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The Journal of Immunology, 2007, 179, 607 -615
Copyright © 2007 by The American Association of Immunologists, Inc.
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Negative Regulation of TLR Responses by the Neuropeptide CGRP Is Mediated by the Transcriptional Repressor ICER1

Marit D. Harzenetter*, Alexander R. Novotny*, Petra Gais*, Carlos A. Molina{dagger}, Felicitas Altmayr* and Bernhard Holzmann2,*

* Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Munich, Germany; and {dagger} Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, NJ 07103


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Communication between the nervous and immune systems involves the release of neuropeptides, such as calcitonin gene-related peptide (CGRP), from sensory nerves during inflammation. CGRP may inhibit the activities of both innate and adaptive immune cells, but the molecular pathways underlying this function are largely unknown. In this study, we identify CGRP as a potent inhibitor of TLR-stimulated production of inflammatory mediators, such as TNF-{alpha} and CCL4, by murine dendritic cells. Inhibition of TLR responses was independent of IL-10 and did not involve perturbation of canonical TLR signaling, including activation of MAPK and NF-{kappa}B. Instead, the inhibitory activity of CGRP was mediated by the cAMP/protein kinase A pathway leading to rapid up-regulation of the transcriptional repressor, inducible cAMP early repressor (ICER). Ectopically expressed ICER directly repressed the LPS-stimulated activity of a synthetic Tnf promoter, as well as TNF-{alpha} protein production driven by the endogenous promoter. Inhibition of dendritic cell gene expression by CGRP was associated with the presence of a composite cAMP response element/{kappa}B promoter element. In a murine model of endotoxemia, CGRP markedly attenuated serum TNF-{alpha} levels, and this effect was associated with the up-regulation of ICER. Together, these results establish a novel pathway for the negative regulation of TLR responses through the nervous system that critically involves induction of the transcriptional repressor ICER by the neuropeptide CGRP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune and nervous systems exhibit extensive bidirectional communication that is mediated by neurotransmitters, cytokines, and endocrine hormones (1). Activation of C-type sensory pain fibers may modulate immune responses through the release of neuropeptides such as calcitonin gene-related peptide (CGRP)3. CGRP-containing C-type sensory nerves are present in most tissues, including blood vessels, the heart, kidney, gastrointestinal tract, and lymphoid organs (2). CGRP may be released in large amounts during the course of severe systemic infections in sepsis patients (3, 4, 5). During inflammatory responses, the release of CGRP from sensory nerves is initiated by mast cells. Mast cells are found in close contact with sensory nerves (6, 7) and, upon activation, may release tryptase, which stimulates nerve fibers through the proteinase-activated receptor 2 (8). In addition, CGRP was reported to be produced by activated T cells (9) and monocytes exposed to nerve growth factor (10). Nevertheless, most of the CGRP present in the circulation is thought to be derived from sensory nerve terminals (2).

The receptor for CGRP is composed of the seven-transmembrane domain protein calcitonin receptor-like receptor (CRLR) and the accessory protein, receptor activity-modifying protein (RAMP) 1 (11). CRLR may also associate with RAMP2 and RAMP3, thereby yielding receptors for adrenomedullin. The members of the RAMP family are required for plasma membrane expression and selective ligand recognition of the CRLR-RAMP heterodimers. In addition, the cytosolic protein CGRP receptor component protein (CGRP-RCP) appears to be required for efficient signal transduction through CRLR-RAMP complexes (12). CRLR-RAMP1 signaling is initiated by activation of receptor-associated heterotrimeric G proteins. In most systems, the CGRP receptor is coupled to G{alpha}s proteins, leading to elevation of cellular cAMP levels (2). Alternatively, the CGRP receptor may activate phospholipase C-beta1 via G{alpha}q/11 proteins, causing calcium mobilization (13). Following prolonged CGRP exposure, internalization of a ternary complex between CRLR-RAMP1 and beta-arrestin through clathrin-coated pit-mediated endocytosis was observed (14).

CGRP is a potent vasodilator and hypotensive agent that has been implicated in migraines and chronic pain (2). Importantly, CGRP also mediates distinct anti-inflammatory and immunosuppressive activities and, therefore, may play a role in neuroimmunological communication. CGRP modulates the adhesion and migration of immune cells including T cells, eosinophilic granulocytes, and dendritic cells (DC) (15, 16, 17). Stimulation of DC with CGRP reduces the expression of MHC class II and costimulatory proteins, augments the production of IL-10, and inhibits the Ag-presenting capacity of these cells (18, 19, 20). Consistent with these in vitro activities, administration of CGRP to mice inhibits delayed-type and contact hypersensitivity responses (19, 21). Furthermore, inflammatory injury in models of acute endotoxemia and chronic colitis (22, 23, 24), as well as expression of immune receptors and mediators in macrophages stimulated with bacteria or LPS, were found to be attenuated by CGRP (25).

In the present study, the molecular pathway(s) were examined by which CGRP inhibits TLR-stimulated production of inflammatory mediators such as TNF-{alpha} and CCL4 by DC. We show that CGRP inhibits the TNF-{alpha} production at the transcriptional level and that this effect is mediated by the cAMP/protein kinase A (PKA) pathway. The inhibitory effect of CGRP is neither mediated by IL-10 nor is it associated with the alteration of canonical TLR signaling pathways. The results are rather consistent with a mechanistic model, indicating that CGRP induces expression of the transcriptional repressor, termed inducible cAMP early repressor (ICER), which was shown to directly suppress LPS-induced transcriptional activity of the Tnf promoter and TNF-{alpha} protein production. Notably, CGRP also attenuated TNF-{alpha} serum levels in a murine model of endotoxemia, providing a mechanistic basis to explain the immunosuppressive activities of CGRP under in vivo conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation and stimulation of bone marrow-derived DC (BMDC)

Femurs of 6- to 10-wk-old C57BL/6 or Il-10-deficient female mice were flushed with PBS, and erythrocytes were lysed by treatment with ammonium chloride. Remaining unfractionated cell populations were plated at a density of 5 x 105 cells/ml. Culture medium consisted of RPMI 1640 supplemented with 10 ng/ml mouse rGM-CSF (PeproTech). Cultures were fed with fresh medium containing rGM-CSF every 3–4 days, and cells were used at day 10. Purity of the BMDC population was assessed by flow cytometry analysis (FACSCalibur; BD Biosciences) using CD11c (HL30) and CD11b (M1/70) Abs (both BD Pharmingen) and was 80–85% in all experiments.

BMDC were stimulated with 10 µg/ml (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH (Pam3Cys) (EMC), 50 µg/ml poly(I:C), 50 ng/ml LPS from Escherichia coli O127:B8 (both from Sigma-Aldrich), or 1 µM CpG-A oligodeoxynucleotide 2216 (5'-GGG GGA CGA TCG TCG GGG GG-3') (TIB MOLBIOL), which engage TLR2, TLR3, TLR4, or TLR9, respectively. CGRP and CGRP8–37 (both from Bachem) were used at 100 nM. This concentration was found to result in maximal cAMP induction by CGRP in BMDC (data not shown). To analyze the cAMP-PKA pathway, BMDC were treated with 50 µM PKA-specific agonist N6-benzoyladenosine-3',5'-cAMP (6-Bnz-cAMP), 50 µM exchange proteins directly activated by cAMP (Epac)-specific agonist 8-(–4-chlorophenyl-thio)-2'-O-methyladenosine-3',5'-cAMP (8-pCPT-2'-O-Me-cAMP) (both from BioLog), 50 µM adenylyl cyclase activator forskolin (Sigma-Aldrich), or 10 µM PKA inhibitor KT5720 (Calbiochem).

Analysis of cytokine mRNA and protein production

Concentrations of TNF-{alpha}, IL-10, CCL4, and CXCL1 proteins in BMDC supernatants were determined by ELISA according to the manufacturer’s instructions (R&D Systems).

BMDC were stimulated as indicated, and RNA extractions were conducted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 µg of total RNA using a mixture of oligo(dT)12–18 and random hexamer primers and Superscript Reverse Transcriptase (Invitrogen Life Technologies). For amplification, the qPCR Mastermix Plus for SYBR Green I without UNG (Eurogentec) was used to detect accumulation of PCR products during cycling on an Applied Biosystems 7300 cycler. RNA expression levels of TNF-{alpha} were normalized to those of beta-actin and were displayed as fold-change relative to samples of unstimulated BMDC used as calibrator (set to 1). The primers were as follows: beta-actin sense, 5'-ACC CAC ACT GTG CCC ATC TAC-3'; beta-actin antisense, 5'-AGC CAA GTC CAG ACG CAG G-3'; TNF-{alpha} sense, 5'-AAA ATT CGA GTG ACA AGC CTG TAG C-3'; and TNF-{alpha} antisense, 5'-AGC CAA GTC CAG ACG CAG G-3'.

For the determination of RNA stability, BMDC were treated with Pam3Cys in the presence or absence of CGRP or CGRP8–37 as indicated. After 3 h, 10 µg/ml actinomycin D (Sigma-Aldrich) was added. Total RNA was isolated at 0, 120, 180, and 240 min after the addition of actinomycin D.

Expression of CGRP receptor components and ICER

Expression of CGRP receptor components was analyzed by RT-PCR using primers specific for RAMP-1 (sense: 5'-GAG ACG CTG TGG TGT GAC TG-3'; antisense: 5'-GTA AGT CAA GGT CAC GTC CCT-3'), CRLR (sense: 5'-AGT TCA TTC ATCTTT ACC TGA TGG-3'; antisense: 5'-CTC AGA ATT GCT TGA ACC TCT CC-3'), or CGRP-RCP (sense: 5'-CAG CCA TTT CCT GGA CGT T-3'; and antisense: 5'-TAT CTC TGT TGT TCT CGA GG-3').

Protein expression of ICER was analyzed by Western blotting of total cellular lysates using a cAMP response element modifier-specific rabbit polyclonal antiserum (26). ICER mRNA was detected by RT-PCR using specific primers (sense: 5'-ATG GCT GTA ACT GGA GAT GAA ACT-3' and antisense: 5'-CTA ATC TGT TTT GGG AGA GCA AAT GTC-3').

For control, mouse GAPDH was amplified (sense primer: 5'-CAA TGC ATC CTG CAC CAC CAA; antisense primer: 5'-GTC ATT GAG AGC AAT GCC AGC-3'). GAPDH primer sequences were separated by introns to control for contaminations with genomic DNA.

Analysis of CGRP receptor signal transduction

BMDC were resuspended in PBS and endogenous cAMP phosphodiesterase was blocked with 300 µM 3-isobutyl-1-methylxanthine (Sigma-Aldrich). Cells were stimulated with 100 nM CGRP for the indicated time periods. Cellular cAMP concentrations were measured by an enzyme immunoassay according to the manufacturer’s protocol (BIOMOL).

BMDC were incubated with 2.5 µM fura 2-AM (Invitrogen Life Technologies) for 30 min. in assay buffer (5 mM HEPES (pH 7.4), 140 mM NaCl, 10 mM glucose, 0.5 mM KCl, 0.12 mM MgSO4, and 1 mM CaCl2). Fluorescence was measured with the Mithras Multimode reader LB 940 (Berthold Technologies). The increase of cellular Ca2+ levels was reported as the ratio of OD 340:380. Cells were measured for 60 s, then 100 nM CGRP was added, and cells were measured for an additional 120 s. Ca2+ flux was stopped by adding 2 mM EGTA. For positive control of Ca2+ flux, BMDC were treated with 1 µg/ml ionomycin.

Analysis of TLR signal transduction

For Western blot analysis, BMDC were stimulated as indicated and lysed in cell extraction buffer containing 50 mM Tris (pH 8.0), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3OV4, and protease inhibitors. Blots were probed with Abs specific for I{kappa}B{alpha}, phosphorylated I{kappa}B{alpha} (Ser32), or p38 MAPK (all from Cell Signaling Technology).

BMDC were stimulated as indicated and washed in ice-cold PBS. Nuclear extracts were prepared using a nuclear extraction kit (Active Motif). The amount of nuclear RelA binding to DNA was quantified using a DNA-binding assay (Active Motif). Specific DNA binding of RelA was determined by competitive inhibition with an oligonucleotide containing a consensus NF-{kappa}B motif as compared with mutated oligonucleotide. Data were normalized against total protein concentrations of nuclear extracts. Total protein concentrations were determined with the BCA Protein Assay (Pierce) according to the manufacturer’s protocol.

For determination of MAPK activation, BMDC were stimulated as indicated and lysed in cell extraction buffer. Total protein and phosphorylated forms of p38 and JNK were determined by specific ELISA according to the manufacturer’s protocols (BioSource International). The amounts of phosphorylated p38 and JNK were normalized against total p38 and JNK proteins, respectively. The results are presented as fold increase of phosphorylated proteins in stimulated BMDC relative to unstimulated cells.

Luciferase reporter assays

HEK293 cells were cotransfected with a NF-{kappa}B-dependent promoter-driven firefly luciferase construct, a reporter plasmid mediating constitutive expression of Renilla luciferase, as well as a CMV promoter-dependent expression plasmid for murine TLR2 by a calcium phosphate method as described elsewhere (27). RAW264 macrophages were transfected by electroporation. Pam3Cys was added to the transfected cells for 16 h. Cells were lysed for measurement of firefly and Renilla luciferase activities using reagents from Promega. Firefly luciferase activities were related to Renilla luciferase activities for normalization.

A fragment of the murine Tnf promoter (nt –562 to –56 relative to transcriptional start site) encompassing the composite cAMP response element (CRE)/{kappa}B site was cloned into the firefly luciferase reporter vector pGL3 promoter (Promega). The vector was transfected into RAW264 macrophages along with a reporter plasmid mediating constitutive expression of Renilla luciferase. Transfected cells were stimulated with 100 ng/ml LPS for 16 h. Firefly luciferase activities were related to Renilla luciferase activities for normalization.

To analyze the effects of ICER on Tnf promoter activity and TNF-{alpha} protein production, an ICER-I encoding cDNA was cloned by RT-PCR using primers 5'-ATG GCT GTA ACT GGA GAT GAA ACT-3' (sense) and 5'-CTA ATC TGT TTT GGG AGA GCA AAT GTC-3' (antisense). The cDNA fragment was subcloned into the vector pEF, allowing for the expression of ICER-I under the control of the elongation factor 1{alpha} promoter.

Mouse model of endotoxemia

C57BL/6 mice (Harlan Sprague Dawley) were housed under specified pathogen-free conditions and used at 8–12 wk of age. Mice were injected i.v. with 10 µg LPS and different doses of CGRP. Serum and liver samples were collected 1 h later. Mouse experiments were approved by the government of Upper Bavaria.

Statistical analysis

Statistical analysis of the data was performed using a two-tailed Student’s t test or Mann-Whitney U test as appropriate. All data are presented as mean values ± SEM, with the number of independent experiments indicated in the figure legends. The mean of the replicate wells of each group in each independent experiment was used as a single value for calculating the mean value. Independent experiments yielded comparable results in all cases. Differences between experimental groups were considered significant for p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CGRP receptor engagement on DC leads to elevation of cAMP and inhibition of TNF-{alpha} production

The neuropeptide CGRP was previously reported to modulate immune responses and to dampen the capacity of APCs to stimulate T lymphocytes (18, 19, 20). To examine the mechanisms by which CGRP may influence gene expression in immune cells, murine DC were generated by culturing bone marrow cells with GM-CSF and analyzed for the expression and activity of the CGRP receptor. As shown in Fig. 1a, BMDC express abundant mRNA for the CGRP receptor subunits RAMP-1 and CRLR, as well as the receptor-associated protein CGRP-RCP. The CGRP receptor complex may couple to G{alpha}s or G{alpha}q/11 proteins, resulting in the activation of adenylyl cyclase and elevation of intracellular cAMP or the activation of phospholipase C-beta1 and release of calcium from internal stores (2, 13). As shown in Fig. 1b, BMDC responded with a rapid and marked elevation of cAMP upon CGRP exposure. In contrast, CGRP did not induce calcium flux in these cells (Fig. 1c), suggesting that the CGRP receptor is coupled to G{alpha}s, but not G{alpha}q/11, proteins in BMDC.


Figure 1
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  		FIGURE 1. Expression and signaling of the CGRP receptor in BMDC. a, Expression of CGRP receptor subunits RAMP-1 and CRLR, as well as of the receptor-associated protein RCP, was determined by RT-PCR from three independent BMDC preparations. BMDC were stimulated with CGRP and cellular cAMP levels (b) or Ca2+ influx (c) were determined.

 
TLRs are crucial for initiating antimicrobial immune responses by activating the expression of inflammatory genes, such as TNF-{alpha} (28). To investigate the influence of CGRP on DC activation, BMDC were stimulated with the TLR agonists P3C (TLR2), poly(I:C) (TLR3), LPS (TLR4), and CpG-A oligodeoxynucleotides (TLR9) for 16 h, and the release of TNF-{alpha} protein was measured. Strikingly, concomitant incubation with CGRP markedly reduced TNF-{alpha} production of BMDC in response to all TLR agonists tested, whereas a N-terminally truncated CGRP peptide (iCGRP), previously shown to act as a nonstimulatory receptor antagonist (29), had no such effect (Fig. 2a). CGRP-mediated inhibition of TNF-{alpha} production was observed as early as 3 h after TLR engagement and was sustained throughout the observation period (Fig. 2b). Flow cytometry analysis of BMDC incubated with P3C and brefeldin A demonstrated that the total cellular content of TNF-{alpha} protein was markedly reduced by CGRP, but not iCGRP, treatment (Fig. 2c), suggesting that de novo TNF-{alpha} protein synthesis may be impaired. To further analyze the effects of CGRP on TNF-{alpha} production, mRNA levels were quantified by real-time RT-PCR after 3 and 6 h of stimulation with P3C. As shown in Fig. 2d, the amount of Tnf transcripts in BMDC were significantly down-regulated by CGRP, but not iCGRP, at both time points. To determine Tnf mRNA stability, BMDC were stimulated with P3C along with CGRP or iCGRP and, 2 h later, gene transcription was blocked with actinomycin D. Tnf mRNA levels were quantified by real-time RT-PCR at 0, 120, 180, and 240 min after actinomycin D treatment. The results in Fig. 2e clearly demonstrate that CGRP treatment of BMDC did not alter the half-life of Tnf mRNA. Collectively, these data suggest that CGRP inhibits TLR-stimulated transcription of the Tnf gene.


Figure 2
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  		FIGURE 2. CGRP inhibits TLR-stimulated production of TNF-{alpha} by BMDC. a, BMDC were left untreated (M) or incubated with CGRP (C) or the nonstimulatory receptor antagonist CGRP8–37 (iC) in the presence or absence of the indicated TLR agonists. TNF-{alpha} release was measured in the supernatant after 16 h (n = 9). b, BMDC were treated as in a using the TLR2 agonist Pam3Cys (P3C), and TNF-{alpha} release was measured after the indicated time periods (n = 9). c, Total cellular TNF-{alpha} content was measured by flow cytometry analyses of fixed and permeabilized BMDC. Histograms depict TNF-{alpha} expression levels of CD11c+ cells. Numbers above gates, mean ± SEM of TNF-{alpha}+ cells and are derived from seven independent experiments. d, BMDC were treated as in b and TNF-{alpha} mRNA levels were determined by quantitative real-time RT-PCR. Relative mRNA expression was calculated using expression levels of unstimulated cells as calibrators (n ≥ 3). e, To determine TNF-{alpha} mRNA stability, BMDC were stimulated as in b for 3 h and subsequently treated with actinomycin D. TNF-{alpha} mRNA levels were measured by quantitative real-time PCR at various time points thereafter (n = 9). *, p < 0.05, **, p < 0.01, ***, p < 0.001.

 
Inhibition of TNF-{alpha} production by CGRP is independent of IL-10

Previous studies have suggested that CGRP may inhibit Ag presentation by increasing the production IL-10 (20, 25). To investigate the role of IL-10 in the CGRP-mediated inhibition of BMDC TNF-{alpha} synthesis, we first measured the influence of CGRP treatment on IL-10 production. We found that BMDC released small amounts of IL-10 in response to P3C. However, CGRP did not significantly increase IL-10 release (Fig. 3a). In additional experiments, we generated BMDC from IL-10–/– mice and observed that CGRP significantly down-regulated P3C-stimulated TNF-{alpha} release in the complete absence of IL-10 (Fig. 3b). Taken together, these experiments clearly indicate that the suppression of BMDC TNF-{alpha} production by CGRP may occur through an IL-10-independent pathway.


Figure 3
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  		FIGURE 3. Inhibition of TNF-{alpha} production by CGRP is independent of IL-10. BMDC were left untreated (Med) or incubated with CGRP (C) or the nonstimulatory receptor antagonist CGRP8–37 (iC) in the presence or absence of Pam3Cys (P3C) for 16 h. Production of IL-10 by wild-type BMDC (a) or release of TNF-{alpha} by Il-10-deficient cells (b) was measured. *, p < 0.05 (n = 5).

 
CGRP inhibits TNF-{alpha} production through the induction of cAMP and PKA

Having established that the engagement of the CGRP receptor on BMDC inhibits TLR-stimulated TNF-{alpha} production, we analyzed the signaling pathway of the CGRP receptor underlying this activity in more detail. As shown in Fig. 4a, the inhibitory effect of CGRP on P3C-stimulated TNF-{alpha} production could be mimicked by the incubation of BMDC with the adenylyl cyclase activator forskolin, suggesting that CGRP exerts its effects through cAMP. Elevation of cellular cAMP levels may activate PKA or two closely related guanine nucleotide exchange factors, Epac-1 and -2 (30, 31). To distinguish between the two pathways, we incubated BMDC with 6-Bnz-cAMP, a specific agonist of PKA, or with 8-pCPT-2'-O-Me-cAMP, a specific agonist for Epac proteins. The results in Fig. 4, b and c, clearly show that the activation of PKA, but not Epac proteins, reduced TNF-{alpha} production of P3C-stimulated BMDC to a similar extent as CGRP treatment. Moreover, pharmacological inhibition of PKA was found to abolish inhibition of TNF-{alpha} release by CGRP (Fig. 4d). These results, therefore, indicate that the inhibition of TLR-stimulated TNF-{alpha} production by CGRP is mediated by cAMP and PKA.


Figure 4
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  		FIGURE 4. Role of the cAMP-PKA pathway for inhibition of TNF-{alpha} production by CGRP. BMDC were left untreated (Med) or incubated with Pam3Cys (P3C) in the presence or absence of CGRP (C). TNF-{alpha} release was measured after 16 h. Additional treatment of cells was performed with the adenylyl cyclase activator forskolin (Fsk) (a), the PKA-specific agonist 6-Bnz-cAMP (PKA-ag) (b), the Epac-specific agonist 8-pCPT-2'-O-Me-cAMP (Epac-ag) (c), or the PKA inhibitor KT5720 as indicated (d). *, p < 0.05; **, p < 0.01 (n ≥ 4).

 
CGRP does not influence the TLR-triggered activation of NF-{kappa}B and MAPK

Engagement of TLRs is known to activate NF-{kappa}B, as well as MAPK, leading to the expression of inflammatory mediators, including TNF-{alpha} (28). To elucidate the mechanism by which CGRP inhibits TNF-{alpha} production in BMDC, we further examined the influence of CGRP on TLR signal transduction. As shown in Fig. 5a, P3C-stimulated phosphorylation and degradation of I{kappa}B{alpha} was not altered by CGRP treatment of BMDC. Moreover, P3C-induced DNA binding of RelA was not influenced by CGRP (Fig. 5b). To investigate the activation of NF-{kappa}B in more detail, HEK293 cells were transfected with a TLR2 expression plasmid along with a NF-{kappa}B-dependent firefly luciferase reporter construct. The results in Fig. 5c reveal that HEK293 cells treated with P3C either in the presence or absence of CGRP activated the transcriptional activity of NF-{kappa}B to a comparable extent. Control experiments demonstrated that, similar to BMDC, HEK293 cells express CGRP receptor and respond to CGRP exposure with a rapid increase in cellular cAMP levels (data not shown). In additional experiments, activation of MAPK was analyzed in BMDC. However, we also did not observe any effect of CGRP treatment on the phosphorylation of JNK and p38 MAPK (Fig. 5d). Thus, these results strongly suggest that the engagement of the CGRP receptor does not influence TLR signaling pathways leading to the activation of NF-{kappa}B and MAPK.


Figure 5
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  		FIGURE 5. CGRP does not influence canonical TLR signaling. BMDC were left untreated (M) or stimulated with Pam3Cys (P3C) in the presence or absence of CGRP (C) or CGRP8–37 (iC). a, Expression of phospho-I{kappa}B{alpha}, I{kappa}B{alpha}, and p38 was analyzed by Western blotting after the indicated time periods. b, Nuclear extracts of BMDC were prepared, and binding of RelA to a consensus DNA motif was measured by an immunoenzyme assay using wild-type and mutated oligonucleotides as competitors. Relative DNA-binding capacity of RelA was calculated using the values of unstimulated BMDC as calibrators (n = 10). c, HEK293 cells were transfected with an expression plasmid encoding TLR2 and a NF-{kappa}B firefly luciferase reporter construct. Cells were left untreated (M) or stimulated with Pam3Cys (P3C) in the presence or absence of CGRP (C) or CGRP8–37 (iC). Firefly luciferase activity was normalized against a constitutively expressed Renilla luciferase reporter (n ≥ 5). d, Phosphorylation of JNK and p38 MAPK was quantified in lysates of BMDC using an immunoenzyme assay. The amount of phosphorylated protein was normalized against total JNK or p38 expression, and relative phosphorylation levels were calculated with unstimulated cells as calibrators (n ≥ 8).

 
Inhibition of TNF-{alpha} production by CGRP involves the transcriptional repressor ICER

The Tnf promoter contains a cis-acting CRE site that is crucial for Tnf gene transcription upon cell stimulation with LPS or mycobacteria (32, 33). The activity of the CRE site was reported to depend on the close proximity to a NF-{kappa}B site, thereby generating a composite regulatory element that binds CREB-c-Jun and RelA-p50 complexes in LPS-stimulated cells (32, 34). ICER is a cAMP-inducible transcriptional repressor that functions as a negative regulator of CRE-binding transcription factors of the CREB and CREM families (26). We, therefore, hypothesized that CGRP treatment of BMDC may induce the expression of ICER, which, in turn, may dampen Tnf transcription through inhibition of the CRE site in the CRE/{kappa}B composite regulatory element. To test this hypothesis, BMDC were stimulated with P3C in the presence or absence of CGRP, and expression of ICER isoforms was determined at the protein level. As shown in Fig. 6a, expression of ICER was induced by CGRP treatment of BMDC as early as 1 h after stimulation, and elevated expression was sustained for at least 6 h. The results of ICER protein expression were confirmed at the mRNA level using RT-PCR. These experiments also revealed a predominant induction of the ICER-I rather than ICER-II isoforms (data not shown). If the inhibition of CGRP on TNF-{alpha} production was indeed mediated by up-regulation of ICER, this effect should be dependent on de novo protein synthesis. We therefore stimulated BMDC with P3C in the presence or absence of CGRP and quantified the effect of cycloheximide treatment on Tnf mRNA induction by real-time RT-PCR 3 h later. We found that inhibition of Tnf mRNA induction by CGRP was completely prevented by cycloheximide (Fig. 6b).


Figure 6
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  		FIGURE 6. Induction of ICER and the presence of a composite CRE/{kappa}B element correlate with the inhibitory activity of CGRP. BMDC were left untreated (M) or incubated with CGRP (C) or CGRP8–37 (iC) in the presence or absence of the indicated TLR agonists. a, Protein expression of ICER and p38 MAPK was determined by Western blot analysis after the indicated time periods. b, BMDC were treated as indicated with or without 10 µg/ml cycloheximide (CHX), and TNF-{alpha} mRNA levels were determined by quantitative real-time RT-PCR. Relative mRNA expression was calculated using expression levels of unstimulated cells as calibrators (n ≥ 5). Production of CCL4 (c) and CXCL1 (d) was determined in BMDC supernatants after 16 h of stimulation (n = 3 for CCL4; n = 7 for CXCL1). *, p < 0.05; **, p < 0.01.

 
Previous studies have shown that, similar to the Tnf promoter, the human Ccl4 promoter also contains a cis-acting CRE site that is located in close proximity to a NF-{kappa}B element (34). The CRE site was found to be crucial for Ccl4 transcription in mitogen-stimulated human T cells, and Ccl4 promoter activity could be suppressed by the ectopic expression of ICER (34). Using the Genomatix database and promoter analysis software (www.genomatix.de), we confirmed the presence of a composite CRE/{kappa}B site in the murine Ccl4 promoter, whereas this regulatory element was not found in the murine Cxcl1 promoter (data not shown). We therefore investigated the influence of CGRP on TLR-stimulated CCL4 and CXCL1 protein production by BMDC. As shown in Fig. 6, c and d, treatment of BMDC with CGRP markedly reduced production of CCL4, but not CXCL1, in response to all of the TLR agonists examined. Thus, the presence of the composite CRE/{kappa}B site correlates with the inhibitory function of CGRP on gene expression.

To directly investigate the influence of ICER on Tnf transcription, we cloned a 506-bp fragment of the murine Tnf promoter encompassing the CRE/{kappa}B composite element and placed it upstream of the minimal SV40 promoter driving expression of the firefly luciferase reporter gene. The Tnf promoter construct was transfected along with a plasmid driving the expression of murine ICER-I into RAW264 macrophages. Expression of ICER-I was confirmed by Western blot analysis (data not shown). The ICER-I isoform was chosen because RT-PCR analyses of CGRP-treated BMDC revealed the predominant induction of this isoform (data not shown), and because previous studies have shown that the different isoforms of ICER exhibit equal capacity to suppress gene expression (35). As shown in Fig. 7a, activity of the wild-type Tnf promoter was stimulated by treatment of RAW264 cells with LPS. Importantly, ectopic expression of ICER-I markedly reduced Tnf promoter activity in a dose-dependent manner (Fig. 7a). Next, the effect of ICER on TNF-{alpha} production driven by the endogenous promoter was examined. The results in Fig. 7b show that transfection with ICER-I also caused a significant inhibition of TNF-{alpha} protein release by LPS-stimulated RAW264 cells. To further examine the role of endogenous ICER, the effects of forskolin treatment of RAW264 cells were analyzed. Forskolin was used because RAW264 cells did not express the CGRP receptor and did not respond to CGRP with elevation of cellular cAMP levels (data not shown). We found that treatment of RAW264 cells with forskolin up-regulated the expression of ICER protein, and that induction of ICER was associated with strongly diminished TNF-{alpha} production of RAW264 cells exposed to LPS (Fig. 7, c and d). Together, these results therefore suggest that up-regulation of ICER attenuates the activity of the Tnf promoter, thereby reducing TNF-{alpha} protein production.


Figure 7
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  		FIGURE 7. ICER represses Tnf promoter activity and dampens TNF-{alpha} protein production. a, RAW264 macrophages were transfected with a plasmid-driving expression of the firefly luciferase reporter under control of a Tnf promoter fragment containing the composite CRE/{kappa}B element. The effect of cotransfection of an ICER-I expression construct on Tnf promoter activity is shown. Firefly luciferase activity was normalized against a constitutively expressed Renilla luciferase reporter (n = 3). b, RAW264 macrophages were transfected with an ICER-I expression construct or empty vector. TNF-{alpha} production was measured in the supernatants after 6 h of stimulation with LPS (n = 4). c, Protein expression of ICER and p38 MAPK was determined 6 h after stimulation of RAW264 macrophages with LPS, forskolin (Fsk), or both. d, RAW264 macrophages were stimulated with LPS in the presence or absence of forskolin (Fsk) and production of TNF-{alpha} was measured in the supernatant 16 h later (n = 7). **, p < 0.01; ***, p < 0.001.

 
CGRP attenuates TNF-{alpha} serum levels and induces ICER protein expression in LPS-treated mice

Having established that the inhibition of TNF-{alpha} production by CGRP and cAMP involves induction of the transcriptional repressor ICER, we next asked whether this process may also be relevant in an in vivo setting. To address this question, mice were injected with LPS along with different doses of CGRP. The results in Fig. 8a demonstrate that, in the absence of exogenous CGRP, high levels of serum TNF-{alpha} were measured 90 min after the injection of LPS. However, when CGRP was administered in combination with LPS, serum TNF-{alpha} levels were reduced in a dose-dependent manner (Fig. 8a). To examine ICER expression in vivo, ICER mRNA levels were determined in a semiquantitative manner using template cDNA titration of liver samples. The results for three individual mice per experimental group are depicted in Fig. 8b. As shown, basal expression of ICER mRNA was observed in PBS-injected control mice. Importantly, injection of CGRP along with LPS resulted in a strong up-regulation of mRNA for ICER-I and ICER-II, with more pronounced expression of the ICER-I isoform. Injection of LPS alone had only a minor influence on ICER mRNA expression. Thus, CGRP also inhibits LPS-triggered TNF-{alpha} release in vivo, and this effect is associated with the up-regulation of ICER.


Figure 8
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  		FIGURE 8. CGRP decreases TNF-{alpha} serum levels in murine endotoxemia. Mice were treated i.v. with 10 µg LPS along with the indicated doses of CGRP. Serum and liver samples were collected 1 h later. a, TNF-{alpha} levels were measured in serum (n = 6). b, Expression of ICER mRNA was determined by RT-PCR from serial 1/5 dilutions of template liver cDNA. Results from three independent mice are shown. **, p < 0.01; ***, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well established that immune responses may be influenced by the nervous system (1, 36, 37). C-type sensory nerve fibers may modulate immune cell functions through the neuropeptide CGRP, which is released upon exposure of pain fibers to mast cell-derived tryptase (8). CGRP inhibits activation of innate and adaptive immune cells in vitro and shows potent immunosuppressive activities in models of delayed-type hypersensitivity, inflammatory bowel injury, and endotoxemia in vivo (18, 19, 20, 21, 22, 23, 24, 25). Despite ample evidence for the negative regulation of immune responses by CGRP, however, the molecular mechanisms of CGRP action on immune cells remain largely elusive. In the present report, we show that CGRP is a potent inhibitor of TLR-stimulated Tnf and Ccl4 gene expression in DC. The inhibitory activity of CGRP was independent of IL-10. CGRP also did not influence canonical TLR signaling pathways involving activation of NF-{kappa}B and MAPK. Instead, the results are consistent with a mechanistic model, indicating that CGRP activates adenylyl cyclase and PKA, leading to the expression of the cAMP-inducible transcriptional repressor ICER, which in turn dampens gene transcription.

The transcription factor CREB was previously found to be crucial for stimulating Tnf promoter activity in response to LPS or mycobacteria (32, 33). The function of CREB for enhancing Tnf transcription is dependent on the binding of CREB/c-Jun heterodimers to a cis-acting CRE site that is part of a composite CRE/{kappa}B element in the Tnf promoter (32). It is thought that activated CREB recruits CREB-binding protein, which acts as a transcriptional cofactor for NF-{kappa}B (38), thereby enabling full transcriptional activity of NF-{kappa}B. The transcriptional repressor ICER is transcribed from an intronic promoter of the CREM gene that is responsive to elevation of cellular cAMP (26). All protein isoforms of ICER contain an intact DNA-binding domain, but no transactivation domain, and therefore function as dominant negative regulators of CRE-binding transcription factors (39, 40). In the present report, we demonstrate that the inhibition of TLR-stimulated TNF-{alpha} production by the neuropeptide CGRP was associated with the rapid up-regulation of ICER at the protein level and that the inhibitory effect of CGRP was dependent on de novo protein synthesis. Moreover, CGRP-attenuated expression of genes that contain a composite CRE/{kappa}B promoter element (e.g., TNF-{alpha}, CCL4), but failed to mediate repression of genes lacking this element (e.g., CXCL1). Importantly, we could show that ectopic expression of ICER directly represses Tnf promoter activity as well as TNF-{alpha} protein production of LPS-stimulated macrophages. These findings are in excellent agreement with transfection studies showing that ectopic expression of ICER dampens the activity of Tnf and Ccl4 promoters in human T lymphocytes stimulated with mitogens or phorbol ester and ionomycin (34, 41). Considered together with these studies, our findings, therefore, indicate that the mechanism, by which the neuropeptide CGRP inhibits inflammatory gene expression, critically involves up-regulation of the transcriptional repressor ICER.

Analysis of CGRP receptor signaling revealed that exposure of BMDC to CGRP results in elevation of cellular cAMP levels, but does not induce Ca2+ flux, suggesting that the CGRP receptor couples to G{alpha}s rather than G{alpha}q/11 proteins in these cells. Several lines of evidence suggest that the inhibitory effects of CGRP on TNF-{alpha} production are mediated by cAMP and PKA. Thus, pharmacological activation of adenylyl cyclase inhibited TNF-{alpha} production and resulted in the up-regulation of ICER protein. In addition, selective activation of the cAMP target kinase PKA attenuated TLR-induced TNF-{alpha} production and the inhibitory effect of CGRP was reversed by the blockade of PKA. Finally, these results are in perfect accordance with previous findings showing that expression of ICER is induced by the cAMP/PKA pathway (26). When we stimulated Epac proteins using a selective cAMP analog, however, we did not observe any influence on the TNF-{alpha} production of BMDC. These data are also consistent with an independent study demonstrating that activation of PKA, but not Epac-1, inhibited the TNF-{alpha} production of alveolar macrophages (42). Instead, activation of Epac proteins was found to inhibit macrophage phagocytosis (42) and to induce integrin-mediated cell adhesion and spreading through Rap1 (43, 44). Collectively, these results therefore strongly suggest that the cAMP/PKA pathway mediates the inhibitory effects of CGRP on TNF-{alpha} production by inducing expression of the transcriptional repressor ICER.

Experiments using BMDC from IL-10-deficient mice clearly established that inhibition of TLR-stimulated TNF-{alpha} production by CGRP is independent of IL-10. Consistent with these observations, we also found that LPS-stimulated IL-10 production by RAW264 cells was not augmented by forskolin (data not shown). However, previous investigations using human PBMC or a murine DC line and using neutralizing Abs have suggested that certain activities of CGRP may be mediated by IL-10 (20, 25). A possible explanation for these divergent results may relate to the different stimulatory conditions used. Whereas in our study BMDC were cultured in the absence of GM-CSF before stimulation, previous studies examined either cells that were not GM-CSF-starved before use (20) or cells that were costimulated with GM-CSF (25). Moreover, previous studies differ from our experiments by applying alternative stimuli such as Staphylococcus aureus Cowan strain and the use of different cell types or cells from different species. Moreover, IL-10 production may be differently influenced when cells are treated with CGRP before or concomitant with TLR stimulation. It should also be noted that Fox et al. (20) reported that the inhibitory effect of CGRP on the release of IFN-{gamma} by PBMC was not reverted by neutralization of IL-10, thereby supporting the notion that IL-10-independent pathway(s) of CGRP action may exist. The results of the present study indicate that CGRP engages the IL-10-independent pathway to dampen TNF-{alpha} and CCL4 production by murine BMDC and provide compelling evidence that up-regulation of the transcriptional repressor ICER represents a mechanistic basis of the IL-10-independent pathway.

Experiments attempting to elucidate the mechanism(s) of CGRP action revealed that concomitant stimulation of BMDC with TLR agonists and CGRP did not alter canonical TLR signaling pathways, including activation of NF-{kappa}B, whereas previous studies using murine thymocytes or type II alveolar epithelial cells reported that CGRP may partially inhibit the nuclear translocation of NF-{kappa}B and degradation of I{kappa}B{alpha} (45, 46). It should be noted, however, that, in addition to using different cell types, the experimental conditions applied in these studies also differ from those of our study by CGRP pretreatment of cells and cell stimulation with phorbol ester and ionomycin or IL-1. Moreover, our failure to find induction of IL-10 by CGRP may also provide a possible explanation for the different results, because IL-10 has been shown to inhibit NF-{kappa}B activation in DC (47).

Administration of CGRP to mice was shown to inhibit LPS-elicited elevation of serum TNF-{alpha} levels in a dose-dependent manner, indicating that CGRP may also control TNF-{alpha} production under in vivo conditions. Previous studies investigating the influence of CGRP on immune responses in mice also have reported potent anti-inflammatory effects, including protection from lethal endotoxemia (22), inhibition of delayed-type hypersensitivity responses (19, 21), and attenuation of the severity of experimental colitis (23, 24). Although protection from lethal endotoxemia has been correlated with reduced serum TNF-{alpha} levels (22), the mechanism(s) of CGRP action in vivo have remained largely unknown. The present report therefore extends previous studies by demonstrating that the inhibition of TNF-{alpha} production by CGRP in mice is clearly associated with enhanced expression of ICER. Thus, up-regulation of ICER may also provide, at least in part, a mechanistic basis to explain the immunosuppressive activities of CGRP under in vivo conditions.

TLR activation is crucial for protecting the host against invading pathogens (28, 48), but immune activation by TLRs has also been implicated in the pathogenesis of acute and chronic inflammatory disorders, as well as autoimmune and infectious diseases (49). It is therefore important that TLR signaling is tightly regulated to ensure a favorable balance between protective and detrimental activities. Multiple cellular mechanisms have been described for down-regulating TLR activation that operate by interfering with signal transduction through TLRs. These mechanisms include perturbation of receptor complexes by soluble TLRs or structurally related membrane proteins, ubiquitin-mediated degradation of TLRs and signaling proteins, sequestration of adapter proteins, expression of dominant negative signaling inhibitors, and deactivation of kinases through phosphatases (50, 51). Our results clearly demonstrate that the engagement of the CGRP receptor does not influence TLR-induced activation of NF-{kappa}B and MAPK, indicating that the mechanism of CGRP action differs from previously described negative regulatory pathways targeting canonical TLR signaling. Instead, we demonstrate the existence of a novel pathway for termination of TLR responses that is engaged by the nervous system. The data support the model that this pathway operates at the level of transcriptional control of inflammatory gene expression and is mediated by up-regulation of the CRE-binding transcriptional repressor ICER through the cAMP/PKA pathway.


   Acknowledgments
 
We thank Dirk Busch and Roland Lang for critically reading this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 576, Project A7) and by the Kommission für Klinische Forschung, Klinikum rechts der Isar. Back

2 Address correspondence and reprint requests to Dr. Bernhard Holzmann, Department of Surgery, Klinikum rechts der Isar, Technische Universität München, Ismaninger Strasse 22, Munich, Germany. E-mail address: holzmann{at}chir.med.tu-muenchen.de Back

3 Abbreviations used in this paper: CGRP, calcitonin gene-related peptide; CRLR, calcitonin receptor-like receptor; RAMP, receptor activity-modifying protein; CGRP-RCP, CGRP receptor component protein; DC, dendritic cell; PKA, protein kinase A; ICER, inducible cAMP early repressor; BMDC, bone marrow-derived DC; 6-Bnz-cAMP, N6-benzoyladenosine-3',5'-cAMP; 8-pCPT-2'-O-Me-cAMP, 8-(-4-chlorophenyl-thio)-2'-O-methyladenosine-3',5'-cAMP; Epac, exchange proteins directly activated by cAMP; CRE, cAMP response element; Pam3Cys, (S)-[2,3-bis (palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH. Back

Received for publication February 13, 2007. Accepted for publication April 17, 2007.


   References
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 Materials and Methods
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 Discussion
 Disclosures
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