Interleukin-lp Induces Production of Granulocyte Colony-Stimulating Factor in Human Hepatoma Cells

By Chun-Fai Lai and Heinz Baumann

Interleukin-1 (IL-1) is a proinflammatory cytokine that partic- ipates in the activation of the acute-phase plasma protein genes in hepatic cells during infection and injury. In human hepatoma HepG2 and Hep3B cells, IL-1fi induced production of the granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner. Activation of G-CSF gene expres- sion was an early and transient response. In HepG2 cells, G- CSF mRNA was strongly upregulated 2 hours after IL-lfi treatment and returned to the pretreatment level by 6 hours. The secreted G-CSF was biologically active, as shown by the

NTERLEUKIN-1 (IL-1) is an early acting proinflamma- I tory cytokine during infection and injury.‘ A wide variety of cell types, including macrophages, fibroblasts, T cells, B cells, neutrophils, mast cells, endothelial cells, smooth mus- cle cells, and astrocytes, are known to secrete IL-1 in re- sponse to inflammatory stimuli? IL-1 exerts diverse effects on cells of hematopoietic and nonhematopoietic origin^.^,^ It stimulates the synthesis of the acute-phase plasma proteins (APP) in liver, induces the expression of adhesion molecules in endothelial cells for enhanced recruitment of leukocytes into the inflammation sites, accelerates bone catabolism, and stimulates prostaglandin synthesis in fibroblasts, macro- phages, and endothelial cells. Additionally, IL- 1 augments the expression of a number of cytokine genes such as IL-6, IL-8, macrophage colony-stimulating factor (M-CSF), gran- ulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF):

Human G-CSF is a 19-kD protein encoded by a single gene’s6 at chromosome 17.7,8 In vitro, the production of G- CSF is stimulated not only by IL-1, but also by tumor necro- sis factor-a (TNF-a) and lipopolysaccharide (LPS).9,10 Some carcinoma cells, such as CHU-2, SK-HEP-1, and U-87MG, produce G-CSF constitutively.” G-CSF regulates the prolif- eration and differentiation of granulocyte progenitors in bone

It also acts on mature granulocytes to stimulate their cytotoxic functions and superoxide release.l3.I4 The re- ceptor for G-CSF (G-CSFR) has been cloned and belongs to the hematopoietin receptor fa mil^.'^ Sequence homology is identified in the cytoplasmic domains of G-CSFR and the IL-6-type receptors, which consist of the IL-6 signal transducer, gp130, and the receptors for the leukemia-inhibi- tory factor and oncostatin M.16 Hepatic cells do not express G-CSFR. However, when G-CSFR is introduced into hepatic cells by stable transfection, it assumes the functions of the IL-6-type receptors in controlling the expression of the APP genes.17 We previously showed that G-CSFR is capable of regulating gene transcription via the IL-6 response element (IL-6RE) found in the APP gene promoters and defined the membrane-proximal 96 amino acids as the minimal region for the regulation of the IL-6RE.I8

In this report, we showed that human hepatoma HepG2 and Hep3B cells secrete biologically active G-CSF in re- sponse to IL-1P. The data suggest that G-CSF is an acute- phase reactant and that the liver, by producing G-CSF, partic- ipates in the control of hematopoiesis required for host defense.

induction of gene transcription through the G-CSF receptor. Maximal G-CSF activity released to culture medium occurred after 8 hours. Previous studies have shown that liver expres- sion of G-CSF was augmented in mice challenged by in- flammatory stimuli. Our data suggest that IL-lp mediates, at least in part, this cytokine activation program in parenchy- mal cells and that liver-derived G-CSF may contribute to the regulation of hematopoiesis during the acute-phase re- sponse. 0 1996 by The American Society of Hematology.

MATERIALS AND METHODS

Cells and treatments. Human hepatoma HepG2I9 and Hep3BZ0 cells were cultured in minimal essential medium (MEM), whereas rat hepatoma H-35’’ cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) media supplemented with 10% fetal calf serum, respectively. For cytokine treatments, cells were incubated with 10 ng/mL IL-lfl and 100 ng/mL G-CSF (Immunex Corp, Seat- tle, WA) or COS cell-derived IL-6 (Genetics Institute, Boston, MA), unless indicated otherwise.

The expression vector for the truncated form of human G-CSFR, G-CSFR(96), which contains membrane-proxi- mal 96 amino acids of the cytoplasmic domain, has been described previously.” The CAT-reporter gene construct, pHpIL-6RE-CAT, was cloned by inserting 5 copies of the oligonucleotides with se- quence of the IL-6RE from rat haptoglobin (Hp) gene promoter.” To monitor the transfection efficiency, the expression vector for the major urinary protein under the control of the immediately-early promoter of adenovirus was cotransfected into the cells.23

HepG2 cells were transfected by calcium phos- phate-DNA pre~ipitate,’~ whereas H-35 cells were transfected using diethyl aminoethyl (DEAE)-dextran-DNA complex.z5 Cells were transfected on IO-cm dishes and, after a recovery period of 16 hours, were redistributed equally into 6-well cluster plates. Twenty-four hours later, cells were treated with cytokines or conditioned media in the presence of 1 pmol/L dexamethasone for 16 hours. CAT activity was determined by incubating cell extracts with I4C-chlor- amphenicol and acetyl-coA. Reaction products were separated by thin-layer chromatography and quantitated by PhosphorImager (Mo- lecular Dynamics, Inc, Sunnyvale, CA).

HepG2 and Hep3B cells were cultured on IO-cm dishes and grown to con- fluency. Cells were treated with IL-lP alone in 10 mL serum-free MEM medium at the indicated dose and the culture media were collected after 1 to 24 hours. The amounts of G-CSF in the condi-

Plasmid constructs.

Transfection.

Preparation of conditioned media and assay for G-CSF.

From the Department of Molecular and Cellular Biology, Roswell

Submitted August 30, 1995; accepted January 9, 1996. Supported by National Cancer Institute Grant No. CA26222. Address reprint requests to Chun-Fai h i , Department of Molecu-

lar and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

Park Cancer Institute, Buffalo, NY.

0 1996 by The American Socieq of Hematology. OOO6-4971/96/8710-O5$3.00/0

Blood, Vol 87, No 10 (May 15), 1996: pp 4143-4148 41 43

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4144 LA1 AND BAUMANN

tioned media were determined by enzyme-Iinked immunosorbent assay (ELISA; R & D Systems, Inc, Minneapolis, MN).

Total RNA from HepG2 and Hep3B cells was prepared by guanidine thiocyanate extraction and cesium chloride gradient?6 Equal amounts of RNA were fractionated on 1% agarose gel and transferred to Nytran+ membrane (Schleicher & Schuell, Keene, NH). The membrane was hybridized with a 32P- labeled cDNA probe for human G-CSF’ (kindly provided by Dr D. Cosman, Immunex COT).

Northem blot analysis.

RESULTS

Detection of G-CSF production by HepG2 and Hep3B cells. Hepatic cells do not express G-CSFR; hence, these cells were used in previous studies to reconstitute the func- tion of G-CSFR and to characterize G-CSF action on gene transcription.” The human hepatoma HepG2 cells transiently transfected with expression vector for G-CSFR responded to G-CSF treatment by a prominent induction of transcription via IL-6RE-containing reporter gene constructs such as pHpIL-6RE-CAT (Fig IA, left panel). The regulation was similar to that mediated by the endogenous ILdR. Surpris- ingly, G-CSFR-transfected cells also gained an IL-1 -medi- ated regulation of the IL-6RE-CAT construct. To determine whether the IL-1 response was mediated by the transfected G-CSFR, the same combination of DNA was also introduced into the rat hepatoma H-35 cells, a cell line that is known for its prominent IL-1 response.” The induction of the CAT construct by IL-6 and G-CSF in the G-CSFR-expressing cells was confirmed (Fig lA, right panel), but no response to IL-1 treatment was observed. We conclude from these results that in HepG2 cells, but not H-35 cells, IL-lp induces the production of a stimulatory activity that acts on the transfected G-CSFR leading to the gene regulation via IL- 6RE.

To determine whether this activity is secretory, condi- tioned media were prepared from control and IL-l@-treated untransfected HepG2 cells and used to treat H-35 cells that had been cotransfected with pHpIL-6RE-CAT and G-CSFR. Only conditioned medium from the E-l,B-treated HepG2 cells did induce CAT activity in the tested H-35 cells (Fig 1B). However, the same medium had no stimulatory effect on H-35 cells lacking transfected G-CSFR. To investigate whether it is a phenomenon peculiar to HepG2 cells, we examine another human hepatoma cell line, Hep3B. Simi- larly, conditioned medium from unstimulated cells did not yield any detectable induction of CAT activity (Fig 1C). However, conditioned medium from cells treated with IL- 1P activated the CAT-reporter gene in cells transfected with G-CSFR. Taken together, these data indicate that the activity from HepG2 and Hep3B cells is highly specific to G-CSFR and likely represents G-CSF.

The suspected induction of G-CSF by IL-10 could be determined by the autocrine effect via G-CSFR. Moreover, the action could be quantitated by comparing it with the G-CSF added exoge- nously. Therefore, we cotransfected the CAT construct with increasing amounts of expression vector for G-CSFR into HepG2 cells. A dose-clependent increase in the CAT gene induction was observed with G-CSF and IL-lp (Fig 2). The IL-lp effect differed from that of G-CSF only by a lower

IL-IP activates the expression of G-CSF.

magnitude of CAT gene regulation, suggesting different con- centrations of ligands present. In the same transfected cells, the IL-6 response remained relatively constant. The IL-1 response was detectable at 0.1 pg/mL of cotransfected G- CSFR and improved by 120-fold when the amount of the cotransfected receptor was increased to 10 pg/mL.

Characterization of the G-CSF production. The amounts of G-CSF produced by HepG2 cells in response to IL-1P were determined by ELISA. G-CSF activity was detected in conditioned medium from HepG2 cells treated with IL-lP as low as 0.1 ng/mL (Fig 3). Maximal amounts of G-CSF (1,550 5 100 pg/ lo6 cells) were produced by cells treated with 1 n@mL IL-Ip. The amounts of G-CSF were also measured by the induction of the CAT gene on G-CSFR-transfected cells and were determined to be 18.8 ? 3.5 ng/mL.

The time course of G-CSF accumulation in conditioned media of HepG2 cells indicated no detectable G-CSF within 1 hour of IL-lp treatment (Fig 4). Prominent increase in G- CSF activity occurred afterward and peaked at 8 hours. The level of G-CSF declined after 8 hours and became stable from 16 to 24 hours. In Hep3B cells, a comparable amount of G-CSF was detected at 6 hours after IL-lp treatment and this level was maintained to 24 hours.

The induction of G-CSF expression was studied by Northern blot analysis of RNA from HepG2 cells treated with IL-1P for various times (Fig 5). G-CSF mRNA was undetectable in untreated cells. After 2 hours of IL-10 treatment, a signifi- cant increase in G-CSF mRNA was observed and the expres- sion declined to the basal level after 6 hours. The induction of G-CSF “A was also detected in Hep3B cells treated with L - l P for 6 hours. The data show that the increase in G-CSF mRNA accounted for the elevated G-CSF activity in the culture supernatant.

IL-lp upregulates the expression of G-CSF mRNA.

DISCUSSION

IL-I is an important proinflammatory cytokine that, in concert with other cytokines, modulates the acute-phase re- sponse in liver.’.28 Together with IL-6 and glucocorticoids, IL- 1 plays an essential role in liver to stimulate the synthesis of APPs, which perform protective functions for the host 0rganism.2~ A subset of the APP genes, including 11.1-acid glycoprotein, Hp, hemopexin, complement C3, and serum amyloid A, require IL-I for maximal transcription.’* IL-1 acts additively or synergistically with IL-6 and glucocorti- coids in coordinating the expression of the APPs. Whereas the synthesis of APPs is well documented as a major function of liver in the acute-phase response, the potential of liver in cytokine production is largely unrecognized. Previous stud- ies have shown that intraperitoneal injection of LPS” or intrapulmonary inoculation of Escherichia coli3’ would in- duce the expression of G-CSF in liver. However, the cell type expressing G-CSF and the mechanism responsible for the induction have yet to be defined. Our present study showed that IL-1P mediates the induction of G-CSF in HepG2 and Hep3B cells, cell types that closely resemble the parenchymal liver cells based on the pattern of plasma pro- tein expression?* Because the production of IL-1 is promi-

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G-CSF PRODUCTION BY HUMAN HEPATOMA CELLS 4145

1 8 5 1 1 1 8 7 1 9 4

A B HepG2 H-35

.4 (Z L .

F i r , ~m, -1 G-CSFR m] G-CSFR 1 1 6 0 1 1 9 3 2 8 6 9 1 1 9 5 1 1 1 7 4 2 3 1 1 1 1 183

- 1 6 G - 1 6 G - 1 6 G - 1 6 G

Treatments

- 1 - 1

Conditioned medium

C I - I + I G-CSFR

Fig 1. lL-lf3 induces the secretion of G-CSFR-activating activity by human hepatoma cells. (A) HepG2 cells (left panel) or H-35 cells lright panel) were transfected with pHplL-6RE-CAT alone or with G- CSFR196). Transfected cells were treated with medium containing dexamethasone alone (4, or with IL-1p (11, IL-6 (61, or G-CSF (GI. CAT activities in each experimental series were determined and expressed as fold induction over control cells indicated above the autoradio- gram. iBI H-35 cells were transfected as in IA) but treated with condi- tioned media from HepG2 cells that had been incubated with medium alone or medium containing 10 ng/mL IL-lp for 24 hours. (C) Similar to IB), transfected H-35 cells were treated with conditioned media from Hep3B cells with or without 11-18 treatment.

c ( ? c c o

- 6 - 1 - 6 - 1 U U " z%F

Treatments

nently induced during bacterial infection or administration of LPS,S3 one predicts that IL-1 produced either exogenously or endogenously in liver may act as an inflammatory media- tor for inducing G-CSF production in hepatic cells.

The regulation of G-CSF gene by IL- 1 has been studied

at the molecular level. The gene elements responsive to IL- 1 are mapped to location at -200 to - 165 relative to the CAP site.34 This region also confers TNF-a responsiveness when linked to a heterologous pr~moter.'~ A decanucleotide CK- 1 element and two NF-IL6 (CEBPO) consensus binding

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LA1 AND BAUMANN 41 46

200

h g 160 3 .= 120 3 Q

;/i G-csF

Fig 2. Action of IL-1p is dosedependent on G-CSFR. HepG2 cells were cotransfected with constant amount of pHplL-GRE-CAT and in- creasing amounts of expression vector for G-CSFR(96). Subcultures of the transfected cells were treated with IL-lp, IL-6, or G-CSF. The stimulation of CAT gene was determined and plotted against the amounts of cotransfected expression vector.

sites have been identified. Moreover, NF-KB p65 and NF- IL6 have been shown to bind cooperatively to this region.35 Based on these observations, p65 and NF-IL6 are postulated to be the transcription factors controlling the regulation of G-CSF promoter. In mice with NF-IL6 gene disrupted by gene targeting, the induction of G-CSF in fibroblasts and macrophages by LPS injection is severely im~aired.~' In- triguingly, the expression of G-CSF in liver remains normal.

I

I

i / 0.001 0.01 0.1 1 10 100

em (ad Fig 3. Dose-response of G-CSF production. Confluent HepGZ cells

were cultured on 10-cm dishes and treated with IL-lp in 10 mL of serum-free medium at the indicated concentrations. Culture superna- tants were collected after 16 hours and assayed for the concentra- tions of G-CSF by ELISA. The production of G-CSF is expressed in picograms per 10' cells.

Hep3B

Fig 4. Activation kinetics of G-CSF production. Confluent HepG2 and Hep3B cells were cultured on 10-cm dishes and treated with 10 n g h L of IL-1p in 10 mL of serum-free medium for the indicated time. Culture supernatants were collected and the levels of G-CSF were determined by ELISA.

These results indicate that the G-CSF production in hepatic cells is controlled by a mechanism distinct from other cell types and NF-IL6 appears dispensable in this signaling path- way. Because the NF-KB family of proteins are also involved in IL-1 signaling, it remains to be determined whether they serve as the mediators for regulating the G-CSF promoter in hepatic cells. It would be of interest to analyze the effects on the hepatic expression of G-CSF in the p65 gene knockout mice. Evidently, the contribution of tissue-specific factors to the IL-1 response cannot be ruled out.

It is noteworthy that the secretion of G-CSF in HepG2 cells was not inhibited by dexamethasone (Fig 1). The pres- ence of dexamethasone neither delayed the activation kinet- ics nor reduced the magnitude of the response (data not shown). Dexamethasone has been shown to potently sup- press the expression of a number of cytokine genes, includ- ing IL-1, IL-2, IL-3, IL-6, interferon-y, TNF-a, and GM- CSF.3" The inhibition of cytokine production is believed to be an important mechanism responsible for immunosuppres- sion. The independence of the glucocorticoids regulation in hepatic cells may allow the expression of G-CSF gene at an increased level of glucocorticoids during the late stage of acute ~hase.~' ,~ ' Nevertheless, its significance in vivo still awaits further exploration.

During infection, neutrophils are recruited into the in- flammation sites.39 Depending on the extent of recruitment, an appreciable decrease in the number of circulating neutro- phils in blood is expected and replenishment from the pro- genitors cells in bone marrow is required. Thus, an elevation of G-CSF production by an organ as massive as liver seems to be critical for host defense. Newborn mice with bacterial infection were found to be more susceptible to neutropenia and prone to death than ad~ l t s .~ ' It correlates with the obser- vation that upregulation of G-CSF mRNA by infection is deficient in the liver of newborn mice. In light of these studies, one has to conclude that liver has a broader func-

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G-CSF PRODUCTION BY HUMAN HEPATOMA CELLS 4147

HepG2 Hep3B I i n

G-CSF

Fig 5. Northern blot analysis of G-CSF expres- sion. Total RNA was prepared from HepG2 and 0 1 Hep3B cells treated with IL-lp for the indicated times. Membrane was hybridized with a cDNA probe for human G-CSF. The ethidium bromide staining pattern of 18s rRNA serves as a control for equal loading.

tional role in the acute-phase response than merely the syn- thesis of APPs. Obviously, the production of G-CSF would contribute beneficially to the control of the activities of cells involved in the inflammatory response at sites distant to liver. In conclusion, our results suggest a potential physiologic link between hepatic cells and hematopoiesis in the context of acute-phase response.

ACKNOWLEDGMENT

We thank Dr D. Cosman and lmmunex Corp for providing IL- ID, G-CSF, and the cDNA for human G-CSF. We also thank Genet- ics Institute for IL-6.

REFERENCES I . Kushner I: Regulation of the acute phase response by cyto-

2. Dinarello CA: Biology of interleukin 1. FASEB J 2: 108, 1988 3. Le J. Vileck J: Biology of disease. Tumor necrosis factor and

interleukin 1 : Cytokines with multiple overlapping biological activi- ties. Lab Invest 56:234, 1987

4. Aloisi F. Cart A, Borsellino G, Gallo P, Rosa S, Bassani A, Cabibbo A, Testa U, Levi G, Peschle C: Production of hemolympho- poietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-ID and tumor necrosis factor-a. J Immunol 149:2358, 1992

5. Nagata S, Tsuchiya M, Asano S, Kaziro Y, Yamazaki T, Yama- moto 0. Hirata Y, Kubota N, Oheda M, Nomura H, Ono M: Molecu- lar cloning and expression of cDNA for human granulocyte colony- stimulating factor. Nature 319:415, 1986

6. Nagata S, Tsuchiya M, Asano S, Yamamoto S, Hirata Y, Ku- bota N, Oheda M, Nomura H, Yamazaki T: The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO J 5575, 1986

7. Simmers RN, Webber LM, Shannon MF, Garson OM, Wong G, Vadas MA, Sutherland GR: Localization of the G-CSF gene on chromosome 17 proximal to the breakpoint in the t(l5; 17) in acute promyelocytic leukemia. Blood 70:330, 1987

8. Le Beau MM, Lemons RS, Carrino JJ, Pettenati MJ, Souza LM, Diaz MO, Rowley JD: Chromosomal localization of the human G-CSF gene to 17ql I proximal to the breakpoint of the t( 15; 17) in acute promyelocytic leukemia. Leukemia I :795, 1987

kines. Perspect Biol Med 36:61 I , 1993

2 4 6 8 1 6 2 4 0 6

Time (h)

9. Koeffler HP, Gasson J, Tobler A: Transcriptional and posttran- scriptional modulation of myeloid colony-stimulating factor expres- sion by tumor necrosis factor and other agents. Mol Cell Biol8:3432. 1988

IO. Ernst TJ, Ritchie AR, Demetri GD, Griffin JD: Regulation of granulocyte-and monocyte-colony stimulating factor mRNA levels in human blood monocytes is mediated primarily at a post-transcrip- tional level. J Biol Chem 2645700, 1989

11. Nishizawa M, Tsuchiya M, Watanabe-Fukunaga R, Nagata S: Multiple elements in the promoter of granulocyte colony-stimulating factor gene regulate its constitutive expression in human carcinoma cells. J Biol Chem 2655897, 1990

12. Begley CG, Nicola NA, Metcalf D: Proliferation of normal human promyelocytes and myelocytes after a single pulse stimula- tion by purified GM-CSF or G-CSF. Blood 71:640, 1988

13. Kitagawa S, Yuo A, Souza LM, Saito M, Miura Y, Takaku F Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemo- tactic peptide. Biochim Biophys Res Commun 144:1143, 1987

14. Lopez AF, Nicola NA, Burgess AW, Metcalf D, Battye FL, Sewell WA, Vadas M: Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors. J lmmunol 13 1 :2983, I983

15. Cosman D: The hematopoietin receptor superfamily. Cyto- kine 5:95, 1993

16. Kishimoto T, Taga T. Akira S: Cytokine signal transduction. Cell 76:253, 1994

17. Nakajima K, Wall R: Interleukin-6 signals activating junB and TIS1 I gene transcription in a B-cell hybridoma. Mol Cell Biol 11:1409, 1991

18. Lai C-F, Morella KK, Wang Y, Kumaki S, Gearing D, Ziegler SF, Tweardy DJ, Campos SP, Baumann H: Function of hematopoie- tin receptor subunits in hepatic cells and fibroblasts. Ann NY Acad Sci 762:189, 1995

19. Aden DP, Fogel A, Plotkin S, Damjanov I, Knowles BB: Controlled synthesis of HBsAg in a differentiated human liver carci- noma-derived cell line. Nature 282:615, 1979

20. Baumann H, Ziegler SF, Mosley B, Morella KK, Pajovic S, Gearing D P Reconstitution of the response to leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in hepatoma cells. J Biol Chem 268:8414, 1993

21. Reuber MD: A transplantable bile secreting hepatocellular carcinoma in the rat. J Natl Cancer lnst 26891, 1961

For personal use only.on April 8, 2018. by guest www.bloodjournal.orgFrom

4148 LA1 AND BAUMANN

22. Morella KK, Lai C-F, Kumaki S, Kumaki N, Wang Y, Blu- man EM, Witthuhn BA, Ihle JN, Giri J, Gearing DP, Cosman D, Ziegler SF, Tweardy DJ, Campos SP, Baumann H: The action of IL-2 receptor subunits defines a new type of signaling mechanism for hematopoietin receptors in hepatic cells and fibroblasts. J Biol Chem 270:8298, 1995

23. Marinkovic S, Baumann H: Structure, hormonal regulation, and identification of the interleukin-6- and dexamethasone-respon- sive element of the rat haptoglobin gene. Mol Cell Biol 10:1573, 1990

24. Graham FL, Van der Eb AJ: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456, 1973

25. Lopata MA, Cleveland DW, Soher-Webb H High level tran- sient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl- sulfoxide or glycerol shock treatment. Nucleic Acids Res 125707, 1984

26. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isola- tion of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 185294, 1979

27. Won K-A, Baumann H: The cytokine response element of the rat a1-acid glycoprotein gene is a complex of several interacting regulatory sequences. Mol Cell Biol 10:3965, 1990

28. Baumann H: Regulation of hepatic acute phase plasma protein genes by hepatocyte stimulating factors and other mediators of in- flammation. Mol Biol Med 7:147, 1990

29. Baumann H, Richards C, Gauldie J: Interaction among hepa- tocyte-stimulating factors, interleukin 1, and glucocorticoids for reg- ulation of acute phase plasma proteins in human hepatoma (HepG2) cells. J Immunol 139:4122, 1987

30. Tanaka T, Akira S, Yoshida K, Umemoto M, Yoneda Y, Shirafuji N, Fujiwara H, Suematsu S, Yoshida N, Kishimoto T: Targeted disruption of the NF-IL6 gene discloses its essential role in bacterial killing and tumor cytotoxicity in macrophages. Cell 80:353, 1995

31. Liechty KW, Schibler KR, Ohls RK, Perkins SL, Christensen RD: The failure of newborn mice infected with Escherichia coli to accelerate neutrophil production correlates with their failure to in- crease transcripts for granulocyte colony-stimulating factor and in- terleukin-6. Biol Neonate 64:331, 1993

32. Won K-A, Campos SP, Baumann H: Experimental systems for studying hepatic acute phase response, in Mackiewicz A, Kush- ner I, Baumann H (eds): Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications. Boca Raton, FL, CRC, 1993, p 255

33. Fenton MJ, Vermeulen MW, Clark BD, Webb AC, Auron PE: H u m pro-IL-lp gene expression in monocytic cells is regulated by two distinct pathways. J Immunol 140:2267, 1988

34. Kuczek ES, Shannon MF, Pel1 LM, Vadas MA: A granulo- cyte-colony-stimulating factor gene promoter element responsive to inflammatory mediators is functionally distinct from an identical sequence in the granulocyte-macrophage colony-stimulating factor gene. J Immunol 146:2426, 1991

35. Dunn SM, Coles LS, Lang RK, Gerondakis S, Vadas MA, Shannon M F Requirement of nuclear factor (NF)-KB p65 and NF- interleukin-6 binding elements in tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter. Blood 83:2469, 1994

36. Taniguchi T: Regulation of cytokine gene expression. Annu Rev Immunol6:439, 1988

37. Woloski BM, Jamieson JC: Rat corticotropin, insulin and thyroid hormone levels during the acute phase response to inflam- mation. Comp Biochem Physiol 86:15, 1987

38. Geisterfer M, Richards C, Baumann M, Fey G, Gywnne D, Gauldie J: Regulation of IL-6 and the hepatic IL-6 receptor in acute inflammation in vivo. Cytokine 5:1, 1993

39. Spertini 0, Kansas GS, MUNO JM, Griffin JD, Tedder TF: Regulation of leukocyte migration by activation of the leukocyte adhesion molecule-1 (LAM-1) selection. Nature 349:691, 1991

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