interleukin-8 gene expression in human bronchial ... · 19612 interleukin-8 expression in bronchial...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 29, Issue of October 15, pp. 19611-19617,1991 Printed in U.S.A.

Interleukin-8 Gene Expression in Human Bronchial Epithelial Cells* (Received for publication, March 6, 1991)

Hidenori Nakamura, Kunihiko Yoshimura, H. Ari Jaffe, and Ronald G . Crystal$ From the Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland20892

The capacity of cells of the human bronchial epithelium to express the gene for interleukin-8 (IL-8) was evaluated in bronchial epithelium derived cell lines, HS-24 and BET-lA, using tumor necrosis factor-a (TNF) as a model inflammatory stimulus. As in other epithelium, TNF markedly increased the level of the 1.8-kilobase IL-8 mRNA transcripts in both bronchial epithelial cell lines. In HS-24 cells, nuclear run-on analyses showed the IL-8 gene transcription rate was dramatically increased, more than 30-fold, after TNF stimulation. The half-life of IL-8 mRNA transcripts in these cells was approximately 40 min and did not change after TNF stimulation, suggesting that TNF up-regulated IL-8 gene expression mainly at the transcriptional level. DNase I hypersensitivity site mapping of chromatin DNA in resting HS-24 cells demonstrated two hypersensitivity sites within 400 base pairs (bp) 5‘ to exon I and one site within exon I. However, after TNF stimulation, the exon I hypersensitivity site disappeared and a new site approximately 120 bp 5’ to exon I emerged. Consistent with these observations, transfection studies with HS-24 cells using fusion genes composed of the 5”flanking sequences of the IL-8 gene and a luciferase reporter gene demonstrated potent promoter activity in a 174-bp segment (-130 to +44 relative to the transcription start site), which also exhibited a response to TNF, while a segment from -112 to +44 showed very low promoter activity and no response to TNF. Thus, human bronchial epithelial cells can express the IL-8 gene, with expression in response to the inflammatory mediator TNF regulated mainly at the transcriptional level, and with elements within the 5”flanking region of the gene that are directly or indirectly modulated by the TNF signal.

The epithelium of the human bronchial tree is a single layer of mostly columnar cells that forms a continuum from the trachea to the alveoli (1,Z). In addition to its role in providing a physiological barrier against contaminants of the ambient air, the bronchial epithelium modulates the continuous clean- sing of the respiratory surface by producing mucus and clear- ing it via the mucociliary escalator (1-4). There is increasing evidence that the bronchial epithelium can also modulate the movement and the function of parenchymal and inflammatory cells, i.e. the airway epithelial cells can influence other components of the bronchi as well as contributing to the defense of the lung (1,4).

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

f Reprint requests should be addressed to: Dr. Ronald G. Crystal, Pulmonary Branch, Bldg. 10, Rm. 6D03, NIH, Bethesda, MD 20892.

One specific mechanism by which bronchial epithelial cells might interact with other cells is by expressing the gene for interleukin-8 (IL-8),‘ an 8.5-kDa inflammatory cytokine known to be produced by mononuclear phagocytes, fibroblasts, endothelial cells, and a variety of epithelial cells, including those of the retina and alveoli (5-9). IL-8 has potent chemoattractive and activating functions for neutrophils, can inhibit neutrophil adherence to the endothelium, modulate chemotaxis for T-lymphocytes, trigger histamine and leukotriene release from basophils, and induce contraction of airway smooth muscle cells (5-7,lO-13). In this regard, and with the knowledge that bovine bronchial epithelium can produce a factor(s) with neutrophil chemotactic activity (1, 14), we have evaluated a variety of human bronchial cell lines for the ability to express the IL-8 gene. The data demonstrate that bronchial epithelial cells are capable of expressing the IL-8 gene and that IL-8 gene expression can be up-regulated in these cells by mediators such as tumor necrosis factor-a (TNF) primarily by increasing the rate of transcription of the gene.

MATERIALS AND METHODS

Cell Cultures-BET-lA, a human bronchial epithelial cell line transformed by the SV40 virus, and BEAS-2B, a human bronchial epithelial cell line transformed by an adenovirus 12-SV40 hybrid virus (both gifts of J. E. Lechner, National Cancer Institute) (15) were cultured in serum-free LHC-9 medium with 25 pg/ml Fungizone, 25 units/ml penicillin, and 25 pg/ml streptomycin (all from Biofluids Inc., Rockville, MD). 4MBr-5, a Rhesus monkey-derived bronchial epithelial cell line (American Type Culture Collection, Rockville, MD; CCL 208) was cultured in Ham’s F12K (Imine Scientific, Santa Anna, CA) supplemented with 10% fetal bovine serum (Biofluids) and epidermal growth factor (30 ng/ml; GIBCO/Bethesda Research Laboratories, Gaithersburg, MD). HS-24, a human bronchial squa- mous carcinoma cell line (provided by w. Ebert, Thoraxklinikum, Heidelberg-Rohrbach, Federal Republic of Germany) (16) was maintained in RPMI 1640 with 25 mM HEPES, pH 7.4, 2 mM glutamine, 100 pg/ml gentamicin (all from GIBCO/Bethesda Research Labora- tories), and 10% fetal bovine serum. All studies with BET-lA, BEAS- 2B, 4MBr-5, and HS-24 were carried out when the cells were 70-80% confluent.

Inflammatory Stimuli-In initial studies to evaluate the response of IL-8 gene expression in bronchial epithelial cells to inflammatory stimuli, BET-lA, BEAS-2B, 4MBr-5, and HS-24 cells were incubated alone or for 3 h with 10 pg/ml lipopolysaccharide (LPS, Sigma), 80 nM phorbol 12-myristate 13-acetate (PMA, Sigma), 10 units/ml human recombinant interleukin-l(3 (IL-1, 10 units/ng; Collaborative Research Inc., Bedford, MA) or 100 units/ml human recombinant TNF (20 units/ng; Genzyme Corp., Boston, MA), and IL-8 mRNA levels evaluated by Northern analysis (see below). After these initial

’The abbreviations used are: IL-8, interleukin-8; TNF, tumor necrosis factor-a; HEPES, 4-(2-hydroxyethyl)-l-piperazineethane- sulfonic acid; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; IL-1, interleukin-l(3; PCR, polymerase chain reaction; pBS, pBluescript I1 SK+; fMLP, formyl-methinyl-leucyl-phenylalanine; CMV, cytomegalovirus; RSV, Rous sarcoma virus; CAT, chlor- amphenicol acetyltransferase; bp, base pair(s); kb, kilobase(s).

19611

19612 Interleukin-8 Expression in Bronchial Epithelial Cells

studies, all further experiments were carried out with HS-24 cells and TNF as the inflammatory stimulus as indicated. IL-8 mRNA Transcript Levels-In the majority of studies, unless

otherwise indicated, the levels of IL-8 mRNA transcripts and, as a control, 0-actin mRNA transcripts were evaluated by Northern analysis (17, 18). Total cellular RNA was isolated by the guanidinium thiocyanate-CsC1 gradient method (19). RNA (10 pg) was subjected to formaldehyde-agarose gel electrophoresis, transferred to a nylon membrane (Nytran; Schleicher and Schuell, Inc., Keene, NH), hybridized with a 3'P-labeled IL-8 or @-actin cDNA probe generated by the random priming method (20), and evaluated by autoradiography. The IL-8 cDNA used as a probe (pPB248) was a 750-bp cDNA segment including the sequence from the PstI site of exon I to the BamHI site of exon IV. It was constructed using polymerase chain reaction amplification of RNA (after conversion to cDNA) from LPS- stimulated human peripheral blood monocytes with Taq polymerase (Perkin-Elmer Cetus Instruments) and primers specific for human

3'; and NAFAS2, 5'-TTGTGGATCCTGGCTAGCAGAC-3') (21- 24), and cloned into pBluescript I1 SK+ (pBS; Stratagene, La Jolla, CA). The cDNA clone pHFpA-1 was used for the 0-actin probe (25). IL-8 Release by Bronchial Epithelial Cells-To demonstrate that

bronchial epithelial cells were capable of releasing functional IL-8 molecules, supernatants of resting and TNF-stimulated HS-24 cells were evaluated for the presence of neutrophil chemotactic activity that could be blocked by an anti-IL-8 antibody. Human neutrophils (>95% pure) were isolated from blood of normal individuals by centrifugation through Mono-Poly-Resolving medium (Flow Labo- ratories, Irvine, CA) (26) and resuspended in Hanks' balanced salt solution (Mediatech, Herndon, VA) at 2 X lo6 cells/ml. Neutrophil chemotaxis was analyzed in multi-well chemotaxis chambers (Neu- roprobe Inc., Cabin John, MD) with 10-pm thick polyvinylpyrroli- done-free 3 hm pore size polycarbonate filters (Nuclepore Corp., Pleasanton, CA) (27). After incubation (40 min, 37 "C), the filters were stained with Diff-Quick (American Scientific Products, McGaw Park, IL), and the number of neutrophils migrating through the filter per high power field was quantified. Chemotactic activity was stand- ardized by expressing it as the percentage of a formyl-methionyl- leucyl-phenylalanine (fMLP, lo-' M; Sigma) positive control after subtracting the value for culture medium alone. Controls included recombinant IL-8 (100 ng/ml; Biosource International, Westlake Village, CA) and culture media with TNF alone (100 units/ml). Serum-free, HS-24-conditioned medium (24 h) was evaluated alone and with TNF (100 uits/ml). Further controls included HS-24 cell supernatants in the presence of various dilutions of rabbit anti-IL-8 IgG or rabbit preimmune IgG (Endogen, Boston, MA). In preliminary chemotaxis experiments using recombinant IL-8, the neutralizing titer of the anti-IL-8 IgG was observed to be relatively low, i.e. concentrations of anti-IL-8 IgG of 1:300 or 1: lO were necessary to block the IL-8 activity of the positive IL-8 control, and thus these concentartions were used for the culture supernatant studies. Similar concentrations of preimmune IgG were used as control.

Modulation of IL-8 Gene Expression in HS-24 Cells-To evaluate the dose-dependency of TNF-induced modulation of IL-8 gene expression, HS-24 cells were stimulated with various concentrations of TNF (0-1000 units/ml) for 3 h. To determine the time dependence of IL-8 gene expression, cells were incubated for various times (1-24 h) in the presence of 100 units/ml TNF. Following incubations, total cellular RNA was isolated, and the levels of IL-8 and, as a control, p- actin mRNA transcripts were evaluated by Northern analysis as described above. The autoradiographic signals were quantified using a laser densitometer (Ultroscan Laser Densitometer; Pharmacia LKB Biotechnology inc.) and the data expressed as -fold over the resting levels.'

IL-8 gene transcription rate was examined by nuclear transcription run-on analysis (28, 29). Nuclei were isolated from 5 X lo7 resting or TNF-stimulated cells (100 units/ml; 1, 3, and 24 h), and incubated (37 "C, 20 min) with 5 mM ATP, 2 mM CTP, 2 mM UTP, 250 pCi of [u-~*P]GTP (>400 Ci/mmol; Amersham Corp.), and 700 units/ml RNase inhibitor (RNasin; Promega Corp., Madison, WI) to label actively transcribed RNA. RNA was recovered by the acid guanidinium thiocyanate-phenol-chloroform method (30), purified by Seph- adex G-50 column chromatography (5 Prim-3 Prime, West Chester, PA) and hybridized to excess amounts (5 pg) of DNA targets (see

IL-8 cDNA (NAFS1,5"ATTTCTGCAGCTCTGTGTGAAGGTGC-

' All data are expressed as mean k standard error of the mean, and all statistical comparisons were done with the two-tailed Student's t test.

below) immobilized on Nytran (18). The membranes were then washed, exposed to RNase A (5 pg/ml) and RNase TI (5 units/ml), followed by proteinase K (50 pg/ml) (all from Boehringer Mannheim), and evaluated by autoradiography. The DNA targets included plasmids containing an IL-8 cDNA (pPB248), a partial c-jun cDNA (kindly supplied by J. D. Minna, National Cancer Institute-Navy Medical Oncology Branch, Bethesda, MD) (31), partial genomic clones for c-fos and c-myc (both from Lofstrand Labs, Gaithersburg, MD), a human @-actin cDNA (pHFoA-l), or, as a negative control, the plasmid pBS containing no human DNA. To determine the relative IL-8 gene transcription rate after TNF stimulation compared to the resting rate, the autoradiograms were quantified using a laser densitometer and expressed as -fold over the resting levels in three individual experiments.

To estimate the stability of IL-8 mRNA transcripts, HS-24 cells were exposed to actinomycin D (10 pg/ml; U. S. Biochemical Corp.) for 1, 3 and 6 h, and the following conditions were evaluated HS-24 cells a t rest, after exposure to TNF (100 units/ml, 1 h), or after the addition of the protein synthesis inhibitor, cycloheximide (10 pg/ml, 3 h, Sigma). In two separate experiments, total cellular RNA was extracted, and IL-8 mRNA levels were evaluated by Northern analysis as described above and quantified by laser densitometry.

DNase I Hypersensitivity Site Mapping-The 5"flanking region of the IL-8 gene was evaluated for DNase I hypersensitivity sites in chromatin DNA isolated from HS-24 cells before and 1 h after TNF (100 units/ml) stimulation (32). Briefly, cells were washed with Hanks' balanced salt solution and incubated for 5 min in lysis buffer (10 mM Tris-HC1, pH 7.4, 10 mM KC1, 3 mM MgC12, 0.1% Nonidet P-40 (Sigma)). Cells were recovered by scraping, centrifuged at 200 X g, 10 min, and resuspended in lysis buffer. Nuclei were then isolated by centrifugation at 200 X g for 5 min and exposed to DNase I (6.25 units of DNase I/2 X lo7 nuclei, Boehringer Mannheim) for 0, 0.5,1, or 4 min. The chromatin DNA was then extracted and purified. DNA (15 pg) from each DNase I treatment was digested with EcoRI (New England Biolabs, Inc., Beverly, MA), subjected to agarose gel electrophoresis, and evaluated by Southern hybridization analysis (33) using a 32P-labeled DNA probe (a 0.8-kb EcoRI-HincII fragment of IL-8 5'- flanking sequence (see below)) (34).

Promoter Activity of IL-8 Gene 5"Flanking Sequences-Transfec- tion vectors containing fusion genes of 5"flanking region sequences of the IL-8 gene and a luciferase reporter gene were constructed from a pUC8-derived vector (pCMV-luciferase) (35). The IL-8 promoter region, a 1525-bp EcoRI-Hind111 fragment spanning -1481 to +44 (numbering based on the reported sequence of IL-8 gene by Mukaida et al. (34)), was prepared by polymerase chain reaction with human genomic DNA as a template, and IL-8 gene specific primers

TTTATC-3'; and NAFPAS1, 5"TTGTCCTAGAAGCTTGTGT- GCTCTGCTGTC-3'). It was then cloned into a luciferase expression vector (pN1481L) by replacing the cytomegalovirus (CMV) promoter in a pCMV-luciferase expression plasmid between unique XhoI and HindIII sites. Sequentially deleted fragments of the IL-8 gene 5'- flanking region (starting from -391, -335, -130, and -112 to +44) were prepared in a similar manner. The sequences of the IL-8 5'- flanking region inserts of all vectors were confirmed by the dideoxy chain-termination method (36). The Rous sarcoma virus (RSV)-long terminal repeat promoter-luciferase construct (pRSVL) (37) was used as the positive control, and a promoterless luciferase plasmid (pLuc0) as the negative control (38).

HS-24 cells were transfected using the technique of electroporation (39). Cells were removed from plates with trypsin, washed twice and resuspended in Dulbecco's phosphate-buffered saline (GIBCO/Be- thesda Research Laboratories) (10' cells in 0.8 ml). Each luciferase expression plasmid vector (15 pg) and a CMV promoter-chloram- phenicol acetyltransferase (CAT) expression plasmid (5 pg; pCMV- CAT (35); as an internal control) were added to cell suspensions. Electroporation was carried out a t 2000 V, 25 pF (Gene Pulser; Bio- Rad), and HS-24 cells were maintained in culture media at 37 "C for 48 h. To evaluate the effects of TNF on reporter gene expression, TNF (100 units/ml) was added to culture media 6 h before harvesting the transfected cells.

To measure reporter gene expression, cells were retrieved by scraping, washed, and resuspended in 150 pl of lysis buffer (100 mM potassium phosphate, pH 7.8, 1 mM dithiothreitol) (37). The cells were then lysed by three freeze-thaw cycles, centrifuged, and 15 pl of supernatant was evaluated for luciferase activity using a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, CA) (37). The protein concentration of supernatants was

(NAFPS1, 5"ATGTCTCGAGAATTCAGTAACCCAGGCATTAT-

Interleukin-8 Expression in Bronchial Epithelial Cells 19613

measured by the Bradford method (40) (Bio-€kd protein assay) with bovine serum albumin as a standard. CAT activity was assayed by standard methods (41). Levels of luciferase expression were normalized by CAT activity and are reported relative to the expression of pRSVL in resting HS-24 cells (defined as 100%). To evaluate the relative increase in IL-8 gene promoter activity after TNF stimulation, luciferase expression was normalized to the total protein concentration.

RESULTS

IL-8 Gene Expression in Bronchial Epithelial Cells-North- ern analyses demonstrated that both the BET-1A bronchial cell line and the HS-24 bronchial carcinoma cell line expressed 1.8-kb IL-8 mRNA transcripts a t a very low level, but accumulated them markedly after exposure to TNF, IL-1, or PMA but not LPS (Fig. 1). In both cell lines, control 8-actin mRNA transcripts were observed in similar amounts in both resting and cells exposed to the various inflammatory mediators. In addition to BET-1A and HS-24, the bronchial epithelial cell lines BEAS-2B and 4MBr-5 expressed IL-8 mRNA transcripts in response to the same stimuli (not shown). In the context of the similar pattern of response among all of these cell lines, HS-24 was chosen for subsequent study along with TNF as the stimulant.

Neutrophil Chemotactic Activity-Supernatants from resting HS-24 cells displayed a low level of neutrophil chemotactic activity, approximately 20% that of the fMLP-positive control (Fig. 2). In contrast, supernatants from TNF-stimulated HS- 24 cells showed approximately $fold greater chemotactic activity for neutrophils that could be blocked by anti-IL-8 antibodies in a dose-dependent fashion, but not by preimmune rabbit IgG. TNF itself had no chemotactic activity. Thus, not only did HS-24 express the IL-8 gene in an exaggerated fashion in response to TNF but in a parallel fashion secreted a neutrophil-chemoattractant with the antigenic properties of

Concentration and Time Course Effects of TNF on IL-8 mRNA Transcript Levels in HS-24 Cells-TNF stimulation caused an increase in the levels of IL-8 mRNA transcripts in dose- and time-dependent fashions (Fig. 3). TNF-induced up- regulation of IL-8 gene expression was observed a t very low concentrations (1-10 units/ml) and IL-8 transcript level con- tinued to increase with concentrations up to 1000 units/ml. In contrast, the level of 8-actin mRNA transcripts in the same cells did not change with increasing concentrations of TNF. Following a fixed dose of TNF (100 units/ml), IL-8 mRNA transcript level rapidly increased to its maximum a t 3 h, and remained elevated at 24 h. In contrast, resting HS-24 cells

IL-8.

BET-1A HS-24

- 0 3 0 3 - Time (hr) + + + +

LPS PMA IL-1 TNF Rest Rest L& PhA IL-1 TNF + +

IL-8

p-actin I

FIG. 1. Effects of LPS, PMA, IL-1, and TNF on IL-8 gene expression in BET-1A and HS-24 bronchial epithelial cells. Data shown are Northern blot analyses of RNA (10 pg each) from resting cells and cells after treatment with LPS (10 pg/ml), PMA (80 nM), IL-1 (10 units/ml), and TNF (100 units/ml) for 3 h hybridized with a 32P-labeled IL-8 cDNA probe (top) and @actin probe (bottom). The sizes of mRNA transcripts are indicated.

L IMLP IL-S IL-8 HS-24 TNF HS-24 HS-24 HS-24 HS-24

+ Anti-IL-8

+ + + + TNF TNF TNF TNF

Anti-IL-8 Anti-IL-8 IgG (1:300) (1:lO) (1 : lO)

(1:lO) + + +

FIG. 2. Neutrophil chemotactic activity released by HS-24 cells following TNF stimulation. Chemotactic activity for neutrophils was measured using modified Boyden chambers as described under “Materials and Methods.” Chemotaxis (determined as numbers of neutrophils/high power field) is expressed as percentage of the positive fMLP control. The media alone control has been subtracted from all data. Shown (left to right) is the chemotactic activity (relative to W L P ) for; IL-8 (100 ng/ml), IL-8 + anti-IL-8 antibody (1:lO dilution), supernatant of resting HS-24 cells, culture media with TNF (100 units/ml) alone, supernatant of HS-24 cells that had been incubated with TNF (100 units/ml) for 24 h, supernatant of HS-24 cells stimulated with TNF evaluated for chemotaxis in the presence of the anti-IL-8 IgG (1:300 and 1:lO) or preimmune IgG (1:lO). Data shown represent triplicate assay determinations of one example of three different experiments.

demonstrated low and constant levels of IL-8 mRNA transcripts during the same period.

Transcription of IL-8 Gene and Its Response to TNF- Isolated nuclei from resting HS-24 cells demonstrated a low rate of transcription of the IL-8 gene (Fig. 4). Following TNF stimulation, however, the IL-8 gene transcription rate increased many-fold over resting levels (p < 0.05 for 1 h and 3 h compared to resting). This increased rate was maintained for a t least 3 h, while in contrast, the transcription rate of the control 8-actin gene was relatively constant.

Stability of IL-8 mRNA Transcripts-Following the inhibition of RNA synthesis with actinomycin D, IL-8 mRNA levels in resting HS-24 cells fell rapidly with a half-life of 40 min, suggesting that IL-8 mRNA transcripts were relatively unstable in the resting state (Fig. 5). In contrast, in the presence of cycloheximide, the half-life of IL-8 transcripts was markedly increased to 6 h. Importantly, the addition of TNF to HS-24 cells did not change the half-life of the IL-8 mRNA transcripts, suggesting that up-regulation of IL-8 transcript levels following TNF stimulation is not modulated by changes in transcript stability.

DNase I Hypersensitivity Sites in the 5’-Flanking Region of the IL-8 Gene-DNase I hypersensitivity site mapping showed, in resting HS-24 cells, a cluster of hypersensitivity sites 5’ to and in exon I of the IL-8 gene at approximately -340, -220, and +70 relative to the transcription start site (shown as 1.1-, 1.3-, and 1.5-kb bands, respectively; Fig. 6). Interestingly, 1 h after TNF stimulation, one site (at +70, shown as the 1.5-kb band) disappeared and a new site (a 1.4- kb band) emerged (at approximately -120), while the two other hypersensitivity sites (the 1.3- and 1.1-kb bands; at approximately -220 and -340) remained unchanged.


A.

40 t T

n 0 0.1 1 10 100 1000

B. TNF TNF (unitslml)

1 r\ ~ Resting ~ ” ” v 0 0 1 3 6 24

Time after addition of TNF (hr)

FIG. 3. IL-8 mRNA levels in HS-24 cells following stimulation by TNF. A, expression of IL-8 mRNA (0) and 8-actin mRNA (0) in response to increasing amounts of TNF in HS-24 cells. HS-24 cells were exposed to TNF (0.1 to 1000 units/ml) for 3 h. B, time course of IL-8 mRNA expression by resting (0) and TNF-treated (0) HS-24 cells before and at various times after exposure to TNF (100 units/ml). Total cellular RNA extracted from HS-24 cells was evaluated by Northern analyses and densitometric scanning of the autoradiograms. The data is presented as the -fold increase over the resting value from three separate experiments.

Evaluution of 5’-Flanking Sequences for Promoter Activ- ity-Transfection studies of HS-24 cells using fusion genes of 5”flanking sequences of the IL-8 gene and a luciferase reporter gene demonstrated promoter activity of these sequences, but with differences among the 5”flanking sequences including differences in response to TNF (Fig. 7). In resting HS-24 cells, the sequence from -1481 to +44 showed relatively potent promoter activity (13% of a RSV promoter). Deletion of the sequence 5’ to -130 caused a mild, but not significant ( p > 0.2), increase in reporter gene expression. Importantly, further deletion of only 18 bp (-130 to -113) caused a dramatic decline of the promoter function to a very low level, only 4% of the RSV control ( p < 0.01, pN130L versus pN112L). TNF stimulation increased luciferase reporter gene expression up to 1.9-3.9-fold in the plasmid constructs which contained sequences 5’ to -130 ( p < 0.05, all constructs 5’ to -130 with TNF compared to resting cells). In contrast, the sequence from -112 to +44 did not respond to TNF ( p > 0.2).

DISCUSSION

By virtue of its anatomic location at the initial interface between the ambient air and the body, the bronchial epithelium has a variety of mechanisms to remove airborne contaminants (1-4,42). The present study demonstrates that among the repertoire of human bronchial epithelial cells is the ability to express the gene for IL-8, a gene coding for an 8.5-kDa protein that can modulate a variety of inflammatory processes

0 1 3 24 Time (hr)

IL-8

c-jun

c-fos

c-myc 4

p-actin 4 PBS

~ Resting ~

1 Y Y ;a 0 1 3 24

Time after addition of TNF (hr)

FIG. 4. Effect of TNF on the transcription rate of the IL-8 gene in HS-24 cells. The cells were incubated in the absence or presence of TNF (100 units/ml) for 1,3, and 24 h. 32P-Labeled nascent nuclear RNA was hybridized to nylon membrane-bound DNA targets (5 pg each) including IL-8, c-jun, c-fos, c-myc, 8-actin, and pBS plasmids. A, example of a representative autoradio-gram of three individual experiments. B, relative increase in rate of IL-8 (0) and 8-actin (0) gene transcription expressed as the -fold increase over that of resting cells.

including the recruitment and activation of neutrophils (5-7, 9-13). IL-8 gene regulation in bronchial epithelial cells is predominantly modulated at the transcriptional level but is complex and involves multiple negative and positive regulatory mechanisms in both the constitutive and TNF-stimulated states.

Parenchymal Cell Modulation of Inflummatory Cells-These observations are consistent with data demonstrating that bovine bronchial epithelial cells can release chemoattractants for inflammatory cells (1,14,43,44), and that epithelial cells derived from the human alveoli can express the IL-8 gene (91, as can a lung giant cell carcinoma cell line (45). Together with the knowledge that fibroblasts (46) and endothelial cells (10,47) can express the IL-8 gene, these observations form a body of evidence suggesting that recruitment of inflammatory cells such a neutrophils to the lung is likely mediated, in part, by parenchymal cells.

The traditional view of acute and chronic inflammatory lung disorders was that the accumulation of neutrophils in the alveoli (as in bacterial pneumonia) and bronchi (as in bronchitis) was mediated by complement components or mediators released by alveolar macrophages (42, 48-50). This concept was expanded to include parenchymal components as chemoattractants, as evidence mounted that fragments of elastin, fibronectin, fragments of al-antitrypsin after prote- olysis and the al-antitrypsin-neutrophil elastase complex were also capable of acting as chemoattractants for inflammatory cells (51-54). With the discovery of IL-8 (also referred to as monocyte-derived neutrophil chemotactic factor, neutrophil-activating factor, or neutrophil attractant/activation pro-


Actinomycin D

2 i 1 1 I I I I I I I

0 1 2 3 4 5 6 7

Time after addition of actinomycin D (hr)

FIG. 5. Stability of IL-8 mRNA transcripts in €IS-24 cells. Shown are effects of inhibition of RNA synthesis on IL-8 mRNA levels in resting, TNF-stimulated (100 units/ml for 1 h) and cycloheximide-treated (10 pg/ml for 3 h) HS-24 cells. Cells were harvested at the indicated times after the addition of actinomycin D (10 pg/ml) and the extracted RNA was evaluated by Northern analysis. Data are a representative of two individual experiments.

tein-1), it was recognized that, in addition to leukotriene B4, alveolar macrophages could release IL-8 in response to activation and thus have the capacity of release at least two defined neutrophil chemoattractants (55, 56). With the rec- ognition that parenchymal cells also release IL-8 in response to inflammatory stimuli (9, 10,47,55,56), it is now apparent that most classes of cells within the lung participate in this form of host defense. With regard to the epithelium, the recent study by Standiford and colleagues (9) has shown that the alveolar epithelium can express the IL-8 gene and release IL- 8 in response to inflammatory stimuli, suggesting a role for IL-8 released by the alveolar epithelium in disorders such as bacterial pneumonia or interstitial disorders in which neutrophils dominate. The present study, together with the observations by others demonstrating that bovine bronchial epithelial cells can release a chemoattractant for monocytes, lymphocytes, and neutrophils (1,14,43,44), suggests that the bronchial epithelium may have an important role in inducing the accumulation of inflammatory cells in the acute and chronic inflammatory disorders of the bronchi such as bronchitis and cystic fibrosis (1,42, 57, 58).

Control of IL-8 Gene Expression in Human Bronchial Epi- thelial Cells-In all classes of cell types evaluated, including mononuclear phagocytes, T-lymphocytes, fibroblasts, keratin- ocytes, endothelial cells, synovial cells, and epithelial cells, IL-8 mRNA transcripts are found in very low levels in the resting state, but accumulate markedly in response to a variety of inflammatory stimuli, typically, IL-1 and TNF (5-9, 23, 24, 34, 46, 47, 55, 56, 59-61). We have observed the same phenomenon in human bronchial epithelial cells, with very low levels of IL-8 transcripts in resting cells, but with a marked increase in transcript levels following exposure of the epithelial cells to TNF. This rapid accumulation of IL-8 transcripts in bronchial epithelial cells responding to TNF appears to be modulated primarily at the transcriptional level.

I 3.4 kb *--7 I 1.5 I

I 1.4 I

I 1.3 t

Resting + TNF (1 hr)

0 0.5 1 4 0 0.5 1 4 Time (min)

1 2 3 4 5 6 7 0

FIG. 6. Evaluation of chromatin DNA for the presence of DNase I hypersensitivity sites in the 5”flanking region of the IL-8 gene in resting and TNF-stimulated HS-24 cells. Top, partial map of the IL-8 gene and 5’-flanking region showing the 3.4- kb EcoRI restriction fragment used for evaluation of DNase I hypersensitivity sites. The location of the 32P-labeled probe, encompassing an EcoRI-Him11 fragment, is indicated as a hatched box. The uertical arrows indicate the approximate locations of the DNase I hypersensitivity sites observed. Bottom, comparison of DNA from untreated and TNF-stimulated (100 units/ml) HS-24 cells for DNase I hypersensitivity sites. Nuclei isolated from HS-24 cells were treated with DNase I for 0 to 4 min; DNA was then extracted, digested with EcoRI, and evaluated by Southern analysis as described under “Materials and Methods.“ Lanes 1-4, chromatin DNA from unstimulated HS- 24 cells. Note the appearance of 1.5-, 1.3-, and 1.1-kb bands indicating

Lanes 5-8, chromatin DNA from TNF-stimulated HS-24 cells. Note DNase I hypersensitivity sites over the period of incubation time.

the disappearance of the 1.5-kb band (shown as a hatched arrow) and the appearance of a 1.4-kb band (a closed arrow).

While resting HS-24 cells transcribe the IL-8 gene at a low rate and the IL-8 transcripts have a short half-life, TNF causes a rapid increase in the rate of IL-8 gene transcription in HS-24 cells, but has no effect on the stability of IL-8 transcripts. These data contrast with the observations in differentiated HL-60 promyelocytic cells in which cytoplasmic stabilization of IL-8 mRNA transcripts, as opposed to transcriptional induction, was a major force in the regulation of IL-8 gene expression in response to inflammatory stimuli such as PMA (62), i.e. the regulatory mechanisms in IL-8 gene expression appear to be cell specific.

While all of the specific controlling elements of IL-8 gene transcription are not defined for any cell type, comparison of our observations in bronchial epithelial cells to those of Mu- kaida et d. (63) in fibrosarcoma cells suggests that at least some elements of control may be cell specific. In fibrosarcoma cells, it appears that two cis-regulatory elements (an NF-KB- like element and cis-regulatory enhancer binding element) in the region of -94 to -71 from the transcription start site are responsible for the up-regulation of IL-8 gene expression by inflammatory stimuli, while other 5“flanking sequences re- main uncharacterized. In contrast, the present study demonstrates that the sequence -112 to +44 relative to the transcription start site supports low level reporter gene expression


Resting Relative Activity

(% RSV)

+TNF Relative Activity

(% RSV in resting cells) Ratio 20 40 60 80 1 0 0 20 40 60 80 100 +TNF’Resting

r“--I“---- -1 RSV LTR H L u c i t e r a s e F p ~ ~ ~ ~ 1 - y 0.9 * 0.1

-335 +44 hl

1.9 f 0.2

2.5 f 0.4

b 3.9 f 1.4

b 1.9 f 0.4

1.1 fO.1

FIG. 7. Promoter activity of the 5”flanking region of the IL-8 gene in HS-24 cells. Promoter activity was evaluated using fusion genes including sequentially deleted 5”flanking regions of the IL-8 gene and a luciferase reporter gene. Levels of luciferase expression by the fusion gene constructs are shown relative to the expression of the positive control, a fusion gene of the RSV promoter and the luciferase reporter gene (pRSVL). pLuc0, a promoterless luciferase plasmid, served as the negative control. Relative values of luciferase activity in the resting state were adjusted by transfection efficiency as determined by CAT expression by cotransfecting cytomegalovirus promoter-CAT plasmid (pCMV-CAT). To evaluate the effects of TNF on IL-8 promoter activity in HS-24 cells, the cells transfected with the fusion genes were stimulated with TNF (100 units/ml) for 6 h. At the far right is shown, for each construct, the relative expression in the presence of TNF to that of resting cells. The data represent three independent transfection studies.

( i e . minimum promoter function) but does not respond to TNF stimulation. The observation that promoter activity in resting cells increases significantly when an additional up- stream 18 bp (-130 to -113) are included suggests the presence of an element(s) within this 18 bp segment that may function constitutively to enhance transcription. Interest- ingly, this segment also responds to TNF by increasing reporter gene expression, implying this segment contains an enhancer sequence responsive to TNF, a possibility also sug- gested by the presence of a DNase I hypersensitivity site emerging after TNF stimulation that is located in this region. Interestingly, nuclear run-on studies demonstrate that transcription of the IL-8 gene is at a very low level in resting HS- 24 cells, but transfection studies indicate active promoter function (13% of a control RSV promoter) with the sequence from -1481 to +44, i.e. there may be a controlling element(s) outside the -1481 to +44 region that may modulate low level transcription in resting HS-24 cells. In this regard, the DNase I hypersensitivity site mapping study implies that TNF causes HS-24 cells to close one site downstream of this region (at approximately +70), suggesting a negative regulatory element in this area which might be inactivated by TNF.

Acknowledgments-We thank Drs. H. Fujii and T. Shimada, Clin- ical Hematology Branch, National Heart, Lung, and Blood Institute, for providing the pCMV-luciferase and the pCMV-cat, and T. Raymer for excellent assistance in preparing the manuscript.

REFERENCES 1. Rennard, S. I., Beckmann, J. D., and Robbins, R. A. (1991) in

The Lung (Crystal, R. G., and West, J. B., eds) pp. 157-167, Scientific Foundations, Raven Press, New York

2. Breeze, R. G., and Wheeldon, E. B. (1977) Am. Reu. Respir. Dis.

3. Clarke, S. W., and Pavia, D. (1991) in The Lung (Crystal, R. G., and West, J. B., eds) pp. 1845-1859, Scientific Foundations, Raven Press, New York

4. Wanner, A., Boushey, H., Junod, A., and Perruchoud, A. (eds) (1988) Am. Rev. Respir. Dis. 138, Sl-S57

5 . Baggiolini, M., Walz, A., and Kunkel, S. L. (1989) J. Clin. Invest.

116,705-777

84,1045-1049

6. Matsushima, K., and Oppenheim, J. J. (1989) Cytokine 1 , 2-13 7. Leonard, E. J., and Yoshimura, T. (1990) Am. J. Respir. Cell Mol.

8. Elner, V. M., Strieter, R. M., Elner, S. G., Baggiolini, M., Lindley, I., and Kunkel, S. L. (1990) Am. J. Puthol. 136 , 745-750

9. Standiford, T. J., Kunkel, S. L., Basha, M. A., Chensue, S. W., Lynch 111, J. P., Toews, G. B., Westwick, J., and Strieter, R. M. (1990) J. Clin. Znuest. 8 6 , 1945-1953

10. Gimbrone, M. A., Obin, M. S., Brock, A. F., Luis, E. A., Hass, P. E., Hbbert, C. A., Yip, K. Y., Leung, D. W., Lowe, D. G., Kohr, W. J., Darbonne, W. C., Bechtol, K. B., and Baker, J. B. (1989) Science 246,1601-1603

11. Larsen, C. G., Anderson, A. O., Appella, E., Oppenheim, J. J., and Matsushima, K. (1989) Science 2 4 3 , 1464-1466

12. Dahinden C. A., Kurimoto, Y., De Weck, A. L., Lindley, I., Dewald, B., and Baggiolini, M. (1989) J. Exp. Med. 170, 1787- 1792

13. Burrows, L. J., Piper, P. J., Lindley, I., Baggiolini, M., and Westwick, J. (1989) Cytokine 1 , 100 (abstr.)

14. Shoji S., Ertl, R. F., and Rennard, S. I. (1987) Clin. Res. 35,539A (abstr.)

15. Reddel, R. R., Ke, Y., Gerwin, B. I., McMenamin, M. G., Lechner, J. F., Su, R. T., Brash, D. E., Park, J-B., Rhim, J. S., and Harris, C. C. (1988) Cancer Res. 4 8 , 1904-1909

16. Appelhans, B., Ender, B., Sachse, G., Nikiforov, T., Appelhans, H., and Ebert, W. (1987) FEBS Lett. 2 2 4 , 14-18

17. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, 0. D., Smith, J. A., Seidman, J. G., and Struhl, K. (1987) Current Protocols

York in Molecular Biology, pp. 4.9.1.-4.9.8, John Wiley & Sons, New

18. Church, G. M., and Gilbert, W. (1984) Proc. Natl. Acud. Sci.

BWl. 2,479-486

u. S. A. 81, im-1995 19. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,

W. J. (1979) Biochemistry 18,5294-5299 20. Feinberg, A. P . , and Vogelstein, B. (1983) Anal. Biochem. 132 ,

21. Roth, M. J., Tanese, N., and Goff, S. P. (1985) J. Biol. Chem. 260,9326-9335

22. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science

23. Schmid, J., and Weissmann, C. (1987) J. Zmmunol. 139 , 250- 256

24. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Koba- yashi, Y., Lew, W., Appella, E., Kung, H. F., Leonard, E. J.,

6-13

239,487-491


and Oppenheim, J. J. (1987) J. Exp. Med. 167 , 1883-1893 46. Strieter, R. M., Phan, S. H., Showell, H. J., Remick, D. G., Lynch, 25. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and J. P., Genord, M., Raiford, C., Eskandari, M., Marks, R. M.,

Kedes, L. (1983) Mol. Cell. Biol. 3 , 787-795 and Kunkel, S. L. (1989) J. Biol. Chem. 264,10621-10626 26. Ferrante, A., and Thong, Y. H. (1982) Immunol. Methods 4 8 , 47. Strieter, R. M., Kunkel, S. L., Showell, H. J., Remick, D. G.,

27. Harvath, L., Falk, W., and Leonard, E. J. (1980) J. Immunol. 243,1467-1469 Methods 37,39-45 48. Warren, J. S, Johnson, K. J., and Ward, P. A. (1991) in The Lung

28. Greenberg, M. E., and Ziff, E. B. (1984) Nature. 311,433-438 (Crystal, R. G., and West, J. B., eds) pp. 1939-1946, Scientific 29. Cohen, R. B., Boal, T. R., and Safer, B. (1990) EMBO J. 9,3831- Foundations, Raven Press, New York

3837 49. Crystal, R. G. (1991) in The Lung (Crystal, R. G., and West, J. 30. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 1 6 2 , B., eds) pp. 527-538, Scientific Foundations, Raven Press, New

31. Schiitte J., Minna, J. D., and Birrer, M. J. (1989) Proc. Natl. 50. Sibille, y., and Reynolds, H. y. (1990) Am. Reu. ResPir. Dis. 141 ,

81-85 Phan, S. H., Ward, P. A., and Marks, R. M. (1989) Science

156-159 York

Acad. Sci. U. S. A. 86, 2257-2261 471-501 32. Sheffery, M., Marks, p, A., and Rifkind, R. A, (1984) J , Mol. Bioi, 5 l . Hunninghake, G. w.9 Davidson, J. M.3 Rennard, s. 1 . 9 Szapiel, s.9

172,417-436 Gadek, J . E., and Crystal, R. G. (1981) Science 212 , 925-927 33. Southern E. M. (1975) J. Mol. Bwl. 98, 503-517 52. Rennard, S. I., Hunninghake, G. W., Bitterman, P. B., and 34. Mukaida, N., Shiroo, M., and Matsushima, K. (1989) J. Immunol. Crystal, R. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 7147-

7151 35. ~ i ~ , J, M., Fujii, H., G ~ ~ ~ ~ , s. w , , ~ ~ ~ ~ t ~ ~ , N., young, N, s., 53. Banda, M. J., Rice, A. G., Griffin, G. L., and Senior, R. M. (1988)

J. Biol. Chem. 263,4481-4484

J. Exp. Med. 167 , 1608-1615 55. Rankin, J. A., Sylvester, I., Smith, S., Yoshimura, T., and Leon-

ard, E. J. (1990) J. Clin. Inuest. 86, 1556-1564 56. Strieter, R. M., Chensue, S. W., Basha, M. A., Standiford, T. J.,

Lynch 111, J . P., Baggiolini, M., and Kunkel, S. L. (1990) Am. J . Respir. Cell Mol. Biol. 2, 321-326

57. Thompson, A. B., Daughton, D., Robbins, R. A., Ghafouri, M. A., Oehlerking, M., and Rennard, S. I. (1989) Am. Reu. Respir. Dis. 140,1527-1537

58. Boat, T. F., Welsh, M. J., and Beaudet, A. L. (1989) in The

Cell. Biol. 2, 1044-1051 Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) 6th Ed., pp. 2649-2680,

B., eds) PP. 1899-1911, Scientific Foundations, Raven Press, 59. Gregory, H., Young, J., Schroder, J”., Mrowietz, u., and Chris- McGraw-Hill, New York

New York tophers, E. (1988) Biochem. Biophys. Res. Commun. 151,883-

R.3 and Robbin% R. A. (1989) Am. J. PhYSiOl. 257, L130- 60. Larsen, C. G., Anderson, A. O., Oppenheim, J. J., and Matsush-

44. Robbins, R. A., Shoji, s., Linder, J., Gossman, G. L., Allington, 61. Watson, M. L., Lewis, G. P., and Westwick, J. (1988) Immunology

62. Kowalski, J., and Denhardt, D. T. (1989) Mol. Cell. Biol. 9,1946-

Kuramoto, A., and Mizuno, S. (1989) J. Exp. Med. 169 , 1895- 63. Mukaida N., Mahe, Y., and Matsushima, K. (1990) J. Biol. Chem.

143,1366-1371

and Shimada, T. (1991) Virology 182, 361-364

Acad. Sci. U. S. A. 74,5463-5467 37. De Wet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., and

Subramani, S. (1987) Mol. Cell. Biol. 7 , 725-737 38. Yoshimura, K., Nakamura, H., Trapnell, B. C., Dalemans, W.,

Pavirani, A., Lecocq, J-P., and Crystal, R. G. (1991) J. Biol. Chem. 266,9140-9144

39. Potter, H., Weir, L., and Leder, P. (1984) Proc. Natl. Acad. Sci.

40. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 41. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol.

42. Reynolds H. Y. (1991) in The Lung (Crystal, R. G., and West, J.

36. sanger, F., ~ i ~ k l ~ ~ , s., and coulson, A. R. (1977) proc, ~ ~ t l . 54. Band% M. J.7 Rice, A. G., Griffin, G. L., and Senior, R. M. (1988)

U. S. A. 81, 7161-7165

43. Koyama, S., Rennard, S. I., Shoji, S., Romberger, D., Linder, J., 890

L136

L. A., Klassen, L. W., and Rennard, S. I. (1989) Am. J. Physiol. 65,567-572

ima, K. (1989) Immunology 68,31-36

257, L109-L115 45. Suzuki, K., Miyasaka, H., Ota, H., Yamakawa, Y., Tagawa, M., 1957

1901 265,21128-21133

interleukin-8 gene expression in human bronchial ... · 19612 interleukin-8 expression in bronchial...

Documents