 B Activation in Alveolar Macrophages
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nitric oxide (NO) is an important endogenous regulatory molecule implicated in both proinflammatory
and antiinflammatory processes in the lung. Previously, we demonstrated that in human alveolar macrophages (AM), NO decreased inflammatory cytokine production, including that of interleukin-1
, tumor
necrosis factor-
and macrophage inflammatory protein-1. One mechanism by which NO could regulate
such diverse cytokine production is through effects on the transcription factor nuclear factor-
B (NF-B),
which controls the expression of the genes for these inflammatory cytokines and growth factors. We therefore investigated whether NO affects NF-B activation in AM in vitro and in vivo. In vitro studies with
AM showed that NF-B activation by lipopolysaccharide (LPS) is decreased by NO in a dose-dependent
manner. NO prevented an LPS-mediated decrease in the NF-B inhibitory protein IB-. In asthma, airway NO levels are increased, whereas in primary pulmonary hypertension (PPH), airway NO levels are
lower than in healthy lungs. In vivo investigations were conducted with freshly isolated AM from healthy controls, asthmatic individuals, and PPH patients. Healthy individuals had airway NO levels of 8 ± 2 ppb
(mean ± SEM), which is associated with low NF-B activation. Asthma patients with airway NO levels > 17 ppb showed minimal NF-B activation, whereas asthmatic individuals with NO levels 
17 ppb
showed greater NF-B activation. PPH patients with low NO (1 ± 1 ppb) had prominent NF-B activation. These in vivo studies in asthma and PPH support the in vitro observation of an inverse relationship
between NO and NF-B activation. One mechanism by which NO blocks cytokine production involves
IB.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nitric oxide (NO) is endogenously produced in the normal
human lung by nitric oxide synthases (1, 2). Multiple physiologic functions are regulated by NO, including smooth-muscle relaxation, neurotransmission, vascular tone, and
host defense (3). Abnormalities of NO levels have been
associated with diseases such as asthma and primary pulmonary hypertension (PPH) (3). PPH is characterized by a progressive increase in pulmonary arterial pressure
(4). Our recent studies have shown decreased NO levels in
PPH patients as compared with healthy controls (5).
Asthma is a chronic inflammatory airway disease characterized by dysregulation of many inflammatory cytokines
(tumor necrosis factor [TNF]-, interleukin [IL]-1, macrophage inflammatory protein [MIP]-1, granulocyte-macrophage colony-stimulating factor, IL-4, and IL-5) (6).
Asthmatic individuals have significantly higher levels of
exhaled NO than do healthy individuals (3).
In the lung, the AM is an important source of cytokines
and growth factors (9). Previously, we have shown that
NO downregulates inflammatory cytokine production
(TNF, IL-1, MIP-1) by human AM in vitro (10). Interestingly, many of the cytokines affected are regulated by the
redox-sensitive transcription factor nuclear factor-B
(NF-B). In unstimulated cells, NF-B resides in the cytoplasm as a dimer of protein components known as rel family members (e.g., p50, p65), which is bound to an inhibitor
(IB) (11). Upon stimulation or activation, IB is phosphorylated and released from the complex, after which the
complex undergoes proteolytic degradation and the rel
proteins migrate to the nucleus and bind to the cognate
sites in the promoter regions of the genes for many inflammatory cytokine and growth factors, resulting in their transcription. We investigated whether NO affects NF-B activation in vitro in LPS-stimulated AM in the absence and
presence of an NO-generating compound (2,2-[hydroxynitrosohydrazono]-bis-ethanamine [DETA NONOate]) and
in vivo by examining freshly isolated AM from asthmatic
subjects and patients with PPH.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents
Salmonella typhimurium lipopolysaccharide (LPS) was obtained from Sigma Chemical Co. (St. Louis, MO) and used at 0.5 µg/ml for all experiments. DETA NONOate was obtained from Cayman Chemical Company (Ann Arbor, MI) and used at indicated concentrations. DETA NONOate releases NO in culture with a t1/2 = 20 h at 37°C. Previous studies showed that NO is generated by DETA NONOate in our culture system and that cell viability is not affected by DETA NONOate at the concentrations used (10). Furthermore, we did not detect endogenous NO production in AM cultures incubated in medium with or without LPS (10).
Study Population and AM Preparation
All volunteers provided written informed consent and the
study was approved by the Institutional Review Board of
the Cleveland Clinic Foundation. Normal individuals had
no history of lung disease and were not taking medication.
Asthmatic subjects satisfied the definition of asthma as
stated by the American Thoracic Society (12) and had to
show a > 14% increase in FEV1 either spontaneously or
after bronchodilator within 1 yr before enrollment in the
study. All asthmatic subjects in the study had mild, stable
asthma and had been free of exacerbations for 
4 wk.
Furthermore, these individuals had not taken oral steroids
within the previous 6 mo and were taking short-acting inhaled 2-agonists on an as-needed basis. Pulmonary hypertension was ascertained in all PPH patients by right heart
catheterization. PPH patients were receiving vasodilators, anticoagulants, diuretics, digitalis, and/or oxygen, and
have previously been described in detail (5). AM were obtained by fiberoptic bronchoscopy with bronchoalveolar
lavage (BAL) from normal volunteers and patients as previously described (10). The tip of the bronchoscope was
wedged into the right middle lobe or the lingula. Saline
warmed to 37°C was instilled by gravity in 50-ml aliquots
(150-300 ml) and immediately withdrawn by gentle aspiration. The lavage fluid was filtered and the cells were
washed with Hanks' balanced salt solution (Gibco, Grand
Island, NY). Cell number was determined with a hemocytometer, and differential cell counts were performed with
a modified Wright's stain (Hema-3 stain; Biochemical Sciences, Inc., Bridgeport, NJ). Cells were resuspended in
RPMI 1640 or Dulbecco's modified Eagle's medium supplemented with 5% human AB serum (Gemini, Calabasas, CA), L-glutamine, and antibiotics. Macrophages were
plated at 4-5 × 106 cells in 100-mm culture plates and allowed to adhere for 1 h at 37°C in a moist 5% CO2 incubator. Nonadherent cells were removed by washing with
warmed RPMI-1640. The adherent cell population was > 99% AM.
Measurement of Airway NO
NO levels were measured in lower airways with a Teflon catheter inserted through the working channel of the bronchoscope. NO levels in the airway gases were determined during a 10- to 15-s expiratory breathhold by chemiluminescence (NOA280; Siever Inc., Boulder, CO) as previously described (13).
Preparation of Whole-Cell Extracts
Extracts were prepared from cells freshly isolated from
bronchoalveolar lavage fluid (BALF). For in vitro experiments, macrophages were allowed to adhere and rest for
24 h before any indicated treatments, after which extracts
were prepared. For extraction, cells were resuspended in
extraction buffer (20 mM Tris, pH 8.0; 150 mM MgCl2; 1%
Triton x100) containing a protease inhibitor cocktail, and
were kept for 20 min on ice. The cell samples were then
centrifuged at 18,000 × g for 20 min at 4°C to clear debris, and supernatants representing whole-cell extracts (WCEs)
were collected. WCEs were aliquoted in small volumes in
order to minimize repeated freeze-thaw damage, and
were kept at 
80°C for further use. The protein content of
WCEs was determined with the bicinchoninic acid (BCA)
protein assay (Pierce, Rockford, IL).
Electrophoretic Mobility Shift Assay
For electrophoretic mobility shift assay (EMSA), 10 µg of the WCEs were incubated in binding buffer (8 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid, pH 7.0; 10% glycerol; 20 mM KCl; 4 mM MgCl2; 1 mM sodium pyrophosphate) containing 1.0 µg of polydeoxyinosine- deoxycytosine and 32P end-labeled probes. The probes had the following sequences: 5'-AACTCCGGGAATTTCCCTGGCCC-3'; 5'-GGGCCAGGGAAATTCCCGGAGTT-3'. For competition experiments, a 1,000-fold excess of cold oligonucleotide was used. For the supershift assay each WCE was incubated with anti-p65 or anti-p50 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at room temperature (RT) before the addition of probe. After incubation with the probe for 20 min, the reaction mixture was analyzed on a 4% nondenaturing acrylamide gel. The gels were then dried and exposed for autoradiography. For in vitro experiments, cells were treated either with LPS or LPS + DETA NONOate, or were left untreated for 4 h before harvesting to make WCEs. EMSA was then done as described. Autoradiograms were quantified through ImageQuant analysis (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis
WCEs containing 10 µg of protein from AM incubated for
45 min in medium alone (unstimulated) or in medium with
LPS ± 1 mM DETA NONOate were analyzed by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis
and transferred to Immobilon-P membranes, where they
were blocked overnight at 4°C with 5% nonfat dry milk in
TBST (10 mM TrisCl, pH 8.0; 150 mM NaCl; 0.1% Tween
20). The blot was rinsed twice with TBST and incubated
for 2 h at RT with anti-IB antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in TBST containing milk. The
membrane was washed for 40 min with TBST and incubated with goat antirabbit IgG conjugated with horseradish peroxidase in 5% milk for 1 h, and then washed four
times with TBST and developed with enhanced chemiluminescence reagent (Amersham, Arlington, IL).
Immunofluorescence Staining
AM obtained as described earlier from normal individuals
were plated (1 × 106 cells per well) in each well of six-well
plates containing glass coverslips. Cells were allowed to
adhere for 1 h, washed with medium, and incubated for 24 h
in medium. The medium was then aspirated and replaced
with medium alone (unstimulated cells), LPS-treated medium, or medium with LPS + 1 mM DETA NONOate for 4 h. Coverslips were rinsed with phosphate-buffered saline
(PBS), and the adherent cells were fixed for 2 min in cold
acetone, and stored at 20°C until stained. Before staining, coverslips were rinsed in PBS (pH 7.4) for 10 min. The
coverslips were then blocked with 2% goat serum for 15 min, briefly rinsed in PBS, and stained with anti-p65 antibody (Santa Cruz Biotechnology) for 60 min in a humid
chamber. Following this, coverslips were washed thrice in
PBS. Fluorescein-conjugated goat antirabbit IgG in 2%
goat serum was added, and the coverslips were incubated
in a humid chamber for 45 min in the dark. The coverslips
were then washed thrice in PBS, mounted on slides with
Vectashield containing propidium iodide (Vector Laboratories, Burlingame, CA), and sealed with clear nail polish.
Simultaneous two-color fluorescence images were recorded with a Leica TCS-NT laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany).
TNF Assay
From the in vitro-stimulated AM cultures, cell-free supernatants were collected and assayed for TNF with an enzyme-linked immunosorbent assay (ELISA) (Endogen, Cambridge, MA). The sensitivity of the assay ranged from 25.6 to 1,000 pg/ml. All samples were assayed in duplicate, and the coefficient of variation for these assays was < 10%.
Statistical Analysis
All values are reported as means ± SEM. Statistical analysis was done through one-way analysis of variance
(ANOVA), using GraphPad Software (GraphPad, Inc.,
San Diego, CA). Significance was defined at P 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In Vitro NF-B Activation
In order to investigate the relationship between NO level
and NF-B activation, we studied the effect of the NO
generator DETA NONOate on LPS-induced NF-B activation in vitro. NF-B activation was determined in WCEs
after 4 h exposure to LPS or LPS + DETA NONOate of
AM from four different isolates, all of which showed decreased DNA binding activity of NF-B at 1 mM DETA
NONOate. Parallel measurements of secreted TNF from
these cultures showed a mean percent TNF inhibition by
DETA NONOate of 51 ± 6% (n = 4). A dose-dependent
inhibition of LPS-mediated NF-B activation by NO is
shown in Figure 1A. DETA NONOate also decreased
TNF secretion by the AM used for the EMSA in a dose-dependent manner (Table 1). Supershift assay revealed
that the complex contained both p50 and p65 rel components (data not shown). Immunoblotting of whole-cell lysates with an anti-IB- antibody showed that LPS stimulation of AM resulted in the loss of IB protein (Figure 1B,
one of three experiments). This loss was not observed in
the presence of DETA NONOate. These results indicate
that NO prevented NF-B activation by maintaining a
steady-state level of IB protein, which may be mediated
by increasing IB synthesis, decreasing IB degradation,
or both. In order to examine whether NO-mediated maintenance of the IB level can prevent nuclear translocation of active NF-B, we cultured AM on coverslips and exposed them to LPS or LPS + DETA NONOate, fixed the
cells after 4 h, and stained them with anti-p65 antibody.
Figure 2 shows results from one of three experiments.
Confocal microscopic analysis showed minimal nuclear
staining in unstimulated AM (Figure 2A). LPS-stimulated macrophages showed intense nuclear staining (Figure 2B),
with decreased nuclear staining in the LPS + DETA
NONOate-treated cultures (Figure 2C), confirming a decrease in NF-B activation. Despite the translocation of
NF-B to the nucleus, the cytoplasm remains green because a large portion of the NF-B complex was retained in the cytoplasm. Recent studies have suggested that in
activation, only 20% of p65 is translocated to the cell nucleus (14).
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In Vivo NF-B Activation
NF-B activation was determined with EMSA of WCEs of
freshly isolated AM from controls, asthmatic subjects, and
PPH patients. The AM population of BALF was not different among healthy controls (94 ± 3% [mean ± SD],
n = 7), asthmatic subjects (95 ± 4%, n = 7), and PPH patients (98 ± 1%, n = 4). Figure 3 shows the status of NF-B
activation in healthy controls, asthmatic subjects, and PPH
patients, as well as their respective airway NO levels. Supershift assay revealed that the complex contained both
p50 and p65 rel components (Figure 4). Control antibody
to an unrelated transcription factor (the c-jun component
of activator protein-1) failed to shift the complex (data not
shown). Mean airway NO level and the arbitrary densitometric units of the EMSA for each group are shown in
Figure 5. The densitometric units of the EMSA for PPH
patients, healthy controls, and asthmatic subjects were
compared through ANOVA (P = 0.05). In healthy individuals (airway NO level = 8 ± 2 ppb), low levels of
NF-B activation were detected. Airway NO levels of
asthmatic subjects varied from 5-36 ppb. Asthmatic subjects with low NO levels ( 17 ppb) showed greater NF-B
activation than did individuals with NO > 17 ppb. Furthermore, despite the known inflammatory milieu in asthmatic
lungs, the NF-B activation in asthma patients with high
NO did not differ from the NF-B activation in controls,
suggesting that increased levels of NO suppressed NF-B
activation. In contrast, PPH patients had low NO levels
(1 ± 1 ppb) and strikingly higher NF-B activation than did controls. These results indicate that NO level is inversely related to NF-B activation in vivo.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Previously, we found that NO decreased cytokine production by human AM in vitro (10). In the present study, we
found that NO decreased NF-B activation in these cells.
IB- protein levels were greater in lystates from LPS + DETA NONOate-treated cells than in those from LPS-treated cells. These results indicate that NO prevented
NF-B activation by maintaining a steady-state level of
IB protein, an effect that may be mediated by increasing
IB synthesis, decreasing IB degradation, or both. Activation of NF-B is controlled by sequential phosphorylation, ubiquitination, and proteasome-mediated degradation of IB (15, 16). The process(es) in the NF-B
activation sequence that is affected by NO has yet to be
determined. Furthermore, whether NO itself or an NO
metabolite affects the activation sequence is unknown.
Previous work by others has also shown that NO donor
agents suppress NF-B activation in human endothelial
cells in vitro by increasing the level of IB (17). In contrast, studies with human peripheral blood lymphocytes have demonstrated activation of NF-B in the presence of
NO donors (18). The reason for these discrepancies is unclear. However, oxidants and antioxidants modulate the
bioavailability of free NO, and cell-type-specific differences in oxidant/antioxidant production may be involved
in the discrepant findings (19).
NO is an important endogenous regulatory molecule
synthesized in the lung and involved in many diverse physiologic processes (1). In asthma, airway NO levels are
increased, whereas in PPH airway NO levels are lower
than in healthy lungs (3, 4). Numerous cytokines and
growth factors appear to be involved in both diseases. Increased circulating levels of proinflammatory cytokines
(IL-1, IL-6) have been reported in PPH patients (20). We
have demonstrated a reciprocal relationship between NF-B
activation and NO in both asthma and PPH. Many of the
cytokine and growth factor genes (e.g., for TNF, IL-1,
MIP-1) linked with these diseases have NF-B elements in
their promoter regions, and NO has been shown to downregulate these factors (10, 11). In the present study, all of
the asthmatic subjects had mild stable disease, yet their
airway NO levels varied, with some patients having NO levels in the normal range (see Figure 1). The variability of NO levels over time in an individual asymptomatic patient
is unknown. If an autoregulatory feedback loop exists for
NO production in vivo (i.e., increased NO production is a
response to inflammatory stimulation, as suggested by the
finding that inflammatory cytokines upregulate inducible
nitric oxide synthase in vitro [8]), and NO downregulates
inflammatory cytokine production via NF-B, the heterogeneity of NO levels in asthmatic individuals may reflect various stages in the autoregulatory loop. Whether NO
has a protective or detrimental role in asthma remains unknown. Inhaled NO has been shown to cause significant
bronchodilation in patients with stable asthma or after
methacholine-induced bronchoconstriction (21). The NO
synthase inhibitor NG-monomethyl-L-arginine (L-NMMA)
has been found to increase bronchoconstriction induced
by bradykinin in asthmatic patients (22). These observations are supportive of an antiinflammatory role for NO in asthma.
Our studies of NF-B levels in AM from asthmatic subjects and PPH patients provide the first evidence that an
inverse relationship of NO levels with NF-B activation
exists in vivo. Our in vitro findings that NO downregulates
NF-B activation and cytokine production (10) indicate
that increased NO is antiinflammatory and therefore protective in asthmatic individuals. Although our studies do
not address the etiology of PPH or asthma, they do support a role for NO in regulating many of the cytokines implicated in the pathophysiology of these diseases. Understanding NO function may lead to ways of modulating the
inflammatory response.
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Footnotes |
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Abbreviations: alveolar macrophages, AM; 2,2-(hydroxynitrosohydrazono)-bis-ethanamine, DETA NONOate; electrophoretic mobility shift
assay, EMSA; nuclear factor-B inhibitor, IB; lipopolysaccharide, LPS;
nuclear factor-B, NF-B; primary pulmonary hypertension, PPH; whole-cell extract, WCE.
(Received in original form November 6, 1998 and in revised form March 24, 1999).
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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1. Gaston, B., J. M. Drazen, J. Loscalzo, and J. S. Stamler. 1994. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149: 538-551 [Abstract].
2. Kobzik, L., D. S. Bredt, C. J. Lowenstein, J. M. Drazen, B. Gaston, D. Sugarbaker, and J. S. Stamler. 1993. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am. J. Respir. Cell Mol. Biol. 9: 371-377 .
3. Gustafsson, L. E. 1998. Exhaled nitric oxide as a marker in asthma. Eur. Respir. J. 11(Suppl. 26):49-52.
4.
Rubin, L. J..
1997.
Primary pulmonary hypertension.
N. Engl. J. Med.
336:
111-117
5.
Kaneko, F. T.,
A. C. Arroliga,
R. A. Dweik,
S. A. Comhair,
D. Laskowski,
R. Oppedisano,
M. J. Thomassen, and
S. C. Erzurum.
1998.
Biochemical
reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.
Am. J. Respir. Crit. Care Med.
158:
917-923
6.
Alam, R.,
J. York,
M. Boyars,
S. Stafford,
J. A. Grant,
J. Lee,
P. Forsythe,
T. Sim, and
N. Ida.
1996.
Increased MCP-1, RANTES, and MIP-1- in
bronchoalveolar lavage fluid of allergic asthmatic patients.
Am. J. Respir.
Crit. Care Med.
153:
1398-1404
[Abstract].
7. Lane, S. J., D. J. Cousins, A. R. Sousa, D. Z. Staynov, and T. H. Lee. 1997. Cytokines and growth factors in asthma. In Inhaled Glucocorticoids in Asthma. R. P. Schleimer, W. W. Busse, and P. M. O'Byrne, editors. Marcel Dekker, New York. 309-347.
8.
Guo, F. H.,
K. Uetani,
S. J. Haque,
B. R. G. Williams,
R. A. Dweik,
F. B. J. M. Thunnissen,
W. Calhoun, and
S. C. Erzurum.
1997.
Interferon-
and interleukin-4 stimulate prolonged expression of inducible nitric oxide synthase
in human airway epithelium through synthesis of soluble mediators.
J. Clin.
Invest.
100:
829-838
[Medline].
9. Kunkel, S. L., S. W. Chensue, N. W. Lukacs, and R. M. Strieter. 1997. Macrophage-derived cytokines in lung inflammation. In Lung Macrophages and Dendritic Cells in Health and Disease. M. F. Lipscomb and S. W. Russell, editors. Marcel Dekker, New York. 183-202.
10.
Thomassen, M. J.,
L. T. Buhrow,
M. J. Connors,
F. T. Kaneko,
S. C. Erzurum, and
M. S. Kavuru.
1997.
Nitric oxide inhibits inflammatory cytokine
production by human alveolar macrophages.
Am. J. Respir. Cell Mol. Biol.
17:
279-283
11.
Baeuerle, P. A., and
T. Henkel.
1994.
Function and activation of NF-B in
the immune system.
Annu. Rev. Immunol.
12:
141-179
[Medline].
12. American Thoracic Society. 1986. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am. Rev. Respir. Dis. 136: 225-244 .
13. Dweik, R. A., D. Laskowski, H. M. Abu-Soud, F. T. Kaneko, R. Hutte, D. J. Stuehr, and S. C. Erzurum. 1998. Nitric oxide synthesis in the lung. J. Clin. Invest. 101: 660-666 [Medline].
14.
Verma, I. M.,
J. K. Stevenson,
E. M. Schwarz,
D. Van Antwerp, and
S. Miyamoto.
1995.
Rel/NF-B/IB family: intimate tales of association and
dissociation.
Genes Dev.
9:
2723-2735
15.
Stancovski, I., and
D. Baltimore.
1997.
NF-B activation: the IB kinase revealed?
Cell
91:
299-302
[Medline].
16.
Thanos, D., and
T. Maniatis.
1995.
NF-B: a lesson in family values.
Cell
80:
529-532
[Medline].
17.
Peng, H.,
P. Libby, and
J. K. Liao.
1995.
Induction and stabilization of IB-
by nitric oxide mediates inhibition of NF-B.
J. Biol. Chem.
270:
14214-14219
18. Lander, H. M., P. Sehajpal, D. M. Levine, and A. Novogrodsky. 1993. Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds. J. Immunol. 150: 1509-1516 [Abstract].
19. Wink, D. A., I. Hanbauer, M. B. Grisham, F. Laval, R. W. Nims, J. Laval, J. Cook, R. Pacelli, J. Liebmann, M. Krishna, P. C. Ford, and J. B. Mitchell. 1996. Chemical biology of nitric oxide: regulation and protective and toxic mechanisms. Curr. Top. Cell. Regul. 34: 159-187 [Medline].
20. Humbert, M., G. Monti, F. Brenot, O. Sitbon, A. Portier, L. Grangeot-Keros, P. Duroux, P. Galanaud, G. Simonneau, and D. Emilie. 1995. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am. J. Respir. Crit. Care Med. 151: 1628-1631 [Abstract].
21. Kacmarek, R. M., R. Ripple, B. A. Cockrill, K. J. Bloch, W. M. Zapol, and D. C. Johnson. 1996. Inhaled nitric oxide: a bronchodilator in mild asthmatics with methacholine-induced bronchospasm. Am. J. Respir. Crit. Care Med. 153: 128-135 [Abstract].
22. Ricciardolo, F. L. M., P. Geppetti, A. Mistretta, J. A. Nadel, M. A. Sapienza, S. Bellofiore, and G. U. Di Maria. 1996. Randomised double-blind placebo-controlled study of the effect of inhibition of nitric oxide synthesis in bradykinin-induced asthma. Lancet 348: 374-377 [Medline].
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T. L. Bonfield, B. Raychaudhuri, A. Malur, S. Abraham, B. C. Trapnell, M. S. Kavuru, and M. J. Thomassen PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor Am J Physiol Lung Cell Mol Physiol, November 1, 2003; 285(5): L1132 - L1136. [Abstract] [Full Text] [PDF]
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 S. B. Khatri, J. Hammel, M. S. Kavuru, S. C. Erzurum, and R. A. Dweik Temporal association of nitric oxide levels and airflow in asthma after whole lung allergen challenge J Appl Physiol, July 1, 2003; 95(1): 436 - 440. [Abstract] [Full Text] [PDF]
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 I. Jibiki, S. Hashimoto, S. Maruoka, Y. Gon, A. Matsuzawa, H. Nishitoh, H. Ichijo, and T. Horie Apoptosis Signal-Regulating Kinase 1-Mediated Signaling Pathway Regulates Nitric Oxide-Induced Activator Protein-1 Activation in Human Bronchial Epithelial Cells Am. J. Respir. Crit. Care Med., March 15, 2003; 167(6): 856 - 861. [Abstract] [Full Text] [PDF]
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 C.-L. Chen, C.-T. Lee, Y.-C. Liu, J.-Y. Wang, H.-Y. Lei, and C.-K. Yu House Dust Mite Dermatophagoides farinae Augments Proinflammatory Mediator Productions and Accessory Function of Alveolar Macrophages: Implications for Allergic Sensitization and Inflammation J. Immunol., January 1, 2003; 170(1): 528 - 536. [Abstract] [Full Text] [PDF]
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 B. Raychaudhuri, A. Malur, T. L. Bonfield, S. Abraham, R. J. Schilz, C. F. Farver, M. S. Kavuru, A. C. Arroliga, and M. J. Thomassen The Prostacyclin Analogue Treprostinil Blocks NFkappa B Nuclear Translocation in Human Alveolar Macrophages J. Biol. Chem., August 30, 2002; 277(36): 33344 - 33348. [Abstract] [Full Text] [PDF]
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D. W. Porter, L. Millecchia, V. A. Robinson, A. Hubbs, P. Willard, D. Pack, D. Ramsey, J. McLaurin, A. Khan, D. Landsittel, et al. Enhanced nitric oxide and reactive oxygen species production and damage after inhalation of silica Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L485 - L493. [Abstract] [Full Text] [PDF]
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 T. Wang, D. El-Kebir, B. Hubert, R. A.W Veldhuizen, D. Gauvin, and G. Blaise EFFECT OF INHALED NITRIC OXIDE (INO) ON SURFACTANT IN PIGS Can J Anesth, June 1, 2002; 49(90001): A99 - 99. [Full Text]
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 J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]
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J. L. Kang, W. Park, I. S. Pack, H. S. Lee, M. J. Kim, C.-M. Lim, and Y. Koh Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation J Appl Physiol, February 1, 2002; 92(2): 795 - 801. [Abstract] [Full Text] [PDF]
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I. M. Robbins, R. J. Barst, L. J. Rubin, S. P. Gaine, P. V. Price, J. D. Morrow, and B. W. Christman Increased Levels of Prostaglandin D2 Suggest Macrophage Activation in Patients With Primary Pulmonary Hypertension Chest, November 1, 2001; 120(5): 1639 - 1644. [Abstract] [Full Text] [PDF]
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R. A. Dweik, D. Laskowski, M. Özkan, C. Farver, and S. C. Erzurum High Levels of Exhaled Nitric Oxide (NO) and NO Synthase III Expression in Lesional Smooth Muscle in Lymphangioleiomyomatosis Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 414 - 418. [Abstract] [Full Text]
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 R. A. Dweik, S. A. A. Comhair, B. Gaston, F. B. J. M. Thunnissen, C. Farver, M. J. Thomassen, M. Kavuru, J. Hammel, H. M. Abu-Soud, and S. C. Erzurum NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response PNAS, February 15, 2001; (2001) 51629498. [Abstract] [Full Text]
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M. J. Coffey, S. M. Phare, and M. Peters-Golden Prolonged Exposure to Lipopolysaccharide Inhibits Macrophage 5-Lipoxygenase Metabolism Via Induction of Nitric Oxide Synthesis J. Immunol., October 1, 2000; 165(7): 3592 - 3598. [Abstract] [Full Text] [PDF]
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S. A. A. Comhair, M. J. Thomassen, and S. C. Erzurum Differential Induction of Extracellular Glutathione Peroxidase and Nitric Oxide Synthase 2 in Airways of Healthy Individuals Exposed to 100% O2 or Cigarette Smoke Am. J. Respir. Cell Mol. Biol., September 1, 2000; 23(3): 350 - 354. [Abstract] [Full Text]
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F. H. Guo, S. A. A. Comhair, S. Zheng, R. A. Dweik, N. T. Eissa, M. J. Thomassen, W. Calhoun, and S. C. Erzurum Molecular Mechanisms of Increased Nitric Oxide (NO) in Asthma: Evidence for Transcriptional and Post-Translational Regulation of NO Synthesis J. Immunol., June 1, 2000; 164(11): 5970 - 5980. [Abstract] [Full Text] [PDF]
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H. E. Marshall and J. S. Stamler Exhaled Nitric Oxide (NO), NO Synthase Activity, and Regulation of Nuclear Factor (NF)-kappa B Am. J. Respir. Cell Mol. Biol., September 1, 1999; 21(3): 296 - 297. [Full Text]
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R. A. Dweik, S. A. A. Comhair, B. Gaston, F. B. J. M. Thunnissen, C. Farver, M. J. Thomassen, M. Kavuru, J. Hammel, H. M. Abu-Soud, and S. C. Erzurum NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response PNAS, February 27, 2001; 98(5): 2622 - 2627. [Abstract] [Full Text] [PDF]
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