INTERLEUKIN-13 INDUCES DRAMATICALLY DIFFERENT TRANSCRIPTIONAL PROGRAMS IN THREE HUMAN AIRWAY CELL TYPES
June H. Lee M.D.*t‡tt, Naftali Kaminski M.D.*t‡§tt, Gregory Dolganov Ph.D. t‡, Gabriele Grunig D.V.M., Ph.D. *‡║, Laura Koth M.D.*t‡, Colin Solomon Ph.D.*‡, David Erle M.D.*t‡, Dean Sheppard M.D.*t‡**
From *Lung Biology Center and Center for Occupational and Environmental Health, tCardiovascular Research Institute and the ‡Department of Medicine, University of California, San Francisco, California. §Functional Genomics and Institute of Respiratory Medicine, Sheba Medical Center, Tel Hashomer, Israel. ║Columbia University, St. Luke's-Roosevelt Hospital New York, NY.
**To
whom reprints and correspondences should be addressed at:
Lung
Biology Center, UCSF Box 0854, San Francisco, California 94143
Telephone:
(415) 206-5901
Fax:
(415) 206-4123
E-mail: deans@itsa.ucsf.edu
Acknowledgements: This work was supported by NIH grants HL 47412, HL53949 and HL56385 (to DS), by training grant HL09961 (to JL) and by the UCSF Sandler Center for Basic Research in Asthma.
ttAuthors denoted contributed equally
Interleukin-13 (IL-13), a cytokine released by T lymphocytes during immediate hypersensitivity responses, is a central mediator of asthma. Because IL-13 induces phenotypic features of asthma in mice deficient in T and B lymphocytes, it is likely that this cytokine contributes to the development of asthma by acting directly on resident airway cells. To analyze the global effects of IL-13 on gene expression in airway cells that could contribute to the phenotypic features of asthma, we used Affymetrix GeneChipâHuGene FL arrays that contain probes for approximately 6,500 human genes. Despite activating a common signaling pathway, IL-13 induced dramatically different patterns of gene expression in primary cultures of airway epithelial cells, airway smooth muscle cells and lung fibroblasts, with little overlap among cell types. The most prominent effects of IL-13 were on airway smooth muscle, but several genes induced in airway epithelial cells and fibroblasts are also candidates to contribute to phenotypic features of asthma. These results suggest that the in vivo response to IL-13 in the airways likely results from a combination of distinct effects on each of several resident airway cell types.
Running title: IL-13 induced transcriptional programs
Asthma
is a complex disease with increasing worldwide incidence and significant
morbidity and mortality. The
disease is characterized by exaggerated narrowing of the conducting airways of
the lung in response to bronchoconstrictor stimuli (airway hyperresponsiveness).
Morphologic changes in the airways of patients with asthma include eosinophilic
inflammation, mucus metaplasia (1),
subepithelial fibrosis (1, 2),
and smooth muscle hypertrophy (3).
T lymphocytes in the airways of patients with asthma are principally of
the T helper 2 (Th2) type and produce the Th2 cytokines, interleukin-4, 5 and 13
(4, 5).
The genes encoding these cytokines are located on human chromosome region
5q25-31, a region mapped by linkage analysis to asthma in several genome-wide
screens (6).
Many of the phenotypic features of asthma can be reproduced by antigen
sensitization and inhalational challenge in mice. Recently, we and others have
shown that inhibition of interleukin 13 (IL-13) blocks antigen-induced effects
on the airways and that administration of IL-13 to the airways of mice can
itself induce mucus metaplasia, eosinophilic inflammation and airway
hyperresponsiveness (7, 8).
These effects of IL-13 do not require the participation of T cells or B cells
since they are retained in recombinase-activating gene-1 null mice (7),
suggesting that they are largely dependent on the effects of IL-13 on resident
airway cells. However, the contributions of various airway cell types to the in
vivo effects of IL-13 are largely unknown. IL-13, like the closely related
cytokine IL-4, binds
to receptors composed of the common IL-4Ra subunit and one of two IL-13a
subunits (a1 or a2). Current evidence suggests that only receptors containing
the IL-13Ra1 subunit are capable of inducing subsequent signals. Ligation of the
IL-13Ra1/ IL-4Ra receptor complex induces tyrosine phosphorylation of a member
of the signal transduction and transactivation (STAT) family, STAT6, which is
essential for many of the known biologic functions of IL-13 and IL-4.
While other signaling pathways including insulin receptor substrate
(IRS)-1 and IRS-2(9) can be
activated by IL-13, they have not been shown to contribute to IL-13 mediated
development of allergic asthma. Mice
lacking STAT6 (10), and mice
lacking STAT6 activation by IL-13, such as mice treated with an IL-13 receptor
antagonist (7, 8), are
protected from many of the phenotypic features of allergic asthma, suggesting
that this pathway is critical to the contribution of IL-13 to asthma. Once
phosphorylated, STAT6 forms a homodimer and translocates to the nucleus, where
it directly binds to regulatory DNA sequences and modulates expression of IL-13
responsive genes. Because IL-13 principally affects cell behavior by altering
gene expression, we initiated exploration of the molecular mechanisms underlying
IL-13's contributions to asthma by examining the global effects of IL-13 on gene
expression in the three major resident cell types in the airway wall: epithelial
cells, smooth muscle cells and fibroblasts.
Cell culture and reagents
Normal
human bronchial epithelial cells (NHBE), bronchial smooth muscle cells (BSMC)
and normal human lung fibroblasts (NHLF) were purchased from Clonetics and grown
at 37 ° C in small airways growth media (SAGM), smooth muscle growth media (SmGM),
and fibroblast growth media (FGM-2), respectively, as recommended. All cells
used for each cell type were obtained from a single donor. Rabbit polyclonal
anti-STAT6 antibody was obtained from Santa Cruz Biotechnology. Mouse monoclonal
anti-phosphotyrosine antibody PY20 was obtained from Transduction Laboratories.
Recombinant human IL-13 was obtained from R & D Systems.
Leupeptin and aprotinin were obtained from Calbiochem-Novabiochem Corp.
and phenylmethylsulfonyl fluoride and pepstatin were obtained from Sigma
Chemical Co.
Western
blotting
Cells were grown to confluence in 100
mm tissue culture plates, placed in basal media overnight and treated with
recombinant IL-13 (100ng/ml) or PBS for 20 minutes.
Total cell extracts were obtained using RIPA buffer (0.1% SDS, 1% sodium
deoxycholic acid, 1% Nonidet P40, 1mM vanadate, 0.5mM molybdate) in the presence
of 10 mg/ml pepstatin 10 mg/ml leupeptin, 5 mg/ml aprotinin and 1 mM
phenylmethylsulfonyl fluoride. Protein concentrations were determined by the BCA
method (Pierce).
To detect tyrosine phosphorylation of STAT6, aliquots of lysates containing equal protein concentrations were precleared with protein G-Sepharose, incubated with anti-STAT6 rabbit polyclonal antibody for 3 hours at 4° C and immune complexes were captured by protein G-Sepharose for 90 minutes at 4° C. The beads were washed and immunoprecipitated proteins were solubilized in 5X reducing Laemmli sample buffer. Samples were separated by SDS-PAGE on 10% polyacrylamide gel and transferred to Immobilon membranes for Western blotting with anti-phosphotyrosine antibody PY20. Membranes were blocked in 3% bovine serum albumin and incubated with primary antibody for 1 hour at room temperature followed by secondary antibody incubation with anti-mouse HRP and developed with ECL (Amersham Pharmacia Biotech).
RNA
Extraction
Cells
were grown to confluence in growth media and placed in basal media overnight
prior to stimulation with recombinant IL-13 (100 ng/ml) or PBS. After 6 hours,
total RNA was isolated using Trizol Reagent (Gibco BRL). RNA quantity was
determined by OD measurement at 260 nm and quality was determined by RNAse
negative 1% agarose gel.
Preparation
of labeled cRNA
10-15
mg of total RNA was used for double stranded cDNA synthesis.
Double stranded cDNA was generated with a cDNA synthesis kit (Life
Technologies Superscript cDNA Synthesis System) using an oligo(dT)24
primer containing a T7 RNA polymerase promoter site at the 3' end (Genset). The
cDNA was extracted with phenol/chloroform, ethanol precipitated and used as a
template for in vitro transcription with biotin labeled nucleotides (BioArrayTM
HighYieldTM RNA Transcript Labeling Kit - Enzo Diagnostics). 12.5 mg
of the labeled cRNA was fragmented at 94o C for 35 min in
fragmentation buffer (40 mM Tris-acetate, pH 8.1/100mM potassium acetate, 30 mM
magnesium acetate) and a hybridization mix was generated by addition of Herring
sperm DNA (0.1 mg/ml, Sigma), sodium chloride (1 M), Tris acetate (10 mM), and
Tween 20 (0.0001%). A mixture of four control bacterial and phage cRNA (1.5 pM
BioB, 5 pM BioC, 25 pM BioD and 100 pM Cre) was included to serve as an internal
control for hybridization efficiency.
Hybridization
of microarrays
Aliquots
of each sample (10 mg
cRNA in 200 ml
hybridization mix) were hybridized to a Genechip Hugene FLâ
array (Affymetrix). After
hybridization, each array was washed, stained with streptavidin phycoerythrin
(Molecular Probes), washed again, hybridized with biotin labeled anti-streptavidin
phycoerythrin antibodies (Vector Laboratories), restained with streptavidin
phycoerythrin and scanned (Hewlett Packard, GeneArrayTM scanner
G2500A) and washed according to procedures developed by manufacturer (Affymetrix).
Samples obtained from four individual dishes of each cell type were included for
each condition examined.
Analysis
of Genechip data
Scanned
output files were visually inspected for hybridization artifacts. Arrays lacking
significant artifacts were analyzed using Genechipâ
3.3 software (Affymetrix).
Arrays were scaled to an average intensity of 150 per gene and analyzed
independently. The expression value for each gene was determined by calculating
the average of differences (perfect match intensity minus mismatch intensity) of
the probe pairs in use for that gene.
The expression analysis files created by Genechipâ 3.3 software were then transferred to a database (Microsoft Access) linked to Internet genome databases (e.g. NHLBI, Swiss Prot, and GeneCards) to update gene definitions. Fold changes were determined by dividing the average difference of each sample by the mean of the average differences of the PBS samples.
Because meaningfulness of low and negative intensity readings are unclear, value of 20 was assigned to all measurements lower than 20 to facilitate calculations. Genes that were either increased at least 1.5-fold or decreased at least by 50% from baseline in 3 or 4 samples were included in subsequent analyses. For further data analysis and data presentation we used Spotfire Pro 4.0 (Spotfire). For cluster analysis we used Gene Cluster and Treeview programs described by Michael Eisen (11).
MCP-1
and IL-6 ELISA
MCP-1
ELISA was performed using monoclonal anti-human MCP-1 antibody and biotinylated
anti-human MCP-1 antibody as described by the manufacturer (R&D Systems,
Minneapolis, MN) with the exception of the detection method. For detection, 40 ml
of 2 mg/ml
alkaline-phosphatase-conjugated streptavidin (Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA) was added and incubated for 1 hour. The
plate was washed 5 times with 0.05% Tween 20 in PBS, and incubated with 100 ml
per well 1 mg/ml p-Nitrophenyl Phosphate (Sigma Diagnostics, Inc, St. Louis, MO)
for 5 minutes. The reaction was
stopped by adding 30 ml
0.5 M NaOH and optical densities were measured at 405 nm. Test
conditions were established using recombinant human MCP-1 (R&D Systems,
Minneapolis, MN) as a standard and positive control. MCP-1 concentrations in the
samples were obtained by using a standard curve of diluted recombinant human
MCP-1 (R&D Systems, Minneapolis, MN).
The IL-6 concentration in conditioned media was measured using ELISA (Pharmacia IL-6 optEIA kit, No. 2645 KI), as described by the manufactures protocol, except for the use of a chemiluminescent developing substrate (Pierce, Pico). The 96 well plates (NUNC, Fluoronunc Module Masisorp Surface, Cat. No. 475515) were coated over-night with the anti-human IL-6 monoclonal capture antibody (Pharmacia) and blocked with 10%FBS-PBS for 1 hour at room temperature. Undiluted samples were incubated in the plates for 2 hours on a shaker. Biotinylated anti-human IL-6 detection antibody conjugated with HRP-Streptavidin (Pharmacia) was incubated in the plates for 1 hour on a shaker. Plates were developed using a chemiluminescent substrate (Pierce, Pico) and measured after 15 minutes using a luminometer (Wallac, Victor2 1420 Multilabel Counter). IL-6 concentrations were determined from a standard curve of diluted recombinant human IL-6 (Pharmacia).
TaqMan
amplification
Aliquots
of RNA used for oligo array experiments were used for preparation of cDNA for
TaqMan amplification. Gene-specific
primers were used in multiplex RT-PCR to generate gene-specific cDNAs. The
"Primer Express" package from Perkin Elmer (Foster City, CA) was used
to design both RT- and TaqMan primers/probes.
The sequences for the primers/probes can be found on the following site http://owl.ucsfmedicalcenter.org.
TaqMan primer-probe sets were designed to span exon-intron junctions
whenever possible. Optimization of multiplex PCR was done as described (12-14)
using KlenTaq DNA polymerase (cDNA Advantage Mix from Clontech, Palo Alto, CA).
The reaction was heated at 94° C for 2 minutes followed by 0-25 cycles
with 94° C for 30 seconds, 55° C for 15 seconds, and 70° C for 45 seconds.
The number of PCR cycles was kept as low as possible to enable efficient gene
quantification. Gene-specific
multiplex RT-PCR was performed several times with different numbers of PCR
cycles, and all data generated were highly reproducible in the range of 0-25
cycles. For each RNA sample, dilution curves were generated to confirm linear
dependence of the signal on the concentration of template RNAs. Cycle thresholds
were determined for each reaction, normalized to the cycle threshold for the
housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
expressed as relative copy number by comparison to a standard curve. Mean values
were calculated from at least 3 separate samples for each condition analyzed and
expressed as fold difference from mean values for the same gene after treatment
with PBS.
IL-13
induces STAT6 phosphorylation in bronchial epithelial cells, bronchial smooth
muscle cells and lung fibroblasts
To
determine whether IL-13 is capable of activating the same signaling pathway in
bronchial epithelial cells, bronchial smooth muscle cells and lung fibroblasts,
we assayed tyrosine phosphorylation of STAT-6 by immunoprecipitation followed by
Western blotting with anti-phosphotyrosine antibody PY20. As shown in Figure 1A,
a band of the appropriate molecular mass to be STAT6 was phosphorylated in
response to IL-13 in all 3 cell types. Similar amounts of STAT6 were present and
immunoprecipitated in equal quantities of cell lysates (Figure 1B).
IL-13
induces different patterns of gene expression in primary airway cells
To determine whether IL-13 had similar
effects on gene expression in each cell type, and to identify the major genes
induced and inhibited, we evaluated the global pattern of gene expression after
6 hours of stimulation with IL-13 with Affymetrix genechips containing probe
sets to detect 6500 human genes. Cluster analysis viewed using the Treeview
program demonstrated different patterns of gene expression in each cell type
tested (Figure 2). In bronchial epithelial cells (NHBE), lung fibroblasts (NHLF),
and bronchial smooth muscle cells (BSMC), and 80, 79, and 204 genes,
respectively, were induced by at least 2 fold in at least 3 of the 4 samples. Of
these genes, only two were common between airway epithelial cells and airway
smooth muscle (TRAIL, lymphoma proprotein convertase), none were common between
airway epithelial cells and fibroblasts, three were common between airway smooth
muscle cells and fibroblasts (Vitamin D receptor, Placental bikunin, SS-A/Ro
ribonucleoprotein autoantigen) and none were induced in all 3 cell types.
Another outstanding feature of this cluster analysis was the large number of
genes that were affected by IL-13 in airway smooth muscle cells, a likely target
for the induction of airway hyperresponsiveness.
Because of the high cost of this approach, we limited this experiment to analysis of a single time point. At this time point, changes in gene expression reflect both genes that are directly modulated by IL-13-induced signals (e.g. through STAT6) and genes that are modulated indirectly through IL-13-induced effects on genes whose products can themselves modulate subsequent gene expression (e.g. transcription factors). Further evidence for the divergence of effects of IL-13 in each of the cell types examined comes from analysis of the specific transcription factors induced by IL-13. As shown in Table 1, IL-13 induced at least a two-fold increase in several transcription factors in each cell type, but there was no overlap between cell types.
Expression data was sorted for genes with >1.5 fold induction over the baseline in at least 3 of 4 treated dishes, and cell-specific lists of genes induced or inhibited by IL-13 are presented in tables 2-5. Induced genes were further evaluated by searching existing databases to assign as many as possible to functional groups.
IL-13
induces expression of several signaling effectors, signaling receptors,
contractile proteins and ion channels in bronchial smooth muscle cells
Among the most dramatic effects of
IL-13 on bronchial smooth muscle cells were induction of a number of signaling
molecules and signaling receptors that could prime these cells for enhanced
responses to a wide array of other stimuli. Notable among these were several
components of MAP kinase signaling pathways (e.g. jnk1b2, jnk2, Mek5c and
MAPkAPK2), phospholipase A2 and diacylglycerol kinase d. Enhanced activity of
these pathways could contribute to airway smooth muscle cell proliferation
and/or hypertrophy and the increase in airway smooth muscle mass that
characterizes asthma. Among the
most highly induced were signaling molecules of the src family, fgr and the CXC
chemokine receptor CXCR2 which are of particular interest since neither of these
proteins has been previously identified on smooth muscle cells. IL-13 also
increased expression of the signaling IL-13Ra1 subunit, an effect that could
result in a positive feedback loop that would amplify and prolong responses to
IL-13 in these cells.
IL-13 also induced expression of several contractile proteins (sarcolipin, dystroglycan associated protein, smooth muscle myosin heavy chain and cardiac b myosin heavy chain) and ion channels (e.g. KCNQ2, KVLQT1 and CLCL3), effects that could alter the contractile response of these cells and thereby contribute to airway hyperresponsiveness. Finally, IL-13 induced expression of secreted factors in airway smooth muscle cells, such as the growth factor bFGF and the IL-6 family cytokine, LIF, which could contribute to the asthma phenotype through autocrine effects or paracrine effects on other airway cells.
Il-13
induces expression of different secreted proteins, signaling molecules and
receptors in lung fibroblasts
As noted in Table 2, distinct members
of some of the same functional classes of genes were induced by IL-13 in lung
fibroblasts. Most noteworthy was induction of secreted cytokines and growth
factors. These include Monocyte Chemotactic Protein-1 (MCP-1), a chemokine that
has been shown to induce collagen production from fibroblasts and contribute to
models of asthma and tissue fibrosis (15-17),
and the growth factor IL-6, whose over-expression has been shown to induce
sub-epithelial fibrosis in transgenic mice (personal communication, Dr. Jack
Elias). To confirm that these effects of IL-13 might be biologically significant
we measured secretion of MCP-1 and IL-6 by IL-13 stimulated lung fibroblasts
with an ELISA assay. As shown in Figure 4, IL-13 induced a more than two-fold
increase in secretion of each of these gene products. IL-13 also increased
expression of the transcript for potent angiogenic factor angiopoietin 1, which
could contribute to the angiogenesis seen in asthmatic airways, and a platelet
derived growth factor isoform that would be expected to induce proliferation of
smooth muscle cells and fibroblasts.
To confirm that genechip analysis was accurately measuring RNA abundance, we utilized the same input RNA used for genechip analysis from treated and untreated fibroblasts to determine RNA abundance with a different technique, TaqMan quantitative PCR (Figure 4). We chose three genes for this analysis (IL-6, MCP-1 and the Vitamin D receptor), that were induced more than two-fold by IL-13, two genes (the chloride channel, CLCN3 and serum response factor (SRF)) that were induced between 1.5 and two fold, and 9 genes that were expressed but unaffected by IL-13. Although there were quantitative differences in the determined fold induction, especially for the 3 most highly induced genes, both methods demonstrated increased expression of the same 5 genes and essentially no change in expression of the other 9 genes.
IL-13
induces expression of secreted extracellular matrix proteins, proteases and
protease inhibitors in bronchial epithelial cells
The most prominent groups of genes
induced by IL-13 on bronchial epithelial cells were genes whose products are
involved in production and turnover of the extracellular matrix (Table 2A).
IL-13 also increased expression of a small number of signaling receptors and
effectors in these cells, but this effect was not as prominent as that seen in
airway smooth muscle cells or fibroblasts. As with induction of angiopoietin-1
in fibroblasts, induction of the gene for the angiogenic agonist placental
growth factor could contribute to airway mucosal angiogenesis in asthma. In each
cell type, IL-13 also inhibited expression of a large number of genes (Tables
3B, 4B, 5B). Analysis of the functional significance of these genes is also
likely to be informative, and is getting underway.
The results of this study demonstrate that a single cytokine, IL-13, using a common signaling pathway that activates a common transcription factor (STAT6) induces different patterns of gene expression in three different primary cell types, with virtually no overlap in the genes that are induced or inhibited in each cell type. The resulting gene expression profile suggests a coordinate and distinct contribution to asthma pathogenesis by each of the cell types examined.
As noted above, IL-13 has been identified as a critical mediator of allergic airway responses in mice and has been suggested to be important in human asthma. In mice, IL-13 has been shown to cause airway hyperresponsiveness, mucus metaplasia, eosinophilic inflammation and sub-epithelial fibrosis (7, 8, 18), which are all features of the human disease (2, 3, 19). The molecular mechanisms underlying these responses are largely unknown. We thus sought to utilize information gained by global analysis of gene expression to generate hypothesis about the cellular targets for IL-13 in the airways and the molecular pathways by which IL-13 might affect the function of these target cells. In that regard, the numerous genes that were affected by IL-13 in airway smooth muscle cells was especially informative and suggests that smooth muscle is likely an important target for IL-13 in the airways. Induction of signaling proteins, including several components of MAP kinase cascades, and of contractile proteins and ion channels suggests that IL-13 could coordinately enhance the contractile (and perhaps, proliferative) response of airway smooth muscle. Among the contractile proteins induced was Dystrophin associated glycoprotein (DAG), a glycoprotein known to be decreased in disorders associated with muscle weakness, such as Duchenne’s muscular dystrophy, with a suspected role in muscle anchoring (20). More recently, DAG has also been found to be increased at sites of muscle injury and muscle regeneration and to contribute to regeneration of tensile strength of the muscle (21). Another induced gene, sarcolipin is a modulator of Ca2+- ATPase (SERCA) type pumps which are known to have a role in regulating the contraction of fast twitch skeletal muscle (22).
The large number of signaling receptors and effectors induced by IL-13 in these cells also suggests that bronchial smooth muscle could be "primed" by IL-13 to enhance responsiveness to a variety of other contractile or proliferative signals. One of the most prominently induced genes in this category was fgr, a Src family tyrosine kinase previously thought to be restricted to mature cells of myeloid lineage. Fgr has been shown to contribute to cell motility in eosinophils (23), neutrophils (24), and macrophages (25) and to adhesion dependent degranulation of neutrophils (26) and is thought to play an important role in cytoskeletal reorganization in these cells. Fgr has not been previously described in smooth muscle cells. By analogy to roles played by other Src isoforms, fgr is a candidate to contribute to smooth muscle cell proliferation in response to IL-13. In addition to effects on individual smooth muscle cells, IL-13 increased expression of genes encoding a number of secreted proteins in bronchial smooth muscle cells. Some of these, such as the growth factor, bFGF and the IL-6 like cytokine LIF could affect other cells, such as fibroblasts, in the airway wall and thereby contribute to the sub-epithelial fibrosis induced by IL-13.
Given the well-established role of IL-13 in inducing fibrosis in the airways and in models of parasitic disease in other organs, we expected to identify dramatic induction of extracellular matrix components such as collagen isoforms in lung fibroblasts. Surprisingly, while IL-13 did increase expression of several matrix proteins, this effect was principally seen in bronchial epithelial cells, not in fibroblasts. However, two of the most highly induced genes in fibroblasts encoded the secreted proteins IL-6 and MCP-1, and we were able to confirm that IL-13 increased the secretion of each these proteins by lung fibroblasts. These findings are noteworthy because both IL-6 and MCP-1 have been implicated in models of tissue fibrosis. Over-expression of IL-6 and the related cytokine IL-11 under the control of the airway epithelial CC10 promoter causes sub-epithelial fibrosis in mice (27). MCP-1 has been shown to induce collagen production from fibroblasts, an effect that appears to involve induction of the potent pro-fibrotic cytokine, transforming growth factor b (TGFb). Recently, studies in mice lacking CCR2, a principal receptor for MCP-1, have also suggested a role for this chemokine in induction of airway hyperresponsiveness in a model of allergic asthma (28).
Several of the genes whose transcriptions were affected by IL-13 were somewhat unexpected. T-cell receptor (TCR) and Myf4 were both decreased in NHBE and Cardiac b myosin heavy chain was induced in BSMC by IL-13 stimulation. These genes were previously thought to be restricted to other cell types. At this time, the significance of the presence of these genes in the cells studied is unclear. Whether the presence of these transcripts translate to the presence of meaningful protein levels has not yet been determined and will require further experiments.
Clearly, analysis of global patterns of gene expression, as described here, is a powerful approach to identify transcriptional programs that could contribute to disease pathogenesis. However, this approach can only identify promising candidates and suggest hypotheses that must ultimately be tested in in vivo models. There are also important limitations to our experimental approach that are worth noting. In the current study, we utilized primary cultures of airway cells. While this approach has the advantage of allowing us to ascribe any changes identified to events that result only from the effects of IL-13 on that cell type, it is likely that transcriptional responses of cells in culture differ considerably from the responses of the same cell in vivo. Since we have shown in this report that cellular differentiation can have enormous effects on these responses, it will ultimately be essential to confirm that genes of interest are also expressed in the relevant cell type in vivo in response to IL-13. Another important limitation to RNA-based analysis of gene expression as described here is the fact that expression of many proteins is regulated at steps downstream of mRNA synthesis. We have begun to address this issue by confirming that at least some of the most interesting changes in gene expression we describe (e.g. induction of IL-6 and MCP-1 in lung fibroblasts) are also associated with similar changes in expression of the encoded proteins.
In this set of experiments, we analyzed the effects of a single cytokine (IL-13) on three functionally diverse but anatomically adjacent cell types. Despite initiation of an identical signaling pathway (STAT-6), IL-13 induced highly distinct transcriptional programs in each of the three cell types. Furthermore, in each cell type, IL-13 induced genes that encode for proteins that may play a significant role in the pathogenesis of chronic asthma, suggesting that the asthma phenotype is likely the result of coordinated effects of IL-13 on the three airway cell types studied. This hypothesis will require specific confirmation and testing in in vivo models. However, the results from our study should facilitate the design and completion of such studies.
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TABLE
2A Selected genes induced in airway epithelial cells
TABLE 2B Selected genes induced in BSMC
TABLE 2C Selected genes induced in NHLF
TABLE 3A Genes most highly induced in airway epithelial
cells
TABLE 3B Genes most decreased in airway epithelial
cells
TABLE 4A Genes most highly induced in BSMC
TABLE 4B Genes most decreased in BSMC
TABLE 5A Genes most highly induced in NHLF
TABLE 5B Genes most decreased in NHLF
Figure 1 -A. Phosphotyrosine immunoblot of cell lysates of normal human lung fibroblasts (NHLF), normal human bronchial epithelial cells (NHBE), and bronchial smooth muscle cells (BSMC) incubated with or without IL-13 for 20 minutes. Lysates were immunoprecipitated with STAT-6 antibody and probed with anti-phosphotyrosine antibody. B. STAT-6 immunoblot of the filter used in Figure 1A.
Figure 2 -Cluster analysis of expression of ~1200 genes with gene expression ratio of >1.5 or <0.5 (in at least n-1 samples) to baseline after 6 hour incubation with IL-13. Each row represents one gene and each column represents one IL-13 stimulated cell sample. For each sample, expression values after IL-13 treatment are compared to the average gene expression of unstimulated (PBS control) samples. Green represents transcript levels below baseline, black represents transcript levels equal to baseline, and red represents transcript levels above baseline. NHBE: normal human bronchial epithelial cells; NHLF: normal human lung fibroblasts; BSMC: bronchial smooth muscle cells. Lines to the right of the cluster analysis represent clusters of genes induced by IL-13 in individual cell types and lines to the left represent cluster of genes inhibited by IL-13. A: Genes induced in airway epithelial cells. B: Genes induced in lung fibroblasts. C: Genes inhibited in bronchial smooth muscle cells. D: Genes induced in bronchial smooth muscle cells
Figure 3- Results of ELISA assays from conditioned media of IL-13 stimulated lung fibroblasts for MCP-1 (Panel A) or interleukin-6 (IL-6, Panel B). The cells were treated with PBS (control) or IL-13 at 100 ng/ml for 24 hours or 48 hours. Error bars represent standard error of the mean for values from 3 dishes analyzed at each time point.
Figure 4 - Comparison of fold induction in selected genes in lung fibroblasts as measured by Taqman method or oligonucleotide array method.
