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Introduction
Asthma is best described as a chronic disease that involves airway inflammation and bronchial hyperrespon-siveness, in
which the inflammatory mediators are produced mainly by mast cells, macrophages, eosinophils, and lymphocytes.
Bronchial epithelial cells directly take part in the pathogenesis of airway inflammation and bronchial
hyperresponsiveness[1].
Stimulated by histamine released from mast cells, airway epithelial cells have been shown to produce numerous
inflammatory mediators, such as platelet activating
factor[2], tumor necrosis factor
(TNF)[2], macrophage chemotactic protein
(MCP)-1[2],
prostaglandins[3],
endothelin-1[4],
interleukin-1[5],
interleukin-6[5],
interleukin-8[5],
interleukin-16[5]. These mediators may affect
the tone of airway smooth muscle, contribute to the local accumulation of inflammatory cells, and act on epithelial cells
themselves, thus promoting the chronic airway inflammation. In patients with bronchial asthma, the airway epithelium
becomes more sensitive to exogenous or endogenous stimuli, which leads to the increased expression of cytokines and
chemoattractants, contributing to exorbitant airway inflammation and bronchial
hyperrespon-siveness[1]. However, the mechanism of the hypersensitization of asthmatic airway epithelium is far from being fully understood.
In 1998, Dolmetsch et al and Li
et al simultaneously reported that "artificial" calcium oscillations could increase or
optimize the gene expression in a frequency-decoding
manner[6-8]. Later, Hu et al proved this in receptor mediated calcium
oscillations[9]. They found that a decrease in the frequency of
[Ca2+]i oscillations during low-level histamine stimulation
resulted in a parallel decrease in transcription activity. The regulation of nuclear transcriptional activity by the frequency of
cytosolic Ca2+ oscillations may provide cells with a specific mechanism to control the gene expression during agonist
stimulation.
These novel findings shed light on the study of diseases related to hypersensitivity. The frequency of calcium oscillation
may therefore be used as an indicator of the inner sensitivity of cells. To testify if hypersensitized epithelium in patients with
bronchial asthma displays a higher calcium oscillation frequency during agonist stimulation, we measured the alterations of
intracellular Ca2+ kinetics caused by different concentrations of histamine in bronchial epithelial cells preincubated with
serum either from sensitized or nonsensitized guinea pigs. To further clarify the relevance of the alterations of calcium
signaling to asthma, the activity of the calcium dependent transcription factor, nuclear factor of
kappaB (NF-kB), also the most important transcription factor involved in airway inflammation, was
examined.
Materials and methods
Airway sensitization of guinea pigs and preparation of sensitized
serum According to the protocol reported previously,
the guinea pigs were sensitized[10]. Briefly, 20 male guinea pigs [400-500 g, conventional animal, certificate
No SCXK(HB2004-0007)], were supplied by the Faculty of Laboratory Animals (Tongji Medical College of HUST, China), and
maintained according to the "Chinese Regulations for Experimental Animals". The animals were randomly divided into 2 groups;
sensitized and nonsensitized group.
In the sensitized group, the animals were sensitized by intraperitoneal injection of 1 mL 10% ovalbumin (Sigma) in
phosphate buffered saline (PBS) on d 1 and d 8. From d 15, the animals were provoked by exposure to 1% ovalbumin aerosol
in PBS generated from supersonic nebulae with a mask for about 30 s until asthma-like attacks occurred for another 2 weeks.
The airway responsiveness to histamine was evaluated in the guinea pigs within 24 h after the last provocation, as previously
reported[11].In the nonsensitized group, the animals were treated with PBS under a same procedure.The sensitized guinea pigs were anaesthetized with 20% urethane with a dose of 6 mL/kg of body weight. Blood was then
collected within 24 h after the last provocation through a carotid intubation. The serum was obtained from the supernatant
of a centrifugation of blood at 93×g for 10 min and was stored -80 ºC. The guinea pigs were then killed with overdoses (1.2
mL/100 g) of 20% urethane, the lungs were fixed with 10% formaldehyde and followed with an HE staining.
Porcine bronchial epithelial cell
culture The method for isolation and culture of porcine bronchial epithelial cells (PBEC)
has been described previously[12,13], but was used with modification. Cells were isolated from bronchia
of 10- to 12-month-old pigs by casein proprotease and cultured in flasks pre-coated with rat-tail collagen. The medium was DMEM/F-12 (GIBCO,
Grand Island, NY, USA) supplemented with 5% fetal bovine
serum (GIBCO), 5 µg/mL insulin (Sigma, St Louis, MO, USA), 10
µg/mL transferring (Sigma), 0.5 µg/mL hydrocortisone (Sigma), 10 ng/mL epidermal growth factor (EGF; Sigma), 100 nmol/L
retinoic acid (Sigma), 0.5 mg/mL bovine serum albumin (Sigma), and antibiotics (100 IU/mL penicillin and
100 µg/mL streptomycin). After incubation for 24 h, primary cultured cells were propagated in monolayer cultures seeded on glass
coverslips pre-coated with rat-tail collagen in culture utensil. The first passages of cells were used in the present study.
Passive sensitization of PBEC When PBEC grew to approximately 50%-60% confluence, the culture medium
DMEM/F-12 was removed and sensitized or nonsensitized serum was added for substitution. Cells were incubated for another 24
h[14]. The PBEC were therefore divided into 2 groups. In the group S, the cells were preincubated with sensitized serum. In the
group N, the cells were preincubated with nonsensitized serum.
Intracellular free Ca2+ measurement
The method for intracellular free
Ca2+ measurement has been described
previously[15]. Briefly, to measure
[Ca2+]i, PBEC monolayers on glass coverslips were incubated with sensitized or nonsen-sitized serum
containing 2 µmol/L Fura-2 (acetoxymethyl ester form; Calbiochem, La Jolla, Calif, USA) in 37 ºC 5 %
CO2 atmosphere for 40 min. The coverslips were then gently washed 3 times with indicator-free Hepes buffered saline (HBS) containing: NaCl 140
mmol/L, KCl 4.5 mmol/L, CaCl2
1.5 mmol/L, MgSO4 1.0 mmol/L, D-glucose 10 mmol/L, and HEPES 21 mmol/L, pH 7.40, at room temperature to allow
deesterification of the indicator. Glass coverslips were gently transferred to a perfusion chamber mounted on the stage of an
inverted epifluorescence microscope (I×70; Olympus, Tokyo, Japan). Monolayers were first exposed to HBS for equilibrium
and then histamine was added to a final concentration of 0.1, 1, or 10 µmol/L. Fura-2 fluorescence was recorded on a field of
1-7 connected cells of a subconfluent PBEC monolayer. Fura-2 fluorescence was alternatively excited at 340 nm and 380 nm
using a polychrome (Photonics, Munich, Germany) corresponding to the
Ca2+-bound and
-free forms of the indicator, respectively. Emitted fluorescence through bandpass interference filters (Photonics) with
selected wavelength bands at 510 nm was captured by a computer coupled device (CCD, Imago-QE; Photonics) and
transferred to Till-vision software (Photonics). Autofluo-rescence from unloaded PBEC was subtracted from Fura-2 fluorescence
recordings before the calculation of the ratio of the emitted fluorescence intensity excited at 340 nm and 380 nm, respectively
(F340/F380). Because the dissociation constant
(Kd) of Fura-2 for
Ca2+ in PBEC may be different from that obtained
in vitro[16] and because of the general uncertainties of the calibration techniques of
[Ca2+]i measure-
ment[17], the absolute amount of
[Ca2+]i was not calculated and therefore
F340/F380 was used as a relative indicator of
[Ca2+]i in this study.
NF-kB activity assay The NF-kB activity assay was carried out according to an ELISA-based method described
previously[18,19]. In brief, after histamine stimulation for 30 min, cells were rinsed twice with cold PBS, detached with trypsin and
centrifuged for 10 min at 93×g. The pellet was then resuspended in 100 µL lysis buffer (20 mmol/L HEPES pH
7.5, 0.35 mol/L NaCl, 20% glycerol, 1% NP-40, 1 mmol/L
MgCl2, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA) containing a protease
inhibitor cocktail (92121; Calbiochem, San Diego, CA, USA). After incubating on ice for 10 min, the lysate was centrifuged for
20 min at 17 968×g. The supernatant constituted the total protein extract. After being quantified with BCA reagent (61105;
Pierce, Rockford, IL, USA), the cell extract was kept frozen at -80 ºC until
NF-kB activity measurement. Cell extracts were incubated in a 96-well plate coated with the oligonucleotide containing the
NF-kB consensus-binding site (5กฏ-GGGACTTTCC-3กฏ). Activated transcription factors from extracts
specifically bound to the respective immobilized oligonucleotide.
NF-kB activity was then detected with the primary antibody to
NF-kB p65 and secondary antibody conjugated to horseradish
peroxidase. NF-kB activity was finally determined as absorbance values measured with a microplate reader at the wavelength
of 450 nm.
Data analysis and statistics All data were expressed as means±SD;
t-test was used to evaluate the significance of
differences in calcium oscillation amplitude and frequency between different groups. ANOVA in SPSS12.0 was used to
evaluate the significance of differences in NF-kB activity between multiple groups, with
P<0.05 as the level of significance.
Results
HE staining and airway responsiveness In sensitized guinea pig lungs, there was increased accumulation of lymphocytes,
eosinophils, neutrophils both in the interstitial and bronchia. Airway smooth muscle became significantly hypertrophic. In
some lungs, the increase in size of the submucosal glands and thickening of basement membrane of bronchial epithelium
could be seen, while others displayed epithelial cell sloughing or cilia cell disruption (Figure 1 A, 1B, 1C).
The baseline of the average intra-airway pressure in sensitized guinea pigs was significantly higher than the
nonsen-sitized guinea pigs (2.41±0.24
vs 1.66±0.20 mmHg, P<0.01,
n=3). The PC20 (the minimum concentration of histamine required
for increasing 20% of the average intra-airway pressure) of nonsensitized guinea pigs did not appear in the concentrations of
histamine used. The PC20 of sensitized guinea pigs appeared at 1.5 mmol/L histamine (Figure 1D).
Effect of passive sensitization on 0.1 µmol/L histamine-induced calcium signaling in PBEC
In the PBEC of group N, 0.1 µmol/L histamine did not induce any oscillation of
[Ca2+]i in all monolayers studied
(n=6). In the PBEC of group S, 0.1
µmol/L histamine induced
[Ca2+]i oscillations in 4 of 6 monolayers studied (Figure 2).
Effect of passive sensitization on 1 µmol/L histamine-stimulated calcium signaling in
PBEC During 1 µmol/L histamine stimulation, repetitive
[Ca2+]i oscillations were
observed in all monolayers studied in the PBEC of group N and group S
(n=6 for each). The average
[Ca2+]i oscillation
amplitude in PBEC of group N and group S was 0.8471±
0.0391 and 0.8700±0.0335, respectively. There was no signifi-cant increase in
[Ca2+]i oscillation amplitude after passive
sensitization (P>0.05). The average
[Ca2+]i oscillation frequ-ency in PBEC of group N and group S was 0.0069±0.0007 and
0.0100±0.0013, respectively. There was a significant increase in
[Ca2+]i oscillation frequency after passive sensitization
(P<0.01) (Figure 3).
Effect of passive sensitization on 10 µmol/L histamine-stimulated calcium signaling in
PBEC When PBEC were stimulated with 10 µmol/L histamine, a transient initial
increase (TII) followed by a sustained elevation of
[Ca2+]i was observed in monolayers of group N and group S
(n=6 for each). There was no significant difference in the amplitude of the TII in PBEC between group N and group S
(0.9532±0.0367 vs 0.9831±0.0318,
P>0.05, n=6); The average amplitude of following sustained elevation (FSE) in PBEC was
significantly increased in group S as compared with that in group N (0.6076±0.0274
vs 0.6559±0.0243, P<0.01,
n=6) (Figure 4).
Histamine-stimulated NF-kB activity in sensitized and nonsensitized PBEC
There was very low level of basic NF-kB activity in the PBEC of groups N and S (0.183±0.016
vs
0.190±0.004, n=3, P>0.05). Histamine 0.1 µmol/L did not increase
NF-kB activity of PBEC in group N (0.183±0.016
vs
0.197±0.013 before and after administration, respectively;
n=3, P>0.05). In contrast, Histamine 0.1 µmol/L significantly
increased the NF-kB activity of PBEC in group S (0.19±0.004
vs 0.412±0.083 before and after administration, respectively;
n=3, P<0.05). Histamine 1 µmol/L significantly increased
NF-kB activity of PBEC in group N and group S, and the
NF-kB activity in group S was significantly higher than that in group N (1.178±0.095
vs 0.873±0.069, n=3, P<0.05). Histamine10 µmol/L also
significantly increased the NF-kB activity of PBEC in group N and group S, and the
NF-kB activity in group S was higher than that in group N, but the difference was not significant (1.226±0.086
vs 1.373±0.11, n=3,
P>0.05) (Figure 5).
Discussion
Lung HE staining and airway responsiveness indicated the existence of airway inflammation and hyperrespon-siveness
in ovalbumin sensitized guinea pigs. This demonstrated that the asthma model employed in the present study was feasible.
Passive sensitization provides a useful model for the understanding of asthma at a cellular level. This technique involves
the incubation of bronchial epithelial or smooth muscle cells in culture with serum from patients with allergic asthma or,
alternatively, serum from nonallergic nonasthmatic
patients[20].
The results in this study showed that histamine could induce calcium oscillations in PBEC. Histamine 0.1 µmol/L could
induce [Ca2+]i oscillations of PBEC in group S, but not in group N. Histamine 1 µmol/L could induce
[Ca2+]i oscillations of PBEC in both group S and group N. The
[Ca2+]i oscillation frequency was significantly higher in PBEC of group S than in
group N. These results demonstrated that passive sensitization might increase histamine-induced calcium oscillation
frequency in PBEC. PBEC becomes more sensitive to histamine after sensitization. The data presented here provide direct
evidence of the existence of hypersensitivity in calcium signaling in passive sensitized PBEC. The subsequent
calcium-regulated NF-kB activity was in accordance to the calcium oscillation frequency in 0.1 µmol/L and 1 µmol/L
histamine-stimulated PBEC.
The allergic serum contains a high concentration of total
IgE[21,22] and cytokines such as
IL-1b[23], TNF-a[24], and so on.
Hakonarson and Grunstein also reported that passive sensitization of human bronchus could markedly increase the
expression of IL-1b as well as the mRNA for the low-affinity
IgE and these cytokines may increase the reactive oxygen species (ROS)
production in PBEC. ROS generation in cells has been observed after stimulation by many cyto-kines, including
IL-1b[26] and TNF-a[27-30]. It has been proved that ROS could increase the sensitivity of endoplasmic reticulum (ER)
Ca2+ stores to inositol 1,4,5-trisphosphate
(Ins-1,4,5-P3)[31], which may lead to an increased calcium oscillation
frequency[32,33].
The finding that passive sensitization may increase histamine-stimulated calcium oscillation frequency in PBEC has
important significance in the understanding of asthma pathophysiology. As gene transcription was regulated by calcium
oscillation frequency[6-9], the increased histamine-induced calcium oscillation frequency led to a parallel elevation of
NF-kB activity in 0.1 µmol/L and 1 µmol/L histamine-stimulated
PBEC. It is known now that NF-kB is the most important
transcription factor involved in the pathogenesis of
asthma[34,35] and that it can drive the expression of cytokines such as
IL-1b, TNF-a, and so on, in airway epithe-lium. This leads to the formation of positive feedback and ultimately contributes to exorbitant
inflammation in asthma. The finding that sensitized cells prone to generate calcium oscillations or displaying higher calcium
oscillation frequency during agonist stimulation might also be observed in other sensitized target cells involved in allergic
diseases such as allergic rhinitis, eczema, and so on, and that they might also be a universal feature of allergic diseases with
hypersensitivity in target cells. Intervention concerning the calcium oscillation frequency might be a kind of strategy to
control allergic diseases in the future.
Histamine 10 µmol/L could increase the
[Ca2+]i to a higher plateau in passive sensitized PBEC than in nonsensitized PBEC.
These results demonstrated that passive sensitized PBEC developed an increasing tendency to form calcium overload during
a higher concentration of agonist stimulation. As calcium overload may mediate injury, sensitized PBEC become more
susceptible to stimuli. This may contribute to the epithelial cell sloughing, cilia cell disruption, which was frequently
observed in late stage asthmatic lung. However, there was no significant difference in
the NF-kB activity of PBEC between the two groups, which might be due to the fact that the frequency-decoding manner of calcium-regulated gene expression
was more effective than the amplitude-decoding manner.
Acknowledgements
We thank Ms Li-hong LONG, Ms Zhong-lin ZHU, and Mr Zhou-huan WU in Department of Pharmacology of Tongji
Medical College for their technical assistance.
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