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Introduction
Asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory lung diseases associated with
progressive airflow limitation. They are characterized by the presence of leukocytes in the
lungs[1]. Type II alveolar epithelial (AEII) cells function in synthesis, secretion, and recycling of all components of the surfactant. Results from a number of
published studies have shown that pulmonary epithelial cells, especially type II cells, help recruit inflammatory cells to the
lungs by generating the chemokines interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) in response to
inflammatory factors such as tumor necrosis factor
(TNF)-a and IL-1β. They have also found that AEII cells can play an
important role in lung inflammatory
diseases[2,3]. As well as expressing surfactants and chemokines, AEII cells also express
adhesive molecules and other immune-related
factors[4]. AEII cells may act as immunoregulatory cells to modulate the
physiological and pathological functions of the lungs.
The suppression of immune responses and improvement of alveolar fluid clearance has received much attention.
Numerous endogenous agents have been verified to protect cell damage from pro-inflammation. In our previous data, we
demonstrated that AEII cells secrete neuropeptide calcitonin gene-related peptide (CGRP) and that
IL-1β induces CGRP secretion[5,6]. Our initial results showed that AEII cell-
derived CGRP could suppress inflammatory chemokine IL-8 secretion induced by IL-1 in an autocrine/paracrine mode. CGRP
is a potent vasodilator neuropeptide localized in peripheral and central nerves. It also has broad anti-inflamatory effects,
especially in the immune and related
systems[6,7]. In the lungs, CGRP-like immunoreactivity is localized in the nerve fibers
of the airway mucosa and around vascular smooth
muscle[8,9]. CGRP modulates airway functions in health and disease. These
functions include vasoregulation, broncho-regulation, anti-inflammatory actions and tissue
repair[10]. It is interesting to note that CGRP is also expressed in AEII cells. Non-specific immune cells and the enhanced release of CGRP under inflammatory
stress may play a negative feedback role in the local immune
reaction[5].
In the pathogenesis of many inflammatory lung diseases such as acute respiratory distress syndrome, COPD and asthma,
neutrophils have been implicated to play an important role. The CXC chemokine IL-8 is a potent neutrophil-recruiting and
activating factor. Although lipopolysac-charide,
IL-1β and TNF-a are IL-8 inducers, IL-8 exerts its effects on neutrophils by
binding with high affinity to 2 chemokine receptors, CXCR1 and CXCR2, on the cell surface. In bronchoalveolar lavage of
children with chronic respiratory diseases, IL-8 levels are significantly associated with
IL-1β, and the detection of IL-8 in clinical samples from
patients with these diseases has led clinicians to believe that the antagonism of IL-8 may be a practicable therapeutic strategy
for disease management[11].
The present study was designed to confirm the inhibitory effect of AEII-derived CGRP on
IL-1β-induced IL-8
secretion in the human AEII cell line (A549) and to investigate the mechanisms of the inhibitory effects.
Materials and methods
Materials A rabbit anti-human CGRP antibody (Ab) and recombinant human
aCGRP, hCGRP8_37 and an anti-CGRP antibody were purchased from Peninsula Laboratory (Belmont, CA, USA). Recombined human
IL-1β was purchased from PeproTech House (W6 Bll, London, United Kingdom). Trizol was purchased from Promega (Madison, WI, USA). Dulbecco's
modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone Co (Logan, UT, USA). All
other chemicals and drugs were purchased from Sigma Chemical Co (St Louis, MO, USA) and Chinese Chemical Co (Beijing,
China).
Cell culture A549 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were
grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (0.1 µL) in a humidified 37
oC incubator. Before being treated with chemicals, the cells were washed with DMEM twice and maintained in DMEM without FBS at 37
oC for 3 to 4 h. To avoid peptide degradation, 1
mg·L-1 aprotinin was added to every experimental group when the cells were incubated
with hCGRP or hCGRP8-37.
ELISA detection of IL-8 Ninety-six well-microtiter plates (Nunc, Gmb H & Co. KG, Wiesbaden, Germany) were coated with
50 µL containing 1 mg·L-1 of the goat anti-mouse IL-8 antibody (R&D Systems, BorsigstraBe, Wiesbaden, Germany) in PBS at
4 oC for 48 h. After removing the excess capture Aβ, the wells were filled with 50 µL of Casine/PBS (Pierce, Biotechnology, Inc,
Rockford, IL, USA) and incubated at room temperature for 1 h to saturate excess binding sites. After being washed 3 times
with wash buffer (0.125% Triton X-100/PBS), serial dilutions of the experimental samples diluted in 10% Casine/PBS were
added to the plates, which were incubated at room temperature for 2 h. After 3 washes, 100 µL of the biotinylated detector
anti-hIL-8 Aβ was added to the wells and incubated for 1 h at room tem-perature. After an additional 3 washes, the plates were
incubated with peroxidase-conjugated streptavidin for 0.5 h. After 3 final washes, the solutions were developed with
hydrogen peroxide and tetramethylbenzidine (Sigma, St Louis, Mo, USA) and then stopped by the addition of 1.5 mol/L
H2SO4. Titrations of recombinant human MCP-1 (R&D Systems, BorsigstraBe, Wiesbaden, Germany) were included in each
experiment for standardization.
Stable transfection of CGRP gene into A549
cells A pcDNA3.1 plasmid containing human CGRP cDNA was constructed
as previously described[12] and transferred to the A549 cells with the use of Lipofectin (Invitrogen, Carlsbad, CA, USA). After
transfection, the cells were cultured with G418 (800
mg·L-1) for 4 d, and a stably transfected cell line was selected by
subculturing the cells from a monoclone.
RNA extraction and RT-PCR
The DMEM was removed and the cells were washed twice with PBS. The RNAs were then
extracted by use of 1 mL Trizol and 0.2 mL chloroform and precipitated with 0.5 mL isopropanol. The suspension was kept at
-70 oC for 30 min and centrifuged at 12
000×g at
4 oC for 15 min. The pellet was washed with 0.5 mL 70% ethanol and dried. The RNA was stored at -70
oC.
As previously
reported[5], the RNA was reverse transcribed with oligo (dT) 15_17. The oligonucleotide primers for IL-8
and β-actin were synthesized on a DNA synthesizer (Model 49a, Applied Biosystems Inc, Foster, CA, USA). The nucleotide
sequences of β-actin were sense, 5'-CATCTCTT-GCTCGAAGTCCA-3' and antisense,
5'-ATCATGTTTGAGA-CCTTCAACA-3'; the PCR product was 300 bp; human IL-8 primers were sense, 5'-ATTTCTGCAGCTCTGTGTGAAGG-TGC-3' and antisense,
5'-TGACCTTTGACCTAGTTTTG-3'; the product was 749
bp[13]. The PCR amplification process consisted of denaturation at
94 oC for 30 s, primer annealing at 58
oC (b-actin) or 60 oC (IL-8) for 30 s, and extension at 72
oC for 1.5 min. PCR was run for 22 cycles for
b-actin and 24 cycles for IL-8.
Inhibition of CGRP expression by small interfering RNA
(siRNA) As we described
previously[14], RNA-interfering plasmid for the
b-CGRP gene was transferred to the A549 cells with the use of Lipofectin (Invitrogen, Carlsbad, CA, USA). For
stable inhibition of CGRP, 2 µg of pSuper-CGRP was cotransfected with 1 µg of pBABE-puro plasmid. The transfected cells
were selected with 1 µg/mL puromycin for
7 d. Monoclones were chosen and expanded. Reverse-transcription PCR and radioimmunoassay showed decreased CGRP
mRNA levels and protein secretion by 50%_70% in transfected cells.
Measurement of intracellular ROS
generation The production of ROS, especially
H2O2, by A549 cells was detected with
luminol plus horseradish peroxide-derived chemiluminescence in a lit box and a luminescence analyzer (BPCL
Ultra-weak, Beijing, China) at 37 oC as previously
described[15]. Photon counts were integrated over 1 s and shown on a computer monitor.
A549 cells were incubated with IL-1β or/and other chemicals for indicated times and the cells were then washed twice with 1
mL 37 oC PBS. Horseradish peroxide 10 µg/mL and 0.5 mmol/L luminol were then added. The luminol plus horseradish
peroxidase-derived chemiluminescence was initiated by adding 3 mmol/L NADH as a substrate. All experiments were
repeated at least 3 times; this reflected the formation of ROS.
Statistics The results are expressed as mean±SD. Data analysis involved the use of GraphPad Prism software (GraphPad
software, Inc, San Diego, CA, USA). One-way ANOVA, Student-Newman-Keuls test (comparisons between multiple groups),
or unpaired Student's t-test (between 2 groups) was used as appropriate.
P<0.05 was considered significant.
Results
Suppression of IL-8 secretion by endogenous CGRP
To investigate the effect of IL-1β-induced endogenous CGRP on
IL-1β-induced IL-8 secretion, we treated A549 cells with
hCGRP8-37 (0.1 nmol·L-1), a CGRP-1 receptor antagonist, simultaneously
with IL-1β (1 ng/mL) for different times. As shown in Figure 1,
hCGRP8-37 at a very low concentration of 0.1_1
nmol·L-1 significantly enhanced the
IL-1β-induced IL-8 secretion, and even increased it to 130% at 24 h.
To further confirm that the endogenous CGRP has an inhibitory effect on
IL-1β-induced IL-8 secretion, we used molecular and genetic manipulation of the CGRP expression.
As previously reported, the knockdown of β-CGRP by
siRNA[15] specifically attenuated both the CGRP mRNA and protein levels in A549 cells (Table 1). As shown in Figure 2, the inhibition of CGRP
expression by siRNA significantly increased IL-8 secretion on
IL-1β stimulation. These data strongly suggest that
endogenous CGRP inhibits chemokine IL-8 expression induced by proinflammatory
IL-1β.
Suppression of IL-8 secretion by exogenous
CGRP Exogenous CGRP (0.1_10
nmol·L-1) reduced IL-1β-induced
IL-8 secretion in a concentration-dependent manner, and the
inhibition reached more than 30% at 24 h (Figure 3). Moreover, A549 cells stably transfected with the CGRP gene showed an
elevated CGRP protein level (Table 2). Compared with the control and empty vector clone, after
IL-1β adminis-tration, both the mRNA and protein levels of IL-8 were greatly inhibited in the CGRP high-expression clones (Figure 4A,4B). The CGRP
inhibitory effect mimicked exogenous CGRP administration in cells.
Effect of CGRP on IL-1β-induced ROS
formation Chemiluminescence assay in A549 cells showed that CGRP (100
nmol·L-1) significantly inhibited
IL-1β-induced ROS production in A549 cells (Figure 5).
N-acetyl-L-cysteine (NAC), a thiol-based
antioxidant, and ROS scavenger or diphenylene-iodonium (DPI), an inhibitor of mitochondrial NADPH-ubiquinone
oxido-reductase, also reduced in part IL-1β-evoked IL-8 production and abolished the CGRP effect, which indicates that CGRP
exerts its inhibitory function via a ROS pathway (Figure 6).
Discussion
In the present study, we demonstrate that endogenously expressed CGRP in the AEII cell line A549 suppresses
IL-1β-induced IL-8 secretion in an autocrine/paracrine mode by inhibiting the ROS pathway.
As an approximately 70 to 72-residue mature protein, IL-8 can be produced by leukocytic cells (monocytes, T cells,
neutrophils, and natural killer cells) and nonleukocytic
somatic cells (endothelial cells, fibroblasts, and epithelial
cells)[16]. IL-8 binds to 2 distinct receptors, CXCR1 and CXCR2, with a similar high affinity, and the receptors are mainly expressed on the
cell surface of neutrophils[17,18]. After ligand binding, the receptors are internalized and subsequently recycled and reappear
on the cell surface rapidly within 60
min[19]. More-over, ligand binding activates pertussis toxin-sensitive and
receptor-coupled G proteins, particularly Gai
proteins[20].
G proteins then activate phosphatidylinositol
3-kinase-g (PI3K-g), which in turn generates phosphatidylinositol
3,4,5-trisphosphate (PIP3)[21]. PIP3 activates protein kinase B (Akt) as well as GTPases, resulting in directed cell migration.
In lung infection, including bacterial and viral infection, lung injury, including reperfusion injury, transplantation and
other physical conditions, and lung acute and chronic
inflammatory diseases, such as acute respiratory distress syndrome, asthma and COPD, IL-8 works as a key mediator in
neutrophil-mediated acute inflammation because of its potent actions on neutrophils. In our previous and present data,
proinflammatory factor IL-1β also stimulated CGRP
secretion from AEII cells and endogenous/exogenous CGRP was found to inhibit
IL-1β-induced IL-8 elevation in an autocrine/paracrine manner. These data indicate that AEII cells, together with neurogenic CGRP, may restrict lung
inflammation at least in part, eliminating injury chemockine secretion.
CGRP is considered an immunomodulatory neuropeptide. We have demonstrated that CGRP can attenuate multiple
low-dose streptozotocin-induced insulitis and reduce the occurrence of diabetes in
mice[12]. As well as this, in vitro
experiments have found that lymphocyte-derived CGRP can inhibit Con A-induced proliferation and IL-2 production in rat thymocytes in
an autocrine/paracrine mode[22]. In the present study, a very low concentration of
hCGRP8_37 (0.1 nmol·L-1), an antagonist of
the CGRP1 receptor, at approximately 10 times more than that of endogenous CGRP, significantly magnified the
IL-1β-induced IL-8 secretion (Figure 1) between 6 and 24 h. The knockdown of
b CGRP gene
expression by siRNA in A549 cells did not change the basal level of IL-8, but greatly potentiated
IL-1β-induced IL-8 secretion. These data indicate that endogenous CGRP may play an inhibitory effect on the inflammatory process. Stably transfected,
high-level CGRP clones seem to attenuate the cell responsibility of IL-8 secretion to
IL-1β (Figure 4). In lung or airway inflammatory diseases, CGRP released from terminal afferents, neuroendocrine cells, and AEII cells might lead to a high local
concentration of CGRP. We predict that CGRP will not only inhibit the immunoreactivity of the lung epithelium to
inflammatory factors by reducing IL-8 secretion, but also enhance the phagocytosis of the peripheral macrophages to attenuate the
inflammation[23].
IL-1β has been shown to stimulate the production of ROS, a class of highly reactive, diffusible, and ubiquitous
mole-cules, in various cell types. ROS are involved in aging and many diseases such as cancer, diabetes mellitus, athero-sclerosis,
neurological degeneration, angiogenesis, and
tumor invasion[24,25]. IL-1β primes and triggers
O2¯· formation by activation of the NADPH oxidase and thereby provides an
oxidative burst[26]. Numerous evidence have
reported that ROS scavenger or NADPH oxidase inhibitor significantly reduces the
IL-1β-induced expression of target
genes[26,27]. IL-8 is one of the target genes of
IL-1β[28]. Our previous report stated that CGRP gene therapy suppressed ROS in the
pancreas and protected mice against autoimmune
diabetes[29], thus, CGRP may protect b cells against ROS damage. In the
present study, our findings that endogenous and exogenous CGRP inhibit
IL-1β-induced ROS production in A549 cells imply that CGRP may have a broad inhibitory effect on ROS formation.
It is interesting to note that
IL-1β simultaneously induces the secretion of IL-8 and CGRP, whereas CGRP itself acts on
A549 cells and inhibits IL-8 production. On the one hand, in a seemingly autocrine feedback loop,
IL-1β, as a stimulatory factor attracts inflammatory cells to the local area via chemo-kine IL-8. On the other hand, it also triggers the protective
mechanism by CGRP release to confine the inflammatory
response and avoid excessive injury. This kind of autocrine feedback loop may be a protective mechanism of tissue.
In conclusion, we have shown that in A549 human AEII cells, intrinsic and extrinsic peptide CGRP inhibits
IL-1β-
induced IL-8 secretion in an autocrine/paracrine mode via ROS suppression. Our data provide a new insight into the
immunomodulatory effect of AEII-derived CGRP and
suggests a novel therapeutic target in lung inflammatory disease.
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