cells/well) were preincubated with cyclo- heximide ( A ) , puromycin ( B ) , and emetine ( C ) in the concentrations indicated in the figure for 1 h, and 500 units of rhIl-lp/ml were then added. 1O"j M dexamethasone was present during the preincubation and the incubation period. After 18 h the cells were lysed, the cytoplasmic RNA extract blotted to a gene screen membrane and hybridized with 32P-labeled a,-AGP cDNA. The radioactivity of each dot was determined in a liquid scintillation counter. Inhibition of protein synthesis was determined in a parallel experiment by measuring the incorporation of 10 pCi of [35S]methionine/5 X lo6 cells into trichloroacetic acid-precipitable material during a 3-h labeling period in methionine-free-medium.

-1

0 5 10 15 2 0 25

Time (h)

FIG. 6. crl-Acid glycoprotein synthesis and secretion at dif- ferent times after rhI1-18 treatment of Fao cells. Fao cells (2 X

sone with 500 units of rhIl-lp/ml for the times indicated in the figure. lo6 cells/well) were incubated in the presence of M dexametha-

3 h prior to the a,-AGP determinations, the medium was changed, and the cells were labeled with 50 pCi of [33S]methionine in the presence of 500 units of rhIl-lfl/ml. The absolute amounts of the secreted a,-AGP were determined by a radioimmunoassay. Increasing amounts of unlabeled al-AGP were mixed with 100 pl of medium containing the [35S]methionine-labeled a,-AGP and allowed to com- pete for binding to a limited amount of a monospecific antiserum against a,-AGP.

inhibitor concentrations than total protein synthesis. Thus, the induction of al-AGP mRNA by rhI1-18 requires ongoing protein synthesis.

Since relative measurements of al-AGP mRNA levels may

be deceiving, we have quantified the amounts of al-AGP secreted at different times after the addition of rhIl-18 to Fao cells. Fig. 6 shows that uninduced Fao cells synthesized and secreted 60.8 ng, whereas 21 h after rhI1-1P 29 ng of al-AGP/ lo6 cells/h (about 36-fold increase) were secreted. I t should be noted that rhI1-1B led to a 2-3-fold stimulation of secretion of total protein into the medium.

DISCUSSION

The comparison of rhI1-la, rh11-18, and rmI1-1 has shown that a,-AGP mRNA is induced by all three species. However, rhIl-lp exhibited a much stronger stimulatory effect than rh11-la or rm11-1. This observation correlates with the fact that mI1-1 and hI1-la have the same isoelectric point of about 5, whereas hI1-18 has an isoelectric point of 7.0 (22). Further- more, the amino acid sequence homologies between hI1-la and mI1-1 are 62%, between hI1-la and hIl-1fl only 26%, and between hI1-18 and mI1-1 only 30% (32). Nevertheless, the difference in the activities of the three 11-1 preparations is surprising, since until now differences in receptor binding could not be observed for 11-la and Il-lP in T lymphocytes (43), fibroblasts (44-46), and EBV-transformed B-iympho- cytes (47). Possible explanations for the different inducing capabilities of the three 11-1 preparations tested in Fao cells could be either different affinities to the same receptor or even the existence of two different receptors.

We estimated half-maximal stimulation of al-AGP mRNA in Fao cells at rhI1-18 concentrations of about 0.6 pM, reflect- ing a specific interaction of rhI1-18 with rat hepatoma cells. Concentrations of human recombinant 11-16 of 0.05-34 phf and 2-50 PM have been determined for T lymphocytes and fibroblasts, respectively (44,45,47-51).

In the present paper we demonstrate that 11-1 induced mRNA for al-AGP in rat hepatoma cells to an extent com- parable to the one found in vivo during inflammation (8, 13). 11-1 was reported to induce several acute-phase proteins in rat hepatocyte primary cultures. Previously, we described a %fold

Induction of crl-Acid Glycoprotein by Recombinant Interleukin-1 7145

stimulation of a,-macroglobulin synthesis by murine recom- binant 11-1 (19). Ramadori et al. (52) described the induction of serum amyloid A and factor B by murine recombinant 11-1 in vivo and in hepatocyte primary cultures from mice. In- creases in the synthesis of a,-antichymotrypsin, factor B and C3, but not C2, C4, and al-proteinase inhibitor were observed in Hep3B cells upon addition of human recombinant 11-1 (24). Using HepG2 cells, Karin et al. (53) induced metallothionein mRNA by recombinant murine 11-1. As in the case of tumor necrosis factor a, Darlington et al. (23) reported that among several acute-phase proteins tested in Hep3B2 cells mouse recombinant 11-1 induced only the synthesis of C3. In rat hepatocytes Fuller et al. (27) did not observe any stimulation of fibrinogen synthesis by murine recombinant 11-1.

From the fact that some acute-phase proteins are inducible by 11-1, whereas others are not, it is evident that 11-1 functions as a mediator in acute-phase protein induction but not as a universal one. Recent work by Baumann et al. (54) has clearly shown that 11-1 induces only a subset of acute-phase proteins, namely C3, haptoglobin, and al-AGP, whereas another subset of acute-phase proteins is regulated by hepatocyte-stimulating factor.

It is of interest to note that in the Fao cells used in this study al-AGP mRNA induction required the combined action of Il-lp and dexamethasone. This is in contrast to the data obtained in other systems, i.e. in rat hepatocyte primary cultures (42), in HTC rat hepatoma cells (55), in L-cells transfected with the a,-AGP gene (56) or in rats in vivo (57) where the sole addition of dexamethasone led to a stimulation of a,-AGP.mRNA synthesis. On the other hand, in the cases of human cell lines such as HepG2 or Hep3B (23,24) as well as in mouse hepatocytes or in mice (52) acute-phase protein induction by Il-lp was achieved without addition of glucocor- ticoids.

Using the combination of 11-la and dexamethasone a 60- fold increase in cytoplasmic mRNA levels of a,-AGP was observed. This increase could not be accounted for by the measurements of transcription rates, where essentially no increases were found (data not shown). Thus, the increase in al-AGP mRNA concentrations must be due to post-transcrip- tional mechanisms, such as stabilization of the pre-mRNA for al-AGP, facilitated nucleocytoplasmic transport, or in- crease in the half-life of cytoplasmic a,-AGP mRNA. We like to favor the first mechanisms, since nuclei from unstimulated cells show high transcription rates, but at the same time no cytoplasmic al-AGP mRNA can be detected. When al-AGP mRNA degradation was measured after blocking RNA syn- thesis by actinomycin D, we did not find any indication for a difference in the degradation rates of al-AGP mRNA in stimulated and unstimulated Fao cells (data not shown). Thus, the elevated al-AGP mRNA levels after stimulation of Fao cells with Il-lp are not due to a reduced degradation. On the other hand, reduction in degradation of mRNA has been described to play a role during the stimulation of several proteins, such as ovalbumin and conalbumin (58), serum amyloid A (59), malic enzyme (60, and insulin (61). Our data are similar to the findings of Vannice et al. (62) who observed essentially no increase in transcription rates in HTC cells after dexamethasone, whereas cytoplasmic RNA increased about 100-fold (55). In contrast to these data Kulkarni et al. (57) observed a 30-40-fold stimulation in the al-AGP tran- scription rate in normal and in adrenalectomized rats after the administration of dexamethasone.

We have demonstrated that the induction of al-AGP mRNA by rhI1-10 is prevented when protein synthesis is inhibited. This finding is an indication 'for the involvement

of a short-lived protein in a,-AGP mRNA induction. Vannice et al. (55), Reinke and Feigelson (56), and Klein et al. (63) have also described the requirement of ongoing protein syn- thesis for the induction of al-AGP mRNA. a,-Uteroglobulin (64), tryptophan oxigenase (65), ovalbumin, conalbumin (66), a-fetoprotein (67) represent further examples, where protein synthesis is needed for induction. On the other hand, the stimulation of fibrinogen and a,-antichymotrypsin by condi- tioned media from human monocytes was unaffected by in- hibitors of protein synthesis (27, 28). Interestingly, various systems have been described, where inhibition of protein synthesis has a stimulatory effect on mRNA synthesis. Prob- ably due to the loss of short-lived repressors, tyrosine ami- notransferase (68), tumor necrosis factor-a and 11-1 (69), interferon-pl (70), and interferon-p2 (71) are well-docu- mented examples for this kind of regulation.

In conclusion, in the present paper a well-defined in vitro system suitable for the study of acute-phase induction of 11- l p has been presented.

Acknowledgments-We are indebted to Dr. F. Wiebel (Munich) for supplying us with the Fao cells. We also thank M. David for excellent technical assistance and H. Gottschalk for her help with the prepa- ration of this manuscript.

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