A cytokine-selective defect in interleukin-1 beta-mediated acute phase gene expression in a subclone of the human hepatoma cell line (HEPG2).

Several well-differentiated human hepatoma cell lines (HepG2, Hep3B) have been used to identify factors which regulate hepatic gene expression during the host response to inflammation/tissue injury (acute phase response). Studies in these cell lines, as well as in primary cultures of rat, rabbit, and mouse hepatocytes, have demonstrated that interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interferon-beta 2 (IFN-beta 2) each mediate changes in expression of several hepatic acute phase genes. In this study we identify a subclone of the HepG2 cell line in which there is a selective defect in IL-1 beta-mediated acute phase gene expression. Recombinant human IL-1 beta mediates an increase in synthesis of the positive acute phase complement protein factor B and a decrease in synthesis of negative acute phase protein albumin in the parent uncloned HepG2 cell line (HG2Y), but not in the subclone HG2N. Recombinant human IFN-beta 2 and TNF-alpha, however, regulate acute phase protein synthesis in the subclone HG2N; i.e. IFN-beta 2 and TNF-alpha increase synthesis of factor B and decrease synthesis of albumin in both HG2Y and HG2N cells. Equilibrium binding analysis with 125I-rIL-1 beta at 4 degrees C showed that both HG2N and HG2Y cells bind IL-1 beta specifically and saturably. HG2N and HG2Y possess 3.8 and 4.0 x 10(3) plasma membrane receptors/cell with affinities of 0.96 and 1.07 x 10(-9) M, respectively. Thus, the defect in this subclone of the HepG2 cell line is likely to involve the signal transduction pathway for the biological activity of IL-1 beta and will be useful in elucidation of this signal transduction pathway.

Several well-differentiated human hepatoma cell lines (HepGZ, Hep3B) have been used to identify factors which regulate hepatic gene expression during the host response to inflammation/tissue injury (acute phase response). Studies in these cell lines, as well as in primary cultures of rat, rabbit, and mouse hepatocytes, have demonstrated that interleukin-18 (IL-lb), tumor necrosis factor (TNF-a), and interferon-82 (IFN-82) each mediate changes in expression of several hepatic acute phase genes. In this study we identify a subclone of the HepGZ cell line in which there is a selective defect in IL-18-mediated acute phase gene expression. Recombinant human IL-18 mediates an increase in synthesis of the positive acute phase complement protein factor B and a decrease in synthesis of negative acute phase protein albumin in the parent uncloned HepGZ cell line (HGZY), but not in the subclone HGZN. Recombinant human IFN-82 and TNF-a, however, regulate acute phase protein synthesis in the subclone HGZN; i.e. IFN-BZ and TNF-a increase synthesis of factor B and decrease synthesis of albumin in both HGZY and HGZN cells. Equilibrium binding analysis with '261-rIL-1/3 at 4 "C showed that both HGZN and HGZY cells bind IL-lD specifically and saturably. HGZN and HGZY possess 3.8 and 4.0 X lo3 plasma membrane receptors/cell with affinities of 0.96 and 1.07 X lo-' M, respectively. Thus, the defect in this subclone of the HepGZ cell line is likely to involve the signal transduction pathway for the biological activity of IL-18 and will be useful in elucidation of this signal transduction pathway.
The host response to inflammation or tissue injury is characterized by a coordinated series of metabolic reactions that * This work was supported in part by an American Gastroenterological Association/Industry (Merck Sharp and Dohme) Research Scholar Award, an RJR Nabisco Research Scholar Award, an American Heart Association Established Investigator Award, the Monsanto Corp., and United States Public Health Service Grants HL-37784, AI24836, and AI24739. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Supported by United States Public Health Service Grant AI-16262, a contract from the National Foundation for Cancer Research, and an Established Investigator Award from the American Heart Association.
5 Clinical Investigator of the National Heart, Lung, and Blood Institutes.
constitute the acute phase response. These reactions include fever, muscle proteolysis, leukocytosis, alteration in fat, carbohydrate and trace mineral metabolism, and marked changes in plasma concentrations of many liver-derived glycoproteins known as the acute phase plasma proteins, or acute phase reactants. Plasma concentrations of some acute phase reactants increase (C-reactive proteins, serum amyloid A, fibrinogen, al-antitrypsin, complement proteins factor B and C3) while others decrease (albumin, transferrin). The changes in absolute concentration are primarily due to changes in synthetic rate, rather than in rate of catabolism. Thus, this acute phase response involves a sequential and orderly activation of multiple genes.
Due to limitations in the availability and long-term cultivation of human hepatocytes, and ethical limitations in studies of humans in vivo, it has been difficult to specifically study the response of human liver during acute inflammation. However, the acute phase response has been recently studied in human hepatoma cell lines (HepG2, Hep3B) using well-defined and highly purified mediators. These studies show that changes in expression of hepatic genes characteristic of the acute phase response may be elicited in these hepatoma cell lines by recombinant human IL-I@,' (1-3), TNF-a, (2, 3) IFN-P2 (4-7), and IFN-7 (8). IL-1P, TNF-a, IFN-P2 also mediate hepatic acute phase gene expression in primary hepatocyte cultures from mouse and rat (5,9,10). Several hepatic acute phase genes are selectively affected by one of these cytokines; e.g. fibrinogen and al-antitrypsin are only affected by IFN-P2 (2)(3)(4)7). Other hepatic acute phase genes are affected by different cytokines; e.g. albumin expression decreases in the presence of IL-lp, TNF-a, or IFN-P2. It is now known that IL-lp, TNF-a, and IFN-P2 each bind to unique cell surface receptors (11)(12)(13)(14). Thus, the possibility that overlapping signal transduction mechanisms are triggered by these cytokines, once engaged by receptor, needs to be examined. In this study we identify a subclone of one of the human hepatoma cell lines which is defective in the signal transduction pathway for IL-lP, but not for IFN42, TNF-a, of IFN-7. This subclone will therefore facilitate further delineation of signal transduction mechanisms for hepatic acute phase gene expression.   (16), were maintained in culture as previously described (17). The a16 subclone of HepG2 was generated from the parent cell line independently by limiting dilution (18). It has been maintained in culture for 7 years. The a16 subclone is referred to in this paper as HG2N to distinguish it from the parent uncloned cell line HG2Y.
Detailed chromosome analysis was made on the parent HepG2 cell line (19) and on the HG2N subclone in 1982 by Dr. Barbara B. Knowles and during these studies by Dr. Michael Watson (Washington University School of Medicine, St. Louis, MO). Techniques for these analyses have been described (19). These analyses show that there are no discernible karyotypic differences between the parent and subclone cell lines. The parent HepG2 cell line is characterized by trisomy or tetrasomy of chromosome 2. The only difference in subclone HG2N is that a higher proportion of cells exhibit trisomy of chromosome 2, leading to a median number of chromosomes of 52 as compared to 54 in the parent cell line. The karotypes of the parent and subclone cell lines have not changed over 7 years.
Metabolic Labeling-Confluent monolayers were rinsed and incubated at 37 "C in the presence of methionine-free medium containing ["S]methionine, 250 @Ci/ml (pulse period). To determine the net synthesis of factor B or albumin, cells were subjected to a short pulse interval (20 min), and radiolabeled proteins were detected in the cell lysate alone. Methods for solubilization of cells and clarification of cell lysates after labeling have been described (17). Total protein synthesis was estimated by trichloroacetic acid precipitation of aliquots of cell lysates (20).
Detection of RNA by RNA Blot Analysis-Total cellular RNA was isolated from adherent monolayers of hepatoma cells by guanidine isothiocyanate extraction and ethanol precipitation (22). RNA was subjected to agarose-formaldehyde gel electrophoresis and transferred to nitrocellulose filters (23). Filters were then hybridized with 32Plabeled cDNA specific for human factor B (24) or albumin (25).
Determination of Cell Surface Receptor Binding-Recombinant IL-10 was iodinated with the Bolton-Hunter reagent. The specific radioactivity of various preparations was 4000-6000 cpm/ng IL-1P. For binding studies, HepG2 cells were washed with phosphate-buffered saline and incubated at 4 "C for 2 h with 12sI-IL-lP diluted in DMEM containing 10 mM Hepes and 0.1 mg/ml cytochrome c. The cells were then rinsed in phosphate-buffered saline at 4 "C and cell-associated radioactivity determined in 1 N NaOH homogenates. Specific binding of iodinated ligand was defined as the difference between total and nonspecific binding. Nonspecific binding was determined by the addition of a 200-fold excess of unlabeled ligand. Scatchard plot analysis of equilibrium binding data was performed as previously described (26,27). Cells were again rinsed and then homogenized. Cell lysates were clarified and subjected to immunoprecipitation with goat anti-human factor B. Immunoprecipitates were subjected to 7.5% SDS-PAGE followed by fluorography. There were no significant differences in the trichloroacetic acid-precipitable radiolabeled proteins under these conditions. Molecular mass markers are indicated at the right. Clarified cell lysates were subjected to sequential immunoprecipitation with goat anti-human factor B and then goat anti-human albumin. Immunoprecipitates were subjected to SDS-PAGE followed by fluorography. Molecular mass markers are indicated at the right.

IL-lP and IFN-P2 Have Independent Regulatory Effects on
2). Each cytokine mediates a concentration-dependent increase in factor B synthesis in HG2Y cells (Fig. 2a, leftpanel). IFN-P2 also mediates a concentration-dependent increase in factor B synthesis in HG2N cells, but IL-1P has no effect on factor B in these cells (Fig. 2a, right panel). IL-1P and IFN-P2 each mediate a decrease in albumin synthesis in HG2Y cells (Fig. 2b, left panel). IFN-P2 also decreases albumin synthesis in HG2N cells but IL-lP is completely defective in down-regulation of albumin synthesis in this subclone (Fig.  2b, right panel).
The difference in response to IL-lP is not due to differences in kinetics as shown in the following experiment. HG2Y and HG2N cells were separately incubated with IL-lP (100 ng/ ml) for 2, 5, 8, 16, and 24 h. Synthesis of factor B in HG2Y cells increases within 2 h, continues to increase from 8 to 16 h, and reaches a plateau between 16 and 24 h. Synthesis of factor B does not increase in HG2N cells at any interval up to 24 h (data not shown).
Although data in Fig. 2a shows a greater effect for IFN-P2 in HG2N cells than in HG2Y cells, data from five separate experiments in each cell line indicate that there is no significant difference in the effect of IFN-P2 on factor B synthesis in these cell lines. Densitometric scanning of fluorograms from these experiments demonstrate increases in factor B synthesis of 3.09 f 0.98-fold in HG2Y cells and of 2.93 f 0.99-fold in HG2N cells.
Similar results are observed in RNA blot analysis (Fig. 3). Steady-state levels of factor B mRNA increase in HG2Y cells incubated with IL-lP or IFN-P2 and in HG2N cells incubated with IFN-P2. However, there is no change in factor B mRNA levels in HG2N cells incubated with IL-1P.
TNF-a, another cytokine with positive regulatory effects on factor B synthesis and negative regulatory effects on albumin synthesis in uncloned HepG2 cells (3), is effective in regulating gene expression in HG2N cells. Recombinant TNF-a mediates a concentration-dependent decrease in synthesis of albumin (Fig. 4a) and an increase in synthesis of factor B (data not shown) in HG2N cells. Furthermore, the effect of IFN-7 on synthesis of C4 in uncloned HepG2 cells (8) is also evident in HG2N cells (Fig. 4b). These data indicate that the defect in IL-lP activity in HG2N cells is cytokinespecific.
One possible explanation for the absence of IL-lP biologic activity in HG2N cells is occupation of IL-1P receptors by endogenous IL-1P in this subclone. This possibility was excluded by subjecting lysates of radiolabeled HG2N cells to immunoprecipitation with anti-IL-lP (Fig. 5a) We also excluded the possibility that the biological activity of IL-1P in the parent uncloned HG2Y cells depends on the induction of endogenous IFN-P2. Lysates of radiolabeled HG2Y and HG2N cells were subjected to immunoprecipitation with anti-IFN-P2 (Fig. 5b). This antibody recognizes the five forms of IFN-P2 of apparent molecular mass in the range of 23-30 kDa in lysates of lipopolysaccharide-activated human monocytes (Fig. 5b, last lane, and Ref. 15). Within the limits of detection of this assay system, no IFN-P2 polypeptides are detected in HG2Y or HG2N cells under control, ILlo-, or IFN-P2-stimulated conditions.
HG2N and HG2Y Cells Bind Iodinated IL-lb-In order to examine whether the defective response in HG2N cells is due to a deficiency in cell surface receptor for IL-10, separate monolayers of HG2N and HG2Y cells were subjected to direct binding assays with radioiodinated IL-1P (Fig. 6). There is specific, saturable binding of IL-1P in each case. Scatchard plot analysis predicts 3.8 x lo3 and 4.0 x lo3 plasma membrane receptors/cell in HG2N and HG2Y cells, respectively.
The k~ for IL-1P binding is 0.96 X lo-' M in HG2N and 1.07 X M in HG2Y cells. Both HG2Y and HG2N Cells-Separate monolayers of HG2Y and HG2N cells were incubated for 18 h with IL-lP alone, IFN-P2 alone, or both IL-lP and IFN-P2 (Fig.  7). Synthesis of factor B was determined by the method described above and is shown graphically on the basis of densitometric scanning of fluorograms for four separate experiments. In these experiments, synthesis of factor B in HG2Y cells increases 2.31 f 0.45-fold in the presence of IL- albumin in the parent uncloned HepG2 cell line, as well as in the Hep3B cell line (1,2). In the current study we show that IL-1p alone has no effect on factor B or albumin in HG2N cells. IFN42 and TNF-a, cytokines with similar effects on factor B and albumin in HG2Y cells, retain their effects in HG2N cells. Likewise, IFN-7 regulates C4 expression similarly in both HG2Y and HG2N cells.

IL-1P and IFN-82 Have an Additive Effect on Synthesis of Factor E in
Two possible mechanisms accounting for the signal transduction defect were considered and excluded by demonstrating the absence of expression of IL-lp and IFN-P2 in either cell line (Fig. 5 ) . In fact, within the limits of detection for our assay system, there was no evidence for synthesis of IL-10 or IFN-P2 in these hepatoma cell lines under constitutive or induced (IL-lp, IFN-P2) conditions. We also excluded the possibility that IL-1p binding to HG2N cells is defective. HG2N cells were similar to HG2Y cells in specific, saturable binding of IL-1 with a kD of -1 X lo-' M and -4 X lo3 receptors per cell. Thus, there are a similar number of IL-lp receptors/cell in HepG2 cells, human dermal fibroblasts (28), human lung fibroblasts (29), human rheumatoid synoviocytes (30), and porcine synoviocytes (31), an order of magnitude greater than IL-lp receptor number for T-lymphocytes (11,28,32). The affinity of IL-1p binding to HepG2 cells is also similar to that of human dermal fibroblasts (28), human lung fibroblasts (29), porcine synoviocytes (31), and human Tlymphocytes (11,28,32), but lower than that of human rheumatoid synoviocytes (30). There has been no explanation for the higher affinity of IL-1p binding sites in rheumatoid synovial cells.
Further evidence that IL-1p is capable of binding to receptors in both HG2N and HG2Y cells is shown in Fig. 7. IL-lp, when together with IFN-@2, is capable of modulating factor B synthesis in HG2N cells: IL-lp and IFN-P2 have an additive effect on factor B synthesis in HG2Y cells; IL-1P and IFN-P2 have a similar additive effect on factor B synthesis in HG2N cells even though IL-1p has no effect by itself. This may mean that the effect of IFN-P2 is permissive for transduction of ILl p biological activity. Alternatively, IL-lp may activate several different signal transduction pathways in HG2Y cells including one responsible for its discrete action and one responsible for an additive effect with IFN-P2. According to this hypothesis, HG2N cells bear a defect in the signal transduction pathway for the discrete biological activity of IL-lp.
In either case, these data indicate that the HG2N subclone bears a defect in the signal transduction pathway for IL-lP. Even though ligand binding is intact, the IL-1p receptor could be defective in activation of the signal transduction pathway. Several examples of this type of receptor defect have been reported, e.g. site-directed mutagenesis of the cytoplasmic domain of the epidermal growth factor and insulin receptors results in defective cellular responses despite normal levels of receptor binding (33,34). Nevertheless, any step within the signal transduction pathway distal to receptor occupancy could be affected in the HG2N cell line. It has been shown that IL-lp does not stimulate phosphatidylinositol turnover or increases in cytosolic-free calcium concentration in Tlymphocytes (35), but there is little additional data on signal transduction mechanisms for its diverse biological activities. Although there are no major differences between the two cell lines by cytogenetic analysis (see "Methods"), we cannot exclude the possibility of one or more subtle genetic differences. The control experiments emphasize, however, that such differences are specific for the transduction pathway activated by IL-lp, one of a group of cytokines which have been shown to modulate acute phase gene expression in human hepatoma cells. Comparisons of the HG2N subclone and HG2Y uncloned parental cell line will therefore provide an opportunity for identification of the systems and mechanisms necessary for IL-1p activity in liver cells.
Results of these experiments also indicate that IL-lp, IFN-p2, and TNF-a have direct and independent effects on acute phase proteins, factor B and albumin. Expression of other acute phase proteins in HepG2 cells, such as fibrinogen (6) and al-antitrypsin (9), is regulated by IFN-P2 but not by ILl p or TNF-a. Still another hepatic acute phase gene, complement protein C4, is regulated only by IFN-7 (10). Initial results suggest that a combination of IL-lp and IFN-P2 may be necessary to induce expression of C-reactive protein and serum amyloid A in human hepatoma cell lines (6). These data, together with the implication from the current study, indicate that discrete and overlapping signal transduction pathways may be elicited by individual cytokines.