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MUSCLE CELL BIOLOGY AND CELL MOTILITY
-induced endogenous monocyte chemoattractant protein-1 secretion in type II alveolar epithelial cells
Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, School of Basic Medical Sciences, Peking University, Beijing, People’s Republic of China
Submitted 24 October 2005 ; accepted in final form 29 March 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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induces CGRP secretion in the A549 human AEII cell line. In the present study, the CGRP-1 receptor antagonist human (h)CGRP8–37 (0.1–1 nM) greatly amplified the production of IL-1-induced monocyte chemoattractant protein (MCP)-1. The inhibition of CGRP expression by small interfering RNA significantly increased MCP-1 secretion on IL-1 stimulation. However, exogenous hCGRP (10–100 nM) suppressed IL-1-evoked MCP-1 secretion in MCP-1 promoter activity, and CGRP gene stably transfected cell clones significantly inhibited both the mRNA and protein levels of MCP-1 induced by IL-1. These data imply that AEII-derived CGRP suppressed IL-1-induced MCP-1 secretion in an autocrine/paracrine mode. Subsequent investigation revealed that CGRP inhibited IL-1-evoked NF-
B activity by suppressing IB
phosphorylation and degradation. Moreover, CGRP attenuated IL-1-induced reactive oxygen species (ROS) formation, the early event in proinflammatory factor signaling. We previously showed that the CGRP inhibitory effect was mediated by elevated intracellular cAMP and show here that analogs of cAMP, 8-bromoadenosine 3',5'-cyclic monophosphothioate and the Sp isomer of adenosine 3',5'-cyclic monophosphothioate, mimicked the CGRP suppressive effect on IL-1-induced ROS formation, NF-B activation, and MCP-1 secretion. Thus increased endogenous CGRP secretion in lung inflammatory disease might eliminate the excessive response by elevating the cAMP level through inhibiting the ROS-NF-B-MCP-1 pathway. reactive oxygen species; lung; inflammation
and IL-1, and AEII cells may play an important role in lung inflammatory diseases (36, 37). The suppression of immune responses and improvement of alveolar fluid clearance have received much attention.
Numerous endogenous agents have been verified to protect cells from proinflammation damage. With our previous data (24), we have shown that AEII cells secrete the neuropeptide calcitonin gene-related peptide (CGRP) and IL-1 induces CGRP secretion. Our initial functional results show that AEII cell-derived CGRP can suppress secretion of the inflammatory chemokine MCP-1 induced by IL-1 in an autocrine/paracrine mode. CGRP is a common neuropeptide with broad anti-inflammatory effects, especially in the immune and relative systems (4, 13). In the lung, CGRP-like immunoreactivity is localized in the nerve fibers of the airway mucosa and around vascular smooth muscle (8, 25) and is also found in pulmonary neuroepithelial cells/bodies and Clara cells (1, 20, 33). Because of this unique distribution, CGRP reacts in response to various respiratory stimuli and is predicted to be important in controlling lung circulation (19) and airway hyperresponsiveness (12). CGRP is secreted from nerve and non-nerve-derived sources, which indicates that it may play an important role in lung inflammatory disease. It is interesting that CGRP is expressed in AEII cells, which are nonspecific immune cells, and the enhanced release of CGRP under inflammatory stress may play a negative feedback role in local immune reactions. It is important to understand the mechanism of the endogenous CGRP feedback role in lung inflammation.
The present study was designed to evaluate the mechanism of AEII cell-derived CGRP in IL-1-induced MCP-1 secretion in the human AEII cell line A549. The results of this study suggest that AEII cell-derived endogenous CGRP could suppress IL-1-induced MCP-1 secretion by inhibiting the reactive oxygen species (ROS)-NF-B pathway through enhanced intracellular cAMP signaling.
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MATERIALS AND METHODS |
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-CGRP, hCGRP8–37, and anti-CGRP antibody were purchased from Peninsula Laboratories (Belmont, CA). Rp and Sp isomers of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS, Sp-cAMPS), 8-bromoadenosine 3',5'-cyclic monophosphothioate (8-BrcAMPS), diphenyleneiodonium chloride (DPI), and phenylarsine oxide (PAO) were from Calbiochem (La Jolla, CA). N-acetyl-L-cysteine (NAC) was purchased from Sigma (St. Louis, MO). Enhanced chemiluminescence (ECL) detection reagents were from Pierce Biotechnology (Rockford, IL). Anti-IB antibody was from Cell Signaling Technology (Beverly, MA). Anti-actin antibody was from Abcam (Cambridge, MA). Cell culture media and supplements were from Hyclone (Logan, UT). Cell culture. A549 cells from the American Type Culture Collection (Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and penicillin-streptomycin (100 U/ml) in a humidified 37°C incubator. When confluent, cells were disaggregated in trypsin solution, washed in DMEM, centrifuged at 125 g for 5 min, and then resuspended and subcultured according to standard protocols. Before being treated with chemicals, the cells were washed with DMEM twice and maintained in DMEM without FBS at 37°C for 3–4 h. To avoid peptide degradation, 1 µg/ml aprotinin was added to every experimental group when the cells were incubated with hCGRP or hCGRP8–37.
ELISA detection for MCP-1. Ninety-six-well microtiter plates (Nunc, Wiesbaden, Germany) were coated with 50 µl of 1 µg/ml goat anti-mouse MCP-1 antibody (R&D Systems, Wiesbaden, Germany) in phosphate-buffered saline (PBS) for 48 h at 4°C. After the excess capture antibody was removed, the wells were filled with 50 µl of casein in PBS (Pierce Biotechnology) and incubated at room temperature for 1 h to saturate excess binding sites. After three washes with buffer (0.125% Triton X-100-PBS), serial dilutions of the experimental samples diluted in 10% casein in PBS were added to the plates and incubated at room temperature for 2 h. After three washes, 100 µl of biotinylated detector anti-MCP-1 antibody was added to the wells and incubated for 1 h at room temperature. After three additional washes, the plate contents were incubated with peroxidase-conjugated streptavidin for 0.5 h. After three final washes, plates were developed with hydrogen peroxide and tetramethylbenzidine (Sigma) and stopped by the addition of 1.5 M H2SO4. Titrations of recombinant human MCP-1 (R&D Systems) were included in each experiment for standardization.
Stable transfer of CGRP gene into A549 cells. A pcDNA3.1 plasmid containing hCGRP cDNA was constructed as previously described (38) and transferred to A549 cells with the use of Lipofectin (Invitrogen). After transfection the cells were cultured in the presence of G418 (800 µg/ml) for 4 days, and a stably transfected cell line was selected by subculturing the cells from a monoclone.
RNA extraction and RT-PCR. Total RNA was extracted from A549 cells with guanidinium isothiocyanate-phenol chloroform solution (TRIzol reagent, Promega), quantified by measuring absorption at 260/280 nm, and subjected to RT-PCR analysis. As previously reported (24), total RNA was reverse transcribed with oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase at 2.0 µg of total RNA per sample.
The oligonucleotide primers for MCP-1 and -actin were synthesized on a DNA synthesizer (model 49a, Applied Biosystems). The nucleotide sequences of -actin were sense 5'-CATCTCTTGCTCGAAGTCCA-3' and antisense 5'-ATCATGTTTGAGACCTTCAACA-3', and the PCR product was 300 bp. The CGRP primers derived from plasmid were sense 5'-GTTCTCCCCCTTCCTGGC-3' and antisense 5'-GGCTTTGGAACCCACATTG-3', and the product was 344 bp. Human MCP-1 primers were sense 5'-ATG AAA GTC TCT GCC GCC-3' and antisense 5'-TTG CTT GTC CAG GTG GTC-3', and the product was 290 bp (47).
Radioimmunoassay. Radioimmunoassay of CGRP followed a procedure used previously in our laboratory (42). Briefly, the culture supernatants were collected and vacuum dried. Samples were incubated with anti-CGRP antibody at 4°C for 24 h. After the addition of 125I-labeled CGRP, samples were further incubated at 4°C for 24 h. The bound radioactivity of calibrators, controls, and samples was measured in duplicate with use of a gamma scintillation counter (Beckman Instruments).
Inhibition of CGRP expression by small interfering RNA.
As we described previously (41), RNA-interfering plasmid for the -CGRP gene was transferred to A549 cells with use of Lipofectin (Invitrogen). 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 days. Monoclones were chosen and expanded. RT-PCR and radioimmunoassay revealed that CGRP mRNA and protein level decreased by 50–70% in transfected cells.
Electrophoretic mobility shift assay.
Briefly, nuclear proteins were extracted with use of NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology). Electrophoretic mobility shift assay (EMSA) involved use of the Gel Shift Assay System (Promega, Madison, WI). Briefly, probes were end-labeled with [
-32P]ATP and T4 polynucleotide kinase. The nuclear extract (5 mg) was incubated at 30°C for 20 min with 1 ng of 32P-labeled probe (40,000–60,000 cpm) in 10 µl of binding buffer. DNA-nuclear protein complexes were separated on a native 6% polyacrylamide gel, and then the gel was vacuum dried and underwent autoradiography with an intensifying screen at –70°C.
Western blot analysis.
A549 cells were cultured in 10-cm petri dishes. After reaching confluence, cells were treated with IL-1 (1 ng/ml) or pretreated with inhibitors and then IL-1 and incubated in a humidified incubator at 37°C. After incubation the cells were rapidly washed with PBS and then lysed with ice-cold lysis buffer (mM: 50 Tris·HCl, pH 7.4, 1 EDTA, 150 NaCl, 1 PMSF, 1 NaF, and 1 Na3VO4, with 1% Triton X-100, 1 mg/ml pepstatin A, and 20 mg/ml aprotinin), and the lysates underwent SDS-PAGE with a 10% running gel. The proteins were transferred to polyvinylidene difluoride membrane. The membrane was incubated with 0.1% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TTBS) at room temperature for 1 h with rabbit antibodies specific for phosphorylated or nonphosphorylated MAPKs at 4°C for 12 h and then horseradish peroxidase-labeled second antibody for 1 h. After each incubation the membrane was washed extensively with TTBS, and the immunoreactive band was detected with use of ECL-detecting reagents (Pierce Biotechnology).
Transient transfection and luciferase reporter assay.
A549 cells were seeded at 80,000 cells/well into 12-well microplates 1 day before transfection, and the cells were transfected with 0.5 µg of 2xNF-B report luciferase plasmid (a kind gift from Dr. Cunyu Wang, Columbia University, New York, NY) together with 0.1 µg of -galactosidase (-gal) expression plasmid (Promega) with the use of jetPEI reagent. After transfection for 24 h, the medium was changed for fresh DMEM without FBS. After incubation with or without IL-1 (1 ng/ml) together with CGRP (10–100 nM) for 6 h, the growth medium was removed and replaced by 200 µl of reporter lysis buffer and the luciferase activity and -gal enzyme activity were measured with the luciferase assay and -gal enzyme assay systems.
Measurement of intracellular ROS generation.
The production of ROS, especially H2O2, by A549 cells was detected by luminol plus horseradish peroxide-derived chemiluminescence with a light-tight box and a luminescence analyzer (BPCL Ultra-weak, Beijing, China) (3). Photon counts were integrated over 1 s and shown on a computer monitor. A549 cells were incubated with IL-1 and/or other chemicals for indicated times, the cells were washed twice with 1 ml of 37°C PBS, and then 10 µg/ml horseradish peroxide and 0.5 mM luminol were added. Luminol plus horseradish peroxidase-derived chemiluminescence was initiated by adding 3 mM NADH as a substrate. All experiments were repeated at least three times, and the sum of counts reflects the formation of ROS.
Transient transfection and luciferase reporter assay.
A549 cells were seeded at 80,000 cells/well into 12-well microplates 1 day before transfection, and the cells were transfected with 0.5 µg of human MCP-1 whole promoter-luciferase reporter plasmid and 0.1 µg of -gal expression plasmid with jetPEI reagent. After transfection for 24 h, the medium was changed to fresh DMEM without FBS. After incubation with or without IL-1 (1 ng/ml) for 6 h the growth medium was removed and replaced by 200 µl of reporter lysis buffer, and luciferase activity was measured with the luciferase assay system.
Statistics. The results are expressed as means ± SE. Data analysis involved use of GraphPad Prism software. One-way analysis of variance, Student-Newman-Keuls test (comparisons between multiple groups), or unpaired Student’s t-test (between 2 groups) was used as appropriate. A P < 0.05 was considered significant.
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RESULTS |
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induced CGRP secretion in human A549 AEII cells and A549 cells expressed a CGRP functional receptor (24). CGRP might play an inhibitory role in the inflammation progress in the lung by suppressing chemokine secretion in an autocrine/paracrine mode (24). To investigate the effect of IL-1-induced endogenous CGRP on IL-1-induced MCP-1 secretion, we treated A549 cells with hCGRP8–37, a CGRP-1 receptor antagonist, simultaneously with IL-1 for different times. As shown in Fig. 1A, hCGRP8–37 at a very low concentration (0.1–1 nM) significantly enhanced the IL-1-induced MCP-1 secretion.
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-induced MCP-1 secretion, we used molecular and genetic manipulation of CGRP expression. Knockdown of -CGRP by small interfering RNA (siRNA) as we previously reported (41) attenuated CGRP mRNA and protein levels in A549 cells (Fig. 1B). As shown in Fig. 1B, the inhibition of CGRP expression by siRNA significantly amplified both basal and IL-1-induced MCP-1 secretion. These data strongly suggest that endogenous CGRP may inhibit chemokine MCP-1 expression induced by proinflammatory IL-1 to reduce the recruitment of inflammatory cells.
Exogenous CGRP suppressed MCP-1 secretion.
To further confirm the effect of CGRP on IL-1-induced MCP-1 secretion, A549 cells were treated with IL-1 in combination with exogenous hCGRP (0.1–10 nM) for different times. Exogenous CGRP (1–10 nM) reduced IL-1-induced MCP-1 secretion in a time- and concentration-dependent manner (Fig. 2A).
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and/or exogenous hCGRP (1–100 nM) for 24 h. The cell lysates were collected, and the luciferase activity was measured. As shown in Fig. 2B, 100 nM hCGRP suppressed MCP-1 promoter activity. These data indicate that exogenous CGRP inhibits MCP-1 secretion by suppressing MCP-1 promoter activity.
Furthermore, A549 cells stably transfected with CGRP showed elevated mRNA and protein levels of CGRP (Fig. 3A and Fig. 4, A and B). Compared with the control and empty vector clone, the CGRP high-expression clone showed greatly reduced mRNA and protein levels of MCP-1 after IL-1 administration (Fig. 3B and Fig. 4, C and D). The inhibitory effect of the CGRP gene mimicked exogenous CGRP administration to the cells.
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-induced NF-B nuclear translocation.
It is well known that nuclear transcription factor NF-B participates in IL-1-induced gene transcription. The promoter of human MCP-1 is the most studied: two NF-B binding sites (A1 and A2) located 
2.6 kb from the transcription initiation site appear to function as the critical elements in MCP-1 induction in response to IL-1 (34). To explore the mechanism of CGRP action, we used EMSA to detect whether CGRP can influence NF-B DNA binding. As shown in Fig. 5A, stimulation with IL-1 for 1 and 4 h with or without 10 nM CGRP significantly attenuated NF-B binding activity. To determine whether CGRP directly inhibits NF-B binding activity or inhibits IB phosphorylation and degradation from upstream signaling, cytoplasmic extracts from A549 cells stimulated with IL-1 in the presence or absence of CGRP were examined by Western blotting. IL-1 cells stimulated for 1–4 h showed lower levels of IB and higher levels of phosphorylated IB, but the presence of CGRP caused a substantial accumulation of IB and reduced IB phosphorylation compared with IL-1 alone at the same time (Fig. 5B). We also detected that at 5–45 min CGRP did not change the level of IB and its phosphorylated form in the short time frame (data not shown). These results therefore indicate that CGRP inhibits NF-B transcriptional activity, at least in part, by increasing IB due to reduced IB phosphorylation and degradation.
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-induced ROS formation.
As is well known, the early event in IL-1 signaling is IL-1 stimulating the generation of ROS, including O2–· and H2O2, via the NAD(P)H oxidase system and/or lipoxygenases (6). We measured IL-1-induced ROS production in A549 cells by chemiluminescence; as shown in Fig. 6A, ROS formation peaked after IL-1 stimulation for 30 min and declined thereafter. To determine the role of ROS in IL-1-invoked MCP-1 secretion, three chemicals were used 30 min before IL-1 stimulation for 24 h: DPI, an inhibitor of mitochondrial NADPH-ubiquinone oxidoreductase; PAO, an inhibitor of NADPH oxidase; and NAC, a thiol-based antioxidant and ROS scavenger. ELISA data showed that NAC, PAO, and DPI all significantly suppressed IL-1-evoked MCP-1 secretion in A549 cells (Fig. 6B). These data imply that NADPH oxidase-derived ROS may play an important role in IL-1-induced MCP-1 production in A549 cells. A549 cell chemiluminescence assay also revealed that CGRP (10–100 nM) inhibited IL-1-induced ROS production in a time- and concentration-dependent manner (Fig. 6, C and D).
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B signaling.
It is well known that the CGRP receptor is coupled to adenylyl cyclase and increased levels lead to elevated levels of intracellular cAMP and its protein kinase (PK) in various cell types. Our previous data (24) showed that Rp-cAMPS, a specific cell-permeant inhibitor of PKA, could partially reverse the inhibitory effect of CGRP. These data indicate that the postsignaling molecules cAMPS might have an important role in the procedure. To explore the mechanism of the suppressive effect of CGRP, the use of Sp-cAMPS and 8-BrcAMPS, two cAMP analogs, showed their mimicking of the exogenous CGRP effect and attenuation of IL-1-induced ROS formation (Fig. 7A), and the CGRP inhibitory effect was reversed by Rp-cAMPS, an antagonist of cAMP (data not shown). Thus CGRP inhibits IL-1-induced NF-B activation, at least in part by increasing the cAMP level and subsequently suppressing IL-1-evoked ROS production.
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B binding/transcription activity and IB degradation in a concentration-dependent manner, and the CGRP inhibitory effect was mimicked by the cAMP analogs 8-BrcAMPS and Sp-cAMPS (Fig. 7, B–D). Similar to CGRP, 8-BrcAMPS and Sp-cAMPS attenuated IL-1-induced MCP-1 secretion by 28.3% and 18.8%, respectively (Fig. 7E). |
DISCUSSION |
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-induced MCP-1 secretion in an autocrine/paracrine mode by enhancing intracellular cAMP signaling and thus inhibiting the ROS-NF-B pathway. CGRP, a 37-amino acid neuropeptide, was discovered in 1982 to be distributed in the nervous system. In the peripheral nervous system, CGRP is present primarily in the sensory ganglia. CGRP-immunoreactive afferent nerve fibers and CGRP receptors are abundant in lung tissue. Except for neural tissue in the lung, CGRP is also in pulmonary neuroepithelial bodies and AEII cells (24). Our previous data showed that the CGRP concentration in the sputum of patients with asthma and chronic obstructive pulmonary disease (COPD) is significantly higher than that in normal subjects (45). Also, CGRP levels in the trachea perfusate, lung tissue, and bronchial tissue of rats with chronic bronchitis are markedly elevated (45, 46). In vitro experiments showed, after inflammatory stimulation, that CGRP is secreted from nerve terminals and AEII cells (16, 17, 24). Therefore, CGRP might play an important role in the pathogenesis of respiratory diseases such as asthma, COPD, or chronic cough.
Many processes believed to be important in the pathogenesis of asthma are due to the activities of IL-1 and TNF- (22). In many inflammatory airway diseases such as asthma and COPD the level of cytokines is increased in the bronchoalveolar lavage fluid and in cultures of peripheral blood mononuclear cells (10, 39). These cytokines induce pulmonary epithelial cells, especially type II cells, to secrete the chemokines IL-8 and MCP-1 to attract inflammatory cells to the local area. In our previous and present data, we found that the proinflammatory factor IL-1 also stimulates CGRP secretion from AEII cells and endogenous/exogenous CGRP can inhibit IL-1-induced MCP-1 secretion in an autocrine/paracrine manner (24). These data indicate that AEII cells derived together with neurogenic CGRP may restrict lung inflammation, at least in part, by eliminating MCP-1 secretion.
CGRP’s effect on immunomodulation has been discussed for a long time. CGRP is an inflammatory mediator, inducing eosinophil migration in rat airways (5, 14) and magnifying IL-1-induced edema in vivo (7). However, CGRP also has anti-inflammatory effects in mouse ear inflammation and rabbit colitis (11, 31, 32). We demonstrated previously (38) that CGRP can attenuate multiple low-dose streptozotocin-induced insulitis and reduce the occurrence of diabetes in mice. Also, in vitro experiments show that lymphocyte-derived CGRP can inhibit Con A-induced proliferation and IL-2 production in rat thymocytes in an autocrine/paracrine mode (44). In the present study, a very low concentration of hCGRP8–37 (0.1 nM), an antagonist of CGRP1 receptor, 10 times more than that of endogenous CGRP, significantly magnified the IL-1-induced MCP-1 secretion (Fig. 1A) between 6 and 24 h. Knockdown of -CGRP gene expression by siRNA in A549 cells does not change the basal level of MCP-1 but greatly potentiates IL-1-induced MCP-1 secretion. These data indicate that endogenous CGRP may have an inhibitory effect on the inflammatory process. Exogenous CGRP reduces IL-1-induced MCP-1 secretion from the promoter level in a concentration-dependent manner (Fig. 2), and stably high levels of CGRP-overexpressed clones seem to attenuate the cell response of MCP-1 secretion to IL-1 (Fig. 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 not only inhibits the immunoreactivity of the lung epithelium to inflammatory factors by reducing MCP-1 secretion but also enhances phagocytosis of the peripheral macrophages to attenuate the inflammation (18).
Little is known about the downstream signaling pathways induced by CGRP to mediate its effects. We have explored the mechanism of CGRP’s effect and found that it inhibits the accumulation of NF-B complexes in the nucleus by preventing phosphorylation and degradation of IB, an NF-B inhibitor. Millet and colleagues (27) showed that stimulated thymocyte-derived CGRP inhibits NF-B activity, beginning at 10 min and lasting up to 4 h. In the present study, we also showed this phenomenon, but these two cell types differ slightly in response.
As is well known, NF-B is an important transcription factor induced by IL-1 and mediates various target chemokine secretions. The chemotactic factors released by A549 cells in response to IL-1 are MCP-1, IL-8, RANTES, and eotaxin. MCP-1 is a proinflammatory chemokine produced by various cell types, especially epithelial cells and macrophages. MCP-1 mRNA and protein are strongly expressed in epithelial cells and macrophages from patients with asthma and COPD. MCP-1 levels in serum and bronchoalveolar lavage fluid were significantly higher than those in healthy volunteers (2, 10). In that it regulates the recruitment of inflammatory cells, especially monocytes/macrophages, MCP-1 is one of the important inflammatory mediators. Many cis elements, including SP-1, AP-1, NF-B, and C/EBP, promote human MCP-1, but two NF-B binding sites function as the critical elements in MCP-1 induction in response to IL-1 and TNF- (26). In the present study, we show that the neuropeptide CGRP inhibits IL-1-induced MCP-1 secretion at the transcriptional level via NF-B suppression. We further searched for other pathways in this system where CGRP might inhibit NF-B activity via the post-receptor signal cAMP pathway. PKA activators such as calcitonin increase the activity of peroxisome proliferator-activated receptors (PPARs), especially PPAR- (23). Many studies have reported that nuclear receptor transcription factor PPAR- and liver X receptor (LXR)- inhibit NF-B by upregulation of IB or directly binding with NF-B p65 (9, 40). To investigate the mechanism mediating the CGRP inhibitory effect on NF-B, a luciferase reporter assay was used to detect the peroxisome proliferator-response elements (PPRE) and LXR- response element activity. In A549 cells, CGRP and cAMP analogs did not significantly increase PPAR and LXR activity (data not shown).
IL-1 primes and triggers O2–· formation by activating NADPH oxidase and thereby produces an oxidative burst (6). Much evidence has revealed that ROS scavengers or NADPH oxidase inhibitors significantly reduce IL-1-induced NF-B activation and the expression of target genes (6, 29). A549 cells have an active redox response and are sensitive to IL-1 stimulation. DPI and PAO, the NADPH oxidase inhibitors, blocked MCP-1 secretion by IL-1, and NAC, the ROS scavenger, also significantly attenuated MCP-1 secretion (Fig. 6B). These data indicate that ROS, primarily from an NADPH oxidase-dependent source, might be the important signal mediating IL-1-induced NF-B activation and MCP-1 expression.
In human airway smooth muscle cells, the use of forskolin results in increased intracellular cAMP concentration that may decrease IL-1-induced eotaxin and MCP-1 expression and production (43). In fact, in other cell types intracellular cAMP-elevating agents such as forskolin and a cAMP analog such as 8-BrcAMPS have cytoprotective effects by reducing ROS production (21, 28), and the accumulated cAMPS results in attenuated NF-B activity by stimuli (30). Because CGRP activates adenylate cyclase, leading to the induction of cAMP production, we detected ROS production in A549 cells and found that CGRP attenuates IL-1-induced ROS production and the cAMP analogs Sp-cAMPS and 8-BrcAMPS mimic the CGRP effect (Fig. 6). The results from our lab and others for other cell types also show that CGRP can inhibit ROS formation by enhancing the cAMP pathway (35). Recently, Yoshimoto et al. (48) reported that adrenomedullin, a 52-amino acid peptide with structural homology to CGRP, directly inhibits intracellular ROS generation via a cAMP-PKA-dependent mechanism in vascular smooth muscle cells.
It is interesting that IL-1 simultaneously induces the secretion of MCP-1 and CGRP, whereas CGRP itself acts on A549 cells and inhibits MCP-1 production. In a seemingly autocrine feedback loop, IL-1, on the one hand as a stimulatory factor, attracts inflammatory cells to the local area via chemokine MCP-1 and, on the other, 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 for tissue.
In conclusion, we have shown that in the A549 human type II alveolar cell line intrinsic and extrinsic peptide CGRP inhibits IL-1-induced MCP-1 secretion in an autocrine/paracrine mode and suppresses the IL-1-evoked ROS-NF-B cascade via cAMP signaling. Our data provide a new insight into the immunomodulatory effect of AEII-derived CGRP and suggest a novel therapeutic target in lung inflammatory disease.
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FOOTNOTES |
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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.
* W. Li and T. Wang contributed equally to this work. 
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2. Alam R, York J, Boyars M, Stafford S, Grant JA, Lee J, Forsythe P, Sim T, and Ida N. Increased MCP-1, RANTES, and MIP-1 in bronchoalveolar lavage fluid of allergic asthmatic patients. Am J Respir Crit Care Med 153: 1398–1404, 1996.[Abstract]
3. Allen RC. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol 133: 449–493, 1986.[ISI][Medline]
4. Bell D and McDermott BJ. Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor populations and their (patho)physiological significance. Pharmacol Rev 48: 253–288, 1996.[ISI][Medline]
5. Bellibas SE. The effect of human calcitonin gene-related peptide on eosinophil chemotaxis in the rat airway. Peptides 17: 563–564, 1996.[CrossRef][ISI][Medline]
6. Brigelius-Flohe R, Banning A, Kny M, and Bol GF. Redox events in interleukin-1 signaling. Arch Biochem Biophys 423: 66–73, 2004.[CrossRef][ISI][Medline]
7. Buckley TL, Brain SD, Collins PD, and Williams TJ. Inflammatory edema induced by interactions between IL-1 and the neuropeptide calcitonin gene-related peptide. J Immunol 146: 3424–3430, 1991.[Abstract]
8. Cadieux A, Springall DR, Mulderry PK, Rodrigo J, Ghatei MA, Terenghi G, Bloom SR, and Polak JM. Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations. Neuroscience 19: 605–627, 1986.[CrossRef][ISI][Medline]
9. Castrillo A, Joseph SB, Marathe C, Mangelsdorf DJ, and Tontonoz P. Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages. J Biol Chem 278: 10443–10449, 2003.
10. Chung KF. Cytokines in chronic obstructive pulmonary disease. Eur Respir J Suppl 34: 50s–59s, 2001.[CrossRef][Medline]
11. Clementi G, Amico-Roxas M, Caruso A, Catena Cutuli VM, Prato A, Maugeri S, de Bernardis E, and Scapagnini U. Effects of CGRP in different models of mouse ear inflammation. Life Sci 54: PL119–PL124, 1994.[CrossRef][ISI][Medline]
12. Dakhama A, Kanehiro A, Makela MJ, Loader JE, Larsen GL, and Gelfand EW. Regulation of airway hyperresponsiveness by calcitonin gene-related peptide in allergen sensitized and challenged mice. Am J Respir Crit Care Med 165: 1137–1144, 2002.
13. Dakhama A, Larsen GL, and Gelfand EW. Calcitonin gene-related peptide: role in airway homeostasis. Curr Opin Pharmacol 4: 215–220, 2004.[CrossRef][ISI][Medline]
14. Davies D, Medeiros MS, Keen J, Turner AJ, and Haynes LW. Eosinophil chemotactic peptide sequences in rat alpha-CGRP. Activation of a novel trophic action by neutral endopeptidase 2411. Ann NY Acad Sci 657: 405–411, 1992.[Abstract]
15. Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2: 33–46, 2001.[CrossRef][ISI][Medline]
16. Hou L, Li W, and Wang X. Mechanism of interleukin-1-induced calcitonin gene-related peptide production from dorsal root ganglion neurons of neonatal rats. J Neurosci Res 73: 188–197, 2003.[CrossRef][ISI][Medline]
17. Hou L and Wang X. PKC and PKA, but not PKG mediate LPS-induced CGRP release and [Ca2+]i elevation in DRG neurons of neonatal rats. J Neurosci Res 66: 592–600, 2001.[CrossRef][ISI][Medline]
18. Ichinose M and Sawada M. Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages. Peptides 17: 1405–1414, 1996.[CrossRef][ISI][Medline]
19. Keith IM. The role of endogenous lung neuropeptides in regulation of the pulmonary circulation. Physiol Res 49: 519–537, 2000.[ISI][Medline]
20. Keith IM, Pelto-Huikko M, Schalling M, and Hokfelt T. Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia. Histochemistry 96: 311–315, 1991.[CrossRef][ISI][Medline]
21. Kim EJ, Kwon KJ, Park JY, Lee SH, Moon CH, and Baik EJ. Neuroprotective effects of prostaglandin E2 or cAMP against microglial and neuronal free radical mediated toxicity associated with inflammation. J Neurosci Res 70: 97–107, 2002.[CrossRef][ISI][Medline]
22. Larrick JW and Kunkel SL. The role of tumor necrosis factor and interleukin 1 in the immunoinflammatory response. Pharm Res 5: 129–139, 1988.[CrossRef][ISI][Medline]
23. Lazennec G, Canaple L, Saugy D, and Wahli W. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators. Mol Endocrinol 14: 1962–1975, 2000.
24. Li W, Hou L, Hua Z, and Wang X. Interleukin-1 induces -calcitonin gene-related peptide secretion in human type II alveolar epithelial cells. FASEB J 18: 1603–1605, 2004.
25. Lundberg JM, Franco-Cereceda A, Hua X, Hokfelt T, and Fischer JA. Co-existence of substance P and calcitonin gene-related peptide-like immunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin. Eur J Pharmacol 108: 315–319, 1985.[CrossRef][ISI][Medline]
26. Martin T, Cardarelli PM, Parry GC, Felts KA, and Cobb RR. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-B and AP-1. Eur J Immunol 27: 1091–1097, 1997.[ISI][Medline]
27. Millet I, Phillips RJ, Sherwin RS, Ghosh S, Voll RE, Flavell RA, Vignery A, and Rincon M. Inhibition of NF-B activity and enhancement of apoptosis by the neuropeptide calcitonin gene-related peptide. J Biol Chem 275: 15114–15121, 2000.
28. Nogueira-Machado JA, Lima e Silva FC, Medina LO, Costa DC, and Chaves MM. Modulation of the reactive oxygen species (ROS) generation mediated by cyclic AMP-elevating agents or interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon. Diabetes Metab 29: 533–537, 2003.[ISI][Medline]
29. Piette J, Piret B, Bonizzi G, Schoonbroodt S, Merville MP, Legrand-Poels S, and Bours V. Multiple redox regulation in NF-B transcription factor activation. Biol Chem 378: 1237–1245, 1997.[ISI][Medline]
30. Rahman A, Anwar KN, Minhajuddin M, Bijli KM, Javaid K, True AL, and Malik AB. cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-B activation and ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol 287: L1017–L1024, 2004.
31. Reinshagen M, Flamig G, Ernst S, Geerling I, Wong H, Walsh JH, Eysselein VE, and Adler G. Calcitonin gene-related peptide mediates the protective effect of sensory nerves in a model of colonic injury. J Pharmacol Exp Ther 286: 657–661, 1998.
32. Reinshagen M, Patel A, Sottili M, Nast C, Davis W, Mueller K, and Eysselein V. Protective function of extrinsic sensory neurons in acute rabbit experimental colitis. Gastroenterology 106: 1208–1214, 1994.[ISI][Medline]
33. Reynolds SD, Giangreco A, Power JH, and Stripp BR. Neuroepithelial bodies of pulmonary airways serve as a reservoir of progenitor cells capable of epithelial regeneration. Am J Pathol 156: 269–278, 2000.
34. Rovin BH, Dickerson JA, Tan LC, and Hebert CA. Activation of nuclear factor-B correlates with MCP-1 expression by human mesangial cells. Kidney Int 48: 1263–1271, 1995.[ISI][Medline]
35. She F, Sun W, Mao JM, and Wang X. Calcitonin gene-related peptide gene therapy suppresses reactive oxygen species in the pancreas and prevents mice from autoimmune diabetes. Sheng Li Xue Bao 55: 625–632, 2003.[Medline]
36. Standiford TJ, Kunkel SL, Basha MA, Chensue SW, Lynch JP 3rd, Toews GB, Westwick J, and Strieter RM. Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung. J Clin Invest 86: 1945–1953, 1990.[ISI][Medline]
37. Standiford TJ, Kunkel SL, Phan SH, Rollins BJ, and Strieter RM. Alveolar macrophage-derived cytokines induce monocyte chemoattractant protein-1 expression from human pulmonary type II-like epithelial cells. J Biol Chem 266: 9912–9918, 1991.
38. Sun W, Wang L, Zhang Z, Chen M, and Wang X. Intramuscular transfer of naked calcitonin gene-related peptide gene prevents autoimmune diabetes induced by multiple low-dose streptozotocin in C57BL mice. Eur J Immunol 33: 233–242, 2003.[CrossRef][ISI][Medline]
39. Townley RG and Horiba M. Airway hyperresponsiveness: a story of mice and men and cytokines. Clin Rev Allergy Immunol 24: 85–110, 2003.[CrossRef][ISI][Medline]
40. Vanden Berghe W, Vermeulen L, Delerive P, De Bosscher K, Staels B, and Haegeman G. A paradigm for gene regulation: inflammation, NF-B and PPAR. Adv Exp Med Biol 544: 181–196, 2003.[ISI][Medline]
41. Wang W, Jia L, Wang T, Sun W, Wu S, and Wang X. Endogenous calcitonin gene-related peptide protects human alveolar epithelial cells through protein kinase C
and heat shock protein. J Biol Chem 280: 20325–20330, 2005.
42. Wang X, Xing L, Xing Y, Tang Y, and Han C. Identification and characterization of immunoreactive calcitonin gene-related peptide from lymphocytes of the rat. J Neuroimmunol 94: 95–102, 1999.[CrossRef][ISI][Medline]
43. Wuyts WA, Vanaudenaerde BM, Dupont LJ, Demedts MG, and Verleden GM. Modulation by cAMP of IL-1-induced eotaxin and MCP-1 expression and release in human airway smooth muscle cells. Eur Respir J 22: 220–226, 2003.
44. Xing L, Guo J, and Wang X. Induction and expression of -calcitonin gene-related peptide in rat T lymphocytes and its significance. J Immunol 165: 4359–4366, 2000.
45. Xu H, Zhao M, and Wang X. Changes of calcitonin gene-related peptide content in induced sputum from patients with COPD and asthma. Zhonghua Jie He He Hu Xi Za Zhi 22: 558–561, 1999.[Medline]
46. Xu H, Zhao M, and Wang X. The levels of calcitonin gene-related peptide in pathogenesis of chronic bronchitis rats. Natl Med J China 79: 385–387, 1999.
47. Yamaguchi N, Yamashita Y, Ikeda D, and Koga T. Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes. Infect Immun 64: 2563–2570, 1996.[Abstract]
48. Yoshimoto T, Fukai N, Sato R, Sugiyama T, Ozawa N, Shichiri M, and Hirata Y. Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells. Endocrinology 145: 3331–3337, 2004.
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