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Abstract
Introduction
Materials and Methods
Results
Discussion
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Alveolar epithelial cells produce many types of chemokines such
as regulated on activation, normal T cells expressed and secreted (RANTES), eotaxin induced by interleukin (IL)-1
, or
tumor necrosis factor (TNF)-
and may contribute to allergic
disease by recruiting eosinophils. However, identification of
the eosinophil chemotacic activity (ECA) release from A549
cells, an alveolar type II cell line, has not yet been completed.
Recently, IL-16 was also reported to be a potent chemotactic
stimulus for CD4+ T lymphocytes and eosinophils in asthma
and other pulmonary diseases. To test the possibility that alveolar epithelial cells produce IL-16, we analyzed RNA and culture supernatant from A549 cells by reverse transcription/
polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The release of ECA from A549 cells
was assessed using a blind-well chemotactic chamber. IL-16
release was increased in a concentration-dependent manner by stimulation with IL-1 or TNF-. A549 cells also expressed IL-16 messenger RNA. The combination of IL-4 and IL-1 or
TNF- had an additive effect on IL-16 production. The release
of ECA was induced by IL-1 or TNF- in a dose-dependent
manner. The combination of these cytokines had a greater effect than one alone. The blockade of eotaxin and IL-16 caused
70% inhibition of ECA, but anti-RANTES antibodies only
caused 30% inhibition and anti-IL-8 antibodies failed to affect
inhibition. These findings suggest a role for chemokines released by alveolar epithelial cells in the recruitment of eosinophils into the lung in pulmonary disorders such as asthma and
interstitial lung diseases, and suggested that eotaxin and IL-16 are potent and effective eosinophil chemoattractants.
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Introduction
Materials and Methods
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Several studies have demonstrated that eosinophils play an important role in the pathogenesis of allergic airway diseases (1, 2). Eosinophil recruitment into the lung is associated with both acute and chronic pulmonary disorders (3). Kraft and coworkers (4) recently studied chronic asthmatics with or without nocturnal asthma by comparing proximal airway endobronchial tissue with distal alveolar tissue and they found that eosinophils and macrophages accumulated to a greater extent in the alveolar tissue and these changes contribute more to the variation in lung function than does inflammation in the more proximal tissue. Despite increasing evidence of an effector role for eosinophils in asthma and other allergic diseases, the mechanisms underlying the recruitment of eosinophils into the airway are still unclear.
Type II alveolar epithelial cells (ATII cells) have been
shown to play a key role in the regulation of the alveolar
space. ATII cells synthesize and secrete surfactant, control
the volume and composition of the epithelial lining fluid,
and proliferate and differentiate into type I alveolar epithelial cells after injury in order to maintain the integrity
of the alveolar wall (5). The alveolar epithelium, which has
been traditionally regarded as a target of the inflammatory
response, may play an important role by its capacity to
modify the development and resolution of the inflammatory reaction into the alveolar space. In previous studies,
A549 cells, an ATII cell line, synthesized the messenger RNA (mRNA) and immunoreactive proteins for several
chemokines, including interleukin (IL)-8, regulated on activation, normal T cells expressed and secreted (RANTES),
and eotaxin, which directly or indirectly influence the activity of eosinophils (6). Production of chemokines by
alveolar epithelial cells may contribute to the local accumulation of inflammatory cells in patients with asthma and
other airway inflammatory diseases. Identification of the eosinophil chemotactic activity (ECA) released from A549
cells in response to pleiotropic proinflammatory cytokines
such as IL-1, tumor necrosis factor (TNF)-, and IL-4 is
not completed.
IL-16 is a chemoattractant cytokine that does not belong to either the CXC or C-C chemokine families. IL-16
was recently reported to be a potent chemotactic stimulus
for CD4+ T lymphocytes and eosinophils (9, 10). Cruikshank and colleagues (11) identified IL-16 and macrophage
inflammatory protein (MIP)1a in the bronchoalveolar lavage fluid (BALF) of asthmatics 6 h after antigen challenge
and showed that the majority of the lymphocyte chemoattractant activity in BALF is attributable to IL-16 and MIP-1a.
We have reported that human bronchial epithelial cells express IL-16 mRNA and protein in a concentration-dependent manner after stimulation with IL-1 and TNF- (12).
To elucidate the potential role of alveolar epithelial
cells in the recruitment of eosinophils, we investigated the
capacity of the alveolar epithelial cell line A549 to release
IL-16 in response to proinflammatory cytokines. The effect of IL-1 and TNF- on the release of ECA from A549
cells was also evaluated.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Cell Culture
A549 cells, human type II-like epithelial lung cells, were obtained from the Japanese Collection of Research Bioresources Cell Bank (National Institute of Health Sciences, Tokyo, Japan). The cells were cultured in tissue flasks incubated in 100% humidity and 5% CO2 at 37°C in Hams' F12K medium (N-3520; Sigma, St. Louis, MO) supplemented with 10% heat-inactived fetal bovine serum (GIBCO BRL, Grand Island, NY) and penicillin-streptomycin (50 µg/ml, GIBCO BRL), at 1 × 106 cells/ml. A549 cells were then plated onto 12-well, flat-bottom tissue culture plates (Falcon 3043; Becton Dickinson and Co., NJ) at a density of 5 × 105 cells/ well in hormonally defined Hams' F12K medium as described previously. The medium was changed every 2 d until the cells became confluent and then the cells were used for the experiments.
Cytokine Assays
A549 cells were exposed to increasing concentrations of IL-1 (0.1 to 10 ng/ml; Genzyme, Cambridge, MA), TNF- (1 to 100 ng/ml), or IL-4 (1 to 100 ng/ml) for 3 h. The cells were also stimulated with
a combination of IL-1 (1 ng/ml), TNF- (100 ng/ml) and IL-4 (100 ng/ml). The epithelial cell layers were then washed three times with
Hanks' balanced salt solution (GIBCO BRL) and were incubated
for 48 h. Cell-free culture supernatants were collected and assayed
for RANTES, eotaxin, IL-8, and IL-16 with enzyme-linked immunosorbent assay kits according to the instructions of the manufacturers. Assay kits for RANTES and eotaxin were purchased from
R&D Systems (Minneapolis, MN), and the minimum detectable concentration of RANTES and eotaxin was 5 pg/ml. The IL-8 kit was purchased from CLB (Amsterdam, the Netherlands), and the
minimum detectable concentration of IL-8 was 1 pg/ml. The IL-16
kit was purchased from Biosource (Camarillo, CA), and the minimum detectable concentration of IL-16 was 5 pg/ml.
Detection of mRNA for IL-16 in A549 Cells
Preparation of mRNA and reverse transcription/polymerase chain
reaction (RT-PCR) analysis.
A total of 5 to 8 × 106 cells was lysed,
and total cellular RNA was exacted from the cells with Trisolv
(Biotecx Laboratories, Houston, TX) by a modified guanidine
thiocyanate phenol chloroform method (13) as recommended by
the manufacturer. The isopropanol-precipitated RNA was washed
three times with 75% ethanol, dried, and resuspended in 30 µl diethylpyrocarbonate-treated water. The total RNA was quantitated by photometric measurment of optical density at 260 nm
and was stored as a 1-µg/µl stock solution at 
80°C. An RT-PCR procedure was performed to determine relative quantities of
mRNA for IL-16 by adapting methods described elsewhere (12,
14). Briefly, the first-strand complementary DNA (cDNA) was
synthesized from mRNA with oligo-dT primer. A 20-µl reaction
volume mix containing 1 µg mRNA, 2.5 pmol oligo-dT primer,
4 µl of 5× synthesis buffer (250 mM Tris-HCI, 375 mM KCl, 15 mM MgCl2, pH 8.3), 10 mM dithiothreitol, 2 mM deoxynucleotide triphosphate (dNTP) mixture, 20 U of RNase inhibitor, and
100 U Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim, Mannheim, Germany) was incubated at
37°C for 30 min in a thermocycler (model 2400; Perkin Elmer,
Norwalk, CT). At the end of this incubation, the reaction was
stopped by heating at 99°C for 5 min, and the reaction mixture
was then cooled to room temperature. Two microliters of cDNA
from the previous reaction were amplified in a 20-µl reaction volume containing 50 mM KCL, 10 mM Tris-HCL (pH 8.3), 1.5 mM
MgCl2, dNTP mixture (0.2 mM for each dNTP), 0.2 µm of IL-16
primers, and 1 U of Taq DNA polymerase (Boehringer Mannheim). Each cycle consisted of 45 s denaturation at 94°C, 45 s annealing at 60°C, and 2 min extension at 72°C in the thermocycler.
-actin were used in each experiment. For IL-16 and -actin gene
products, the optimum number of cycles was determined experimentally, and was defined as the number of cycles that would
produce a detectable concentration that was well below saturating conditions. The IL-16 PCR primers consisted of base pairs
1,504 to 1,893 of the coding region of the IL-16 gene and had the
sequences 5'-ATGCCCGACCCTCAACTCC-3' and 5'-CTAGGAGTCTCCAGCAGC-3'; the expected PCR product size was 389 bp. The primers for -actin were 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3'; the expected PCR product size
was 600 bp. The PCR products were subjected to electrophoresis at 100 V on a 1.5% agarose gel in Tris-acetate EDTA buffer. The PCR products were detected by ethidium bromide staining. PCR
products in electrophoresis analysis were quantified by video densitometry using a gel documentation system configured by UVP
(San Gabriel, CA) interfaced with a Macintosh PowerPC containing Image 1.53 software (NIH Public Software; National Institutes
of Health, Bethesda, MD). The level of IL-16 expression was
quantified by calculating the ratio of densitometric reading of the
bands for IL-16 and -actin from the same cDNA. -actin values
did not usually differ more than 1.5-fold.
Assessment of expression of IL-16 gene in A549 cells.
To assess the
effect of some stimulants on IL-16 gene expression, we treated
epithelial cell monolayers with control medium, 100 ng/ml TNF-
10 ng/ml IL-1 100 ng/ml IL-4, or with combinations of these
stimuli. Specific IL-16 and -actin mRNAs were visualized as
PCR products in electrophoresis gels appropriate for the expected molecular sizes of these mRNAs. When IL-16 or -actin
cDNA was amplified through 28 cycles, a linear correlation was
observed between the quantity of input RNA and the optical
density of the PCR product (data not shown).
Preparation of Eosinophils
Eosinophils were isolated by the method described previously (15), with minor modifications, by using immunomagnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and a magnetic cell separation system (MACS; Miltenyi Biotec GmbH) from the peripheral blood of four healthy individuals (16). We used anti-CD16 and anti-CD3 immunomagnetic beads. After isolation, eosinophils were stained with Randolph's stain and counted in a hemocytometer. Cytospins of each preparation were also stained with Diff-Quik (International Reagent Corp., Green Cross, Osaka, Japan). The mean percentage of eosinophil purity was 99.4 ± 0.15%. The viability by trypanblue dye exclusion was consistently greater than 98.5%.
Eosinophil Chemotaxis Assay
Eosinophil chemotaxis assay was measured by the Boyden's blind-well chamber technique (17) using a 48-well, multiwell chamber (NeuroProbe Inc., Bethesda, MD). The bottom wells of the chamber were filled with 26.5 µl of the A549 cell supernatant stimulated by various cytokines, as described previously, in triplicate. A polycarbonate filter with a pore size of 5 µm (Nucleopore, Pleasanton, CA) was placed over the bottom wells above the filter, and isolated eosinophils, prepared as described previously and suspended at 1 × l06 cells/ml in Hams' F12K medium supplemented with 0.2% bovine serum albumin, were placed into each of the top wells. The chambers were then incubated at 37°C, 5% CO2 for 90 min. After incubation, eosinophils in the top wells were removed by scraping. The filter was then stained with Diff-Quik. Eosinophil chemotaxis activity is shown as the total number of migrated eosinophils counted in 10 high-power fields under a light microscope (Olympus, Lake Success, NY) at ×400 magnification.
To confirm that cell migration was chemotactic, "checkerboard" analysis was performed (18). In the top wells of the chemotactic chamber, various dilutions of A549 supernatant (TNF- + IL-4 stimuli ) fluid (1:1, 1:2, 1:4, and 1:8) were placed with eosinophils. In the bottom wells, the same concentrations of A549 supernatants were placed so that all combinations of concentrations were tested. Each combination was assayed in triplicate.
Effects of Polyclonal Antibodies on Chemotactic Activitiy
The neutralizing antibodies to RANTES, eotaxin, IL-8, (R&D Systems), and IL-16 (PeproTech EC Ltd., London, UK) were added to the supernatant fluids harvested at 48 h at the saturated concentrations to inhibit these cytokines and were incubated for 30 min at 37°C. Anti-RANTES monoclonal antibody (mAb) (5 µg/ml), with a Neutralization Dose (ND50) of 200 ng/ml for recombinant RANTES (rRANTES); anti-eotaxin mAb (5 µg/ml), with an ND50 of 50 ng/ml for reotaxin; anti-IL-8 mAb (50 µg/ml), with an ND50 of 1,000 ng/ml for rIL-16, and anti-IL-8 mAb (5 µg/ml), with an ND50 of 100 ng/ml for rIL-16, were used to neutralize these cytokines. In parallel, the supernatant was preincubated with goat immunoglobulin (Ig)G (Dako, Kyoto, Japan) as a control, which is of the same isotype as these antibodies. Then these samples were used for chemotactic assay.
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Materials and Methods
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Effects of IL-1, TNF-, and IL-4 on IL-16 Release
from A549 Cells
IL-16 protein was not detected in the absence of stimulation. IL-1 or TNF- increased IL-16 release in a concentration-dependent manner (Figure 1). IL-4 had no effect on
IL-16 release (data not shown). We further examined the
effects of combinations of IL-1 (10 ng/ml), TNF- (100 ng/ml), and IL-4 (100 ng/ml) (Figure 2). IL-4 significantly
enhanced IL-16 release stimulated by IL-1 or TNF-.
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Effect of Cytokines on IL-16 Expression by A549 Cells
To determine whether the production of IL-16 is accompanied by transcription of the corresponding genes, we
used semiquantitative RT-PCR to examine IL-16 mRNA
expression in A549 cells (n = 5). A549 cells were stimulated with IL-1 (10 ng/ml), TNF- (100 ng/ml), IL-4 (100 ng/ml), or a combination (Figure 3). IL-16 was not detected under basal conditions but was detected after stimulation with IL-1, TNF-, and IL-4. The combination of
TNF- and IL-4 greatly enhanced expression of IL-16
mRNA than did the combination of IL-1 and TNF-2.
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Concentration of IL-8, RANTES, and Eotaxin in the Supernatant
IL-1 and TNF- stimulated the release of IL-8 (Figure 4A),
RANTES (Figure 4B), and eotaxin (Figure 4C) significantly.
IL-8 and RANTES were noticeably enhanced by the combination of IL-1 (10 ng/ml) and TNF- (100 ng/ml). In
contrast, IL-1 or TNF--induced IL-8 or RANTES production was significantly suppressed by IL-4 (Figure 4A
and 4B), although IL-4 (100 ng/ml) significantly enhanced IL-1 and TNF- induced eotaxin production. The combination of IL-1 and TNF- induced only a small amount
of eotaxin.
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Release of Eosinophil Chemotactic Activity from A549 Cells in Response to Various Cytokines
IL-1 and TNF- stimulated the release of ECA from
A549 cells in a concentration-dependent fashion (Figures
5A and 5B). We also demonstrated the effect of a cytokine
combination on the release of ECA from A549 cells (Table 1). Combinations of IL-1 (10 ng/ml) and TNF- (100 ng/ml) had a greater effect on ECA release than did either
alone. The TNF- (100 ng/ml) and IL-4 (100 ng/ml) combination also exhibited the same effect as an IL-1 and
TNF- combination.
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Checkerboard analysis confirmed that cell migration was due to chemotaxis (directed migration) and not to chemokinesis (Table 2).
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Contribution of Eotaxin, RANTES, IL-16, and IL-8 to ECA from A549 Cells
We compared the contribution of eotaxin, RANTES, IL-16,
and IL-8 in ECA with A549 cells stimulated with TNF-
and IL-4 (Table 3). The addition of antibodies to eotaxin
or IL-16 alone resulted in a significant decrease in ECA of
approximately 70%. Anti-eotaxin plus anti-IL-16 caused
an inhibition of ECA that exceeded 80% compared with
the ECA of supernatants without antibodies. This inhibition was significant compared with the inhibitory effect of
either antibody alone, but anti-RANTES treatment only
resulted in a 30% inhibition and anti-IL-8 treatment failed
to affect ECA. Control IgG did not have any effect on ECA
release from A549 cells.
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Materials and Methods
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In this study, we have characterized ECA released by A549
cells stimulated with IL-1, TNF-, and IL-4. The release
of ECA was induced by IL-1 or TNF-, and a combination of the two cytokines had a greater effect than did either alone. The addition of IL-4 also enhanced the release
of ECA. The released activity from A549 cells was chemotactic, as certified by checkerboard analysis. In the TNF-
and IL-4-stimulated supernatant, the blocking of eotaxin
caused an inhibition of ECA that exceeded 70%. However, ECA was inhibited by 30% with anti-RANTES antibodies, and anti-IL-8 antibody failed to affect the ECA.
We also showed that cultured A549 cells express IL-16 mRNA
and protein stimulated by IL-1 or TNF-. Furthermore,
the release of IL-16 protein from A549 cell supernatant was
confirmed in culture supernatants that had a strong chemoattractant action on eosinophils. Our findings suggest that alveolar type II cells may modify the eosinophil recruitment in allergic disease by releasing ECA.
A number of processes believed to be important in the
pathogenesis of asthma have been ascribed to the activities of IL-1 and TNF- (19). These cytokines are found at
increased concentrations in lung lavage fluid from patients
with asthma, and their spontaneous release is augmented
in alveolar macrophages from adult patients with asthma
and wheezy infants (20, 21). It has also been reported that
the number of alveolar tissue eosinophils and macrophages is increased in patients with nocturnal asthma (4). Although IL-1 and TNF- can cause the airway hyperresponsiveness and eosinophilia that characterize asthma,
neither is a potent chemoattractant for eosinophils. The
chemotactic factors released by A549 cells in response to
IL-1 and TNF- included IL-8, RANTES, and eotaxin. We have shown that significant amounts of ECA were released from A549 cells in response to IL-1 and TNF- in
the present study. These findings suggest that IL-1 and
TNF- may play a key role in eosinophil recruitment in alveolar tissue by stimulating release of ECA from alveolar
type II cells.
Important T helper 2 cytokines such as IL-4 induce IgE
synthesis (22). It has also been suggested to be involved in
selective tissue recruitment of eosinophils. Intradermal
and intraperitoneal injection of IL-4 results in marked
eosinophil infiltration in mice (23). In a murine model of
asthma with antigen-induced eosinophilic inflammation,
IL-4 production was positively correlated with eosinophil
recruitment (24). In the current study, we found that in
A549 cells, IL-4 enhanced eotaxin production stimulated with IL-1 or TNF-. In contrast, IL-4 suppressed IL-8
production significantly. The ECA stimulated by IL-1 or
TNF- was also increased by the combination with IL-4,
although IL-8 protein was released in much greater quantities than was eotaxin. IL-8 production was significantly
decreased by IL-4, perhaps to an extent that would inhibit
neutrophil recruitment. These results suggest that IL-4 may be involved in selective recruitment of eosinophils by
increasing eotaxin and reducing IL-8.
A549 cells have the potential to release many chemokines, including IL-8, RANTES, and eotaxin. Among them,
eotaxin was a predominant eosinophil chemotactic factor
in the present study. The blocking of eotaxin caused a decrease in ECA of more than 70% under TNF- and IL-4
stimulation, this is in contrast to findings indicating that
ECA was inhibited by 30% with anti-RANTES antibodies, and anti-IL-8 antibodies failed to affect ECA. Eotaxin
is unique among eosinophil-active chemoattractants in
that it specifically attracts eosinophils. This specificity has
also been demonstrated in vitro for both mouse and human eotaxin in chemotaxis assay (25). Intratracheal instillation of eotaxin in rodents is followed by a marked
lung eosinophilia. Similarly, injection of eotaxin in the skin
of animals is associated with the rapid accumulation of
eosinophils. Although a number of chemokines are likely
to participate in ECA from A549 cells. Eotaxin appears to
be the predominant ECA secreted by A549 cells rather
than IL-8 and RANTES. It has been reported that IL-8 has chemotactic activity on eosinophils; however, in our
study anti-IL-8 treatment failed to affect eosinophil chemotaxis. The eosinophils used in the chemotaxis assay were
obtained from healthy individuals. Sehmi and co-workers
(29) demonstrated that IL-8 is a chemoattractant for eosinophils purified from subjects with a blood eosinophilia
but not from normal healthy subjects. Our findings also
confirm previous reports showing the lack of responsiveness of eosinophils from normal subjects to IL-8 (30, 31).
IL-16 has been described as a potent human eosinophil
chemoattractant in vitro (10). Previous independent reports have shown that IL-16 was secreted in several cell
types, including CD8+ T cells (32, 33), eosinophils (34),
and bronchial cells (12). To the best of our knowledge, this
is the first study that reports alveolar epithelial cells expressing IL-16 mRNA and protein stimulated by IL-1
and TNF-. We have also observed that anti-IL-16 antibodies inhibit ECA strongly under TNF- and IL-4 stimulation. Although the same isotype IgG antibodies have no effect on ECA, this finding confirmed that IL-16 was responsible for part of the epithelia-derived chemoattractant
activity for eosinophils. This blocking effect is the same as
that of the eotaxin antibody. These data indicate that
IL-16 has a potency comparable with that of eotaxin in
causing eosinophil migration. IL-16 is present in increased
amounts in BALF from antigen-challenged asthmatic subjects (11). Treatment of mice with antibodies to IL-16 during the challenge period significantly inhibited the development of airway hyperresponsiveness after repeated
ovalbumin inhalation in asthmatic animals (35). Alveolar
type II cell-derived IL-16 may be involved in airway inflammation and especially in the accumulation of eosinophils in asthma.
In conclusion, we have found that A549 cells can release ECA stimulated by IL-1 or TNF-. A549 cells also
have the capacity to produce IL-16. The ECA was strongly
inhibited by addition of anti-eotaxin and anti-IL-16 antibodies. These findings support the concept that alveolar
epithelial cells can participate in the pathogenesis of eosinophilic inflammatory diseases such as asthma and interstitial lung diseases and that eotaxin and IL-16 are potent
and effective eosinophil chemoattractants that are produced
by alveolar epithelial cells.
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Footnotes |
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Address correspondence to: Gang Cheng, M.D., Dept. of Pulmonary Medicine and Clinical Immunology, Dokkyo University School of Medicine, Mibu-machi, Shimotsuga-gun, Tochigi, Japan. E-mail: cheng{at}dokkyomed.ac.jp
(Received in original form June 19, 2000 and in revised form October 7, 2000).
Abbreviations: bronchoalveolar lavage fluid, BALF; complementary DNA, cDNA; deoxynucleotide triphosphate, dNTP; eosinophil chemotactic activity, ECA; immunoglobulin, Ig; interleukin, IL; monoclonal antibody, mAb; messenger RNA, mRNA; regulated on activation, normal T cells expressed and secreted, RANTES; reverse transcription/polymerase chain reaction, RT-PCR; tumor necrosis factor, TNF.| |
References |
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