1 Departments of Pulmonary and Critical Care Medicine, 2 Cancer Biology, and 3 Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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Respiratory epithelium
expresses nitric oxide synthase 2 (NOS2) continuously in vivo; however,
mechanisms responsible for its expression are only partially
understood. We definitively identify an autocrine mechanism of
induction and maintenance of NOS2 in human airway epithelial cells
through the synthesis and secretion of a soluble mediator. Short
exposure of human airway cells to interferon (IFN)- leads to
prolonged NOS2 expression. Transfer of the overlying culture medium
(conditioned medium) induces NOS2 expression in other airway epithelial
cells, suggesting the presence of an intermediary substance regulating
NOS2 expression in an autocrine loop. Characterization of the soluble
mediator reveals that it is stable and transferable in conditioned
medium for up to 7 days. However, soluble mediator does not induce NOS2 mRNA in human alveolar macrophages, indicating that the response to
soluble mediator is unique to human respiratory epithelium. Soluble
mediator is heat labile but is not inactivated by acid treatment,
unlike IFN-
itself. Importantly, IFN regulatory factor-1, which is
critical for murine NOS2 expression, is expressed and activated by
soluble mediator through the signal transducer and activator of
transcription-1-dependent pathway. Based on these findings, we propose
novel regulatory mechanisms for NOS2 expression in human airway epithelium.
lung; nitric oxide; inflammatory mediators; gene regulation; signal transduction
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INTRODUCTION |
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NITRIC OXIDE (NO) has been proposed as a mediator of vital biological functions in the lung, including modifying airway tone, regulating pulmonary vascular tone, stimulating mucin secretion, modulating mucociliary clearance through effects on ciliary beat frequency, and immune surveillance for tumoricidal and bactericidal effects (25). Furthermore, NO mediates inflammatory responses relevant to host defense mechanisms and perhaps to lung injury and edema (25). Three types of nitric oxide synthase (NOS), the enzyme responsible for endogenous NO production, have been described in human cells: inducible NOS (NOS2), which produces high levels of NO, and two constitutive NOSs, which produce low levels of NO (27, 28).
NO is detected in exhaled air of all individuals in a pattern
localizing formation of NO to airways (23). We have shown previously that normal human airway epithelial cells (HAEC) in vivo
produce NO through the continuous expression of NOS2 (7). Expression of NOS2 mRNA and protein is detected in epithelial cells in
the normal, noninflamed upper and lower airway by a variety of
techniques, including Northern blotting, in situ hybridization, Western
blotting, and immunohistochemistry (13, 14). The
continuous NOS2 expression in HAEC is in contrast to other cell types
that do not express NOS2 unless induced by cytokines such as interferon (IFN)-, interleukin (IL)-1
, and/or tumor necrosis factor
(TNF)-
(20, 28). Although human airway epithelium
expresses abundant levels of NOS2 in vivo, culture of the human airway
cells ex vivo leads to loss of NOS2 gene expression (7).
These findings suggest that the control of NOS2 expression in human
airway epithelium is dependent on a combination of in vivo factor(s) or
exposures to which airway epithelial cells are uniquely responsive.
However, the mechanisms responsible for maintenance of expression are
not fully understood. We have previously demonstrated that a
combination of IFN-
and IL-4, which occur naturally in the lung
epithelial lining fluid, stimulates prolonged expression of NOS2 in
human airway epithelium and is important for maintaining the continuous expression of NOS2 through a protein synthesis-dependent mechanism (8). In the present study, we show that a 1-h incubation
of HAEC with IFN-
alone followed by removal of IFN-
by washing and further incubation in fresh medium without cytokines induces prolonged NOS2 expression for at least up to 6 days. Transfer of the
overlying culture medium induces NOS2 expression in other HAEC. These
unusual patterns of induction have led us to reason that IFN-
induces NOS2 in human airway epithelium by an autocrine loop
mediated by an unknown soluble mediator. We describe and characterize
the soluble mediator responsible for the induction and maintenance of
NOS2 expression in HAEC and investigate the mechanisms regulating expression.
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MATERIALS AND METHODS |
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Primary HAEC and Alveolar Macrophages
HAEC and alveolar macrophages (AM) were obtained through bronchoscopy with a flexible fiber-optic bronchoscope (Olympus BS-IT10; Olympus Optical, Tokyo, Japan) from normal nonsmoking volunteers with no history of lung disease and no medications. Informed consent was obtained under a protocol approved by the Institutional Review Board at Cleveland Clinic Foundation. For some experiments, HAEC were isolated from surgical specimens of tracheae and main bronchi as previously described (3). Bronchoscopic brush samplings of airway epithelial cells were taken from second- and third-order bronchi as previously described (7). For the preparation of AM, the tip of the bronchoscope was wedged into the right middle lobe or into the lingula. A total of 300 ml of saline was instilled by gravity in 50-ml aliquot portions and was withdrawn by gentle aspiration. Lavage fluid was passed through a blood filter (Baxter Scientific Products, Chicago, IL), and the cells were washed with Hanks' balanced salt solution (GIBCO BRL, Grand Island, NY). Cell number was determined on a hemocytometer, and differential cell counts were performed with a modified Wright's stain (Hema-3 stain; Biochemical Sciences, Bridgeport, NJ).Cell Culture and Treatments
HAEC obtained by bronchial brushing were cultured in serum-free Lechner and LaVeck medium (LHC-8; Biofluids, Rockville, MD) on plates precoated with coating medium containing 29 µg/ml collagen (Invitrogen; Palo Alto, CA), 10 µg/ml BSA (Biofluids), and 10 µg/ml fibronectin (Calbiochem, La Jolla, CA) for 5 min (7). The cells were passaged at 60-80% confluence by dissociation from plates with 0.02% trypsin (E-PET, Biofluids), which was neutralized with soybean trypsin inhibitor (Biofluids). Primary cultures of passages 0-2 were used in experiments. The epithelial nature of primary and cultured cells was confirmed by immunocytochemical staining as previously described (7). AM were resuspended in RPMI 1640 medium (GIBCO BRL) supplemented with 5% human AB serum (Gemini, Calabasas, CA), L-glutamine, and antibiotics. Macrophages were plated and allowed to adhere for 1 h. Nonadherent cells were removed by washing with warm RPMI. The adherent cell population was >99% AM. BEAS-2B and BET-1A cells (24), human bronchial cell lines transformed by Ad12-SV40 virus and SV40 T antigen, respectively, were cultured in serum-free LHC-8 with the additives 0.33 nM retinoic acid and 2.75 µM epinephrine on precoated plates similar to HAEC. A549 cells, an epithelial cell line derived from lung adenocarcinoma (American Type Culture Collection, Manassas, VA), were cultured in MEM (GIBCO) with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (8).Human IFN- was a gift from Genentech (South San Francisco, CA) or
was purchased from R&D Systems (Minneapolis, MN).
RNA Extraction and Northern Analysis
Total RNA was extracted and evaluated by Northern analysis as previously described using a 32P-labeled 1.9-kb NOS2 cDNA probe (pCCF21) or as a control 2-kbWestern Analysis
Cell lysate was prepared by freeze-thaw of cells, cultured for the indicated times with IFN-Electrophoretic Mobility Shift Assay
Whole cell extracts were prepared by a modification of a previously described method (5). In brief, adherent cells were harvested by a cell lifter, and the cell suspensions were centrifuged, washed with PBS, and resuspended in ice-cold low-salt buffer (in mM): 10 HEPES, pH 7.9, 1.5 MgCl2, 10 KCl, 0.5 PMSF, and 0.5 DTT. After a 5-min incubation on ice, cells were washed in the same buffer and then pelleted. A volume of high-salt extraction buffer equal to the volume of the cell pellet was added (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 5 µg/ml leupeptin, 2 µg/ml aprotinin and 1 µg/ml pepstatin) and the mixture was placed on ice for 30 min. Whole cell extracts were clarified by centrifugation at 12,000 g for 20 min at 4°C. The protein concentration was measured by bicinchoninic protein assay (Pierce).For electrophoretic mobility shift assay (EMSA) experiments, the
IFN- activation site oligonucleotide
(5'-tcgaGCCTGATTTCCCCGAAATGACGGC-3') (22) or the
multimerized hexamer probe (AAGTGA)4, originally used to clone IRF-1
(19), was used. These synthetic oligonucleotides were
either end-labeled with [
-32P]ATP by polynucleotide
kinase or fill-in labeled with [
-32P]dCTP by Klenow.
For binding reactions, whole cell extracts (5 µg of protein) were
incubated in 24 µl of total reaction volume containing 20 mM HEPES,
pH 7.9, 10% glycerol, 60 mM NaCl, 5 mM MgCl2, 4 mM
Tris · HCl, 1 mM DTT, 0.6 mM EDTA, 200 µg/ml BSA, and 2 µg
of poly(dI-dC) (Amersham) for 15 min at 4°C. The
32P-labeled oligonucleotide (0.2 ng, 2 × 105 counts/min) was then added to the reaction mixture and
incubated for 20 min at room temperature. To specifically identify
signal transducer and activator of transcription (STAT)-1 and IRF-1
proteins, 2-4 µg of rabbit anti-STAT1
polyclonal antibody or
rabbit anti-IRF-1 or IRF-2 antibody (Santa Cruz Biotechnology) were
added to the binding reaction mix and incubated for 30 min at 4°C
before the 32P-labeled oligonucleotide was added. The
reaction products were analyzed by electrophoresis in a 6%
polyacrylamide gel with 0.4× TBE buffer (36 mM Tris, 36 mM borate, and
8 µM EDTA) for STAT1 or a 4% polyacrylamide gel containing 50 mM
Tris · HCl, pH 7.5, 0.38 M glycine, and 2 mM EDTA for IRF-1.
The gels were dried and analyzed by autoradiography.
Characterization of Soluble Mediator in Conditioned Medium
Wash-off experiments.
After a 1-h incubation of human respiratory epithelial cells with
IFN-, the cells were vigorously washed with HEPES-buffered saline to
remove residual IFN-
and cultured in fresh medium without cytokines.
After the indicated times, the overlying culture medium (conditioned
medium) was transferred to unstimulated fresh cells that were harvested
at 24 h for the detection of NOS2 by either Northern or Western
analysis. All IFN-
exposures were performed following this method of washoff.
Heat stability. To examine whether the soluble mediator in the overlying tissue culture medium was heat labile, the conditioned medium was heated at 95°C for 5-15 min. The heated conditioned medium was cooled on ice and resuspended in an equal volume of fresh LHC-8 medium. Respiratory epithelial cells were exposed to either control or heat-treated medium for 24 h, at which time the cells were harvested for Western and Northern analyses to detect NOS2 expression.
pH stability. To examine whether the soluble mediator in the conditioned medium was acid or base labile, the conditioned medium was adjusted to pH 2 or pH 10 with concentrated HCl or 40% saturated NaOH and maintained at 4°C for 2 h. The pH of the acid- or base-treated conditioned medium was readjusted to pH 7.4 with 40% saturated NaOH or concentrated HCl, sterilized by passage through a 0.45-µm filter and resuspended in an equal volume of fresh LHC-8 medium. Respiratory epithelial cells were exposed to either control or acid- or base-treated medium for 24 h, at which time the cells were harvested for Western analysis to detect NOS2 expression.
Ultrafiltration separations. The conditioned medium was filtered by using an Amicon centrifugal ultrafiltration device (Millipore, Bedford, MA) equipped with Amicon YM cellulose ultrafiltration membrane at 10-kDa molecular mass cutoff. Of the initial volume, 15 ml were concentrated to approximately 1.5 ml of final volume by centrifugation at 3,000 g. Retentate and filtrate were resuspended in an equal volume of fresh LHC-8 medium. Respiratory epithelial cells were exposed to either control or retentate or filtrate medium for 24 h, at which time the cells were harvested for Western analysis to detect NOS2 expression.
Statistical Analysis
Data in the text and figures are expressed as means ± SE. ![]() |
RESULTS |
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Prolonged NOS2 mRNA Expression in HAEC in Response to IFN-
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Transfer of Conditioned Medium to Other HAEC in Culture Induces NOS2 Gene Expression
After 1 h in the absence or the presence of IFN-
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Determination of Cell Type-Specific NOS2 mRNA Expression in Response to Transferable Soluble Mediator
To determine whether the conditioned medium induction of NOS2 is limited to primary HAEC or expandable to other kinds of lung cells, BEAS-2B cells were exposed to IFN-
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Soluble Mediator Is Relatively Stable and Transferable in the Conditioned Medium for up to 7 Days
To further understand the mechanism by which the soluble mediator is produced in the conditioned medium, BEAS-2B cells were stimulated with IFN-
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NOS2 Protein Induction by the Conditioned Medium
To investigate NOS2 protein induction by the conditioned medium, BEAS-2B cells were incubated in the absence or the presence of IFN-
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Characterization of Respiratory Epithelial Cell-Derived Soluble Mediator
BEAS-2B cells were incubated in the absence or the presence of IFN-
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STAT1 Activation and IRF-1 Expression by the Conditioned Medium
Although the cellular signaling mechanisms by which cytokines induce the expression of NOS2 by human respiratory epithelial cells are complex (4, 15, 20), we have previously shown that the expression of NOS2 in HAEC is dependent on Janus kinase (JAK)-STAT1 signaling pathway (8). Nuclear factor (NF)-
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DISCUSSION |
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The airway epithelium not only serves as a barrier to isolate the airway microenvironment from external stimuli but also communicates and regulates the function of neighboring cells, including adjacent airway epithelial cells, bronchial smooth muscle cells, fibroblasts, mast cells, and inflammatory cells. It is well established that airway epithelial cells produce and release signaling molecules including proteins, small peptides, amino acids, nucleotides, fatty acid derivatives, and even dissolved gases such as NO and carbon monoxide (10).
We show here that the continuous NOS2 expression in human airway
epithelium in vivo is mediated through an autocrine signaling mechanism. The delayed and prolonged induction of NOS2 mRNA
in IFN--treated HAEC, which peaks at 24-48 h and
lasts up to at least 6 days, suggests the presence of a stable
intermediary soluble substance regulating the expression of NOS2.
Furthermore, persistence of NOS2 expression in HAEC in culture is
dependent on maintaining the overlying tissue culture medium containing
soluble mediator. Transfer of the soluble mediator to other HAEC
in culture induces NOS2 gene expression, whereas transfer of
soluble mediator to AM does not lead to NOS2 induction. In this
context, the soluble mediator is specific for human airway epithelium
and may help to explain the previous finding of NOS2 expression in HAEC
in vivo, without expression in other resident lung cells including human macrophages (7). In contrast, NOS2 mRNA could not be induced by the soluble mediator in the transformed human airway epithelial cell lines BEAS-2B and BET-1A, whereas they retain the
strong potential to produce soluble mediator in response to IFN-
.
Based on the knowledge that BEAS-2B and BET-1A cells express chemokines
and adhesion molecules in response to IFN-
(18, 26) and
have an intact IFN-
signaling pathway, a defect in a specific
receptor or signaling component is more likely the reason for lack of
NOS2 induction in response to soluble mediator. On the other hand,
defects in the NOS2 gene may also be present in these transformed
cells, which make a response to soluble mediator impossible.
Several facts regarding the soluble mediator emerge from this study.
First, the majority of the mediator synthesis or secretion occurs early
after initiating IFN- stimulation. In contrast to known cytokines,
soluble mediator is stable and transferable in the overlying culture
medium, with full activity for at least 7 days. The soluble mediator is
an IFN-
-stimulated gene product and induces signaling events similar
to IFN-
. For example, STAT1 activation and IRF-1 induction and
activation by the soluble mediator are similar to induction and
activation by direct addition of high levels of IFN-
.
However, soluble mediator is not IFN-
as judged by several findings.
First, levels of NOS2 expression in HAEC by direct addition of IFN-
were markedly lower than those induced by the conditioned medium. In
fact, NOS2 induction in A549 cells by conditioned medium was greater
than that observed in the A549 cells from which the conditioned medium
was derived. Thus A549 cells producing soluble mediator were relatively
refractory to its effects, perhaps due to downregulation of receptor
and/or signaling pathways by IFN-
. Importantly, the soluble mediator retained its activity after acid treatment, whereas IFN-
is
inactivated under these conditions. Finally, IFN-
is not produced by
HAEC exposed to IFN-
in culture, and pretreatment with neutralizing antibodies to IFN-
does not affect the ability of conditioned medium
to induce NOS2 (8).
Based on our findings, we propose a novel mechanism for NOS2 induction
and maintenance in human airway epithelium in which IFN- stimulates
epithelial production and secretion of a soluble mediator. We have
shown previously that activation of STAT is required for the
conditioned medium activation of NOS2, using an inhibitor of tyrosine
kinase (6). Thus soluble mediator induces airway
epithelial cells in an autocrine fashion to express NOS2, likely
through STAT1 activation and IRF-1 induction and activation (Fig.
12). Notably, the levels of IFN-
used in this study are within the levels found in human lung epithelial
lining fluid of healthy (~50 U/ml) or asthmatic (~125 U/ml)
individuals (6). Thus NOS2 expression in the human airway
in vivo may also be through this autocrine mechanism. Although NF-
B
activation is important in multiple cytokine induction in A549 cells
(15), NF-
B is not activated in human respiratory
epithelial cells by IFN-
or soluble mediator. This finding excludes
multiple known cytokines that signal through NF-
B as candidates for
soluble mediator, including IL-1, TNF, or IL-18. On the other hand,
STAT1 tyrosine phosphorylation and translocation to the nucleus occur in response to soluble mediator and, indeed, to many growth factors and
cytokines including IL-10, IFN-
/
, epidermal growth factor, platelet-derived growth factor, granulocyte-macrophage
colony-stimulating factor, IL-6, IL-11, leukemia inhibitory factor,
ciliary neurotrophic factor, oncostatin M, growth hormone, prolactin,
and colony-stimulating factor 1 (2, 9, 16). Epidermal
growth factor and growth hormone are less likely candidates for soluble
mediator because airway epithelial cells are grown in medium containing
considerable levels of these humoral factors and yet do not express
NOS2 under basal culture conditions. IFN-
/
are
acid-stable interferons but are inhibitors of NOS2 induction in
epithelial cells (9) and thus are also not likely
candidates for soluble mediator. Similarly IL-10, a cytokine with
anti-inflammatory properties, inhibits NOS2 expression
(1). The STAT1 activation resulting from the conditioned
medium may be due to one of these known mediators or to an as yet
unidentified mediator. Nevertheless, in this study, we provide strong
evidence for the existence of an autocrine loop regulating NOS2
expression in the human airway through an IFN-
-inducible respiratory
epithelial cell-derived soluble mediator and define the signaling
events in response to the soluble mediator.
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ACKNOWLEDGEMENTS |
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We thank C. C. Harris for BEAS-2B and BET-1A cells, J. L. Humes for anti-NOS2 (NO53) antibody, L. Kedes for pHFA-1, Genentech for human IFN-
, and R. A. Dweik for assistance in bronchoscopic sampling of airways.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-60917.
Address for reprint requests and other correspondence: S. C. Erzurum, Dept. of Pulmonary and Critical Care Medicine and Cancer Biology, Cleveland Clinic Foundation, 9500 Euclid Ave./A90, Cleveland, OH 44195 (E-mail: erzurus{at}ccf.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 August 2000; accepted in final form 15 December 2000.
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