INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: calcitonin gene-related peptide; airway hyperresponsiveness; allergen

Sensory neuropeptides play an important role in the pathogenesis of several airway diseases such as allergic rhinitis and asthma (1-4). The most studied lung neuropeptides are the tachykinins, substance P, and neurokinin A, which are released from sensory C-fiber afferents by a variety of stimuli including organic irritants (5), ozone (6), and allergens (7). These neuropeptides bind to their receptors present on a variety of cell types in the upper and lower airways and mediate effects that contribute to asthmatic airway dysfunction. Such effects include bronchoconstriction (4), mucus hypersecretion (8), increased vascular permeability (9, 10), chemoattraction and activation of inflammatory cells (11- 13), and stimulation of cytokine production (14, 15).

Calcitonin gene-related peptide (CGRP) is another neuropeptide that colocalizes with substance P in sensory C-fiber afferents in the airways (16, 17). CGRP is a 37-amino acid peptide that results from alternative processing of the precursor mRNA encoded by the peptide hormone calcitonin gene (18, 19). CGRP is a potent vasodilator with long-lasting effects (20). Unlike substance P, CGRP does not induce mucus hypersecretion (21) or plasma protein extravasation in the airways (17). CGRP has been described earlier as a bronchoconstrictor of human airways (22). This effect, however, is discordant with the ability of this neuropeptide to activate adenylate cyclase, which results in increased cellular levels of cyclic AMP (23), a pathway usually associated with bronchodilation. Other studies performed with isolated guinea pig or human airways found no bronchoconstrictive effects with CGRP (16, 24, 25).

In normal airways, CGRP is also found in pulmonary neuroepithelial bodies, consisting of innervated clusters of neuroepithelial cells localized within the mucosa, mostly at the branching points of intrapulmonary airways (26, 27). In the submucosa, CGRP-immunoreactive nerve fibers can be found in close contact with the epithelium and smooth muscle (28, 29). Based on this unique distribution, we postulated that CGRP could play a potential role as a modulator of airway function during exposure to allergens and environmental irritants. The present study was undertaken to identify the changes that occur in the distribution and expression of CGRP in the airways following sensitization and airway challenge and to investigate the potential role of CGRP in modulating airway function in a mouse model of ovalbumin-induced allergic airway inflammation and hyperresponsiveness.

	METHODS

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Reagents

Synthetic human CGRP and its receptor antagonist CGRP(8-37) were obtained from Sigma Chemical Co. (St. Louis, MO), and were administered at the optimal doses of 4 and 20 nmol/kg (respectively), selected from preliminary dose-response experiments. Rat antibodies to very late antigen (VLA)-4 and interleukin (IL)-5 were purified from PS/2 (30) and TRFK-5 (31) hybridoma cell cultures, respectively. Nonimmune rat immunolglobulin (Ig) G (Sigma Chemical Co.) was used as control antibody. Mice were treated by a single intravenous injection of antibody (2 mg/kg), 2 hours before the first challenge.

Animals

BALB/c mice were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were sensitized to ovalbumin on days 0 and 14 and challenged with aerosolized ovalbumin (1% in saline) via the airways on Days 28, 29, and 30, as previously described (32, 33). Controls consisted of nonsensitized mice that were exposed to saline or ovalbumin and sensitized mice that were exposed to saline. The study was conducted under a protocol that was approved by the local institutional animal care and use committee.

Airway Function

Two days after the last ovalbumin challenge, airway function was assessed in vivo as previously described by measuring changes in lung resistance and dynamic compliance in response to intratracheal administration of aerosolized methacholine (32, 33). The data are presented as a percent of change from baseline lung resistance and dynamic compliance, obtained after challenge with saline. Baseline lung resistance varied between 0.58 and 0.67 cm H₂O/ml/second, and dynamic compliance values were between 0.046 and 0.054 ml/cm/H₂O, with no significant differences between the different groups and treatments.

Airway smooth muscle responsiveness was assessed in vitro through electrical field stimulation and by exposure to carbachol, as previously described in detail (34). Acetylcholine release was measured by using high-performance liquid chromatography, as previously described (35).

Immunohistochemistry

Forty-eight hours after the last ovalbumin challenge (24 hours in some experiments), the lungs were removed, fixed in formalin, and processed into paraffin. Tissue eosinophils, CGRP, and airway nerve fibers were detected by immunoperoxidase, using polyclonal rabbit antibodies to mouse eosinophil major basic protein (generously provided by Dr. J.J. Lee, Mayo Clinic, Scottsdale, AZ), human CGRP (both - and -forms), and pan-neuronal marker PGP9.5 (Biogenesis Inc., Sandown, NH), respectively.

The immunohistochemistry data were quantified by digital image analysis using NIH Scion Image software (version 1.62, developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). All measurements (the groups were blinded to the observer) were performed on at least 3 serial tissue sections cut from the paraffin blocks every 50 µm and were normalized to the BM perimeter for the corresponding airways. The measured values were averaged for each animal, and the mean values were determined for each group.

Statistical Analysis

Data (n = 8 per group) were analyzed by analysis of variance with the Tukey-Kramer HSD test for multiple comparisons of the means to determine significant differences (p value  0.05) between the groups.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CGRP Is Depleted in the Airways after Allergen Challenge

Mice sensitized and exposed to ovalbumin developed characteristic airway tissue eosinophilic inflammation (Figure 1H) and goblet cell metaplasia/hyperplasia (Figure 1I). In control mice, CGRP was detected in neuroepithelial bodies, localized mostly at the branching points of central intrapulmonary airways with a lower expression in peripheral airways (Figures 1C and 1D). CGRP-immunoreactive nerve fibers were detected in the submucosa of normal airways showing intimate contact with neuroepithelial bodies and smooth muscle bundles (Figures 1E and 1F). In sensitized/challenged mice, CGRP expression was considerably diminished in the bronchial epithelium and submucosal nerve plexuses in intrapulmonary airways (Figures 1J-1L). Immunostaining for the pan-neuronal marker PGP9.5 revealed no differences in the overall density of airway nerve fibers between control mice and sensitized/challenged mice (Figure 1G and 1N).

View larger version (53K): [in this window] [in a new window]   Figure 1.   Effect of sensitization and allergen challenge on airway inflammation and CGRP expression. Lung tissue sections from nonsensitized control mice (A-G) and from ovalbumin sensitized/challenged mice (H-N ) were stained for eosinophil major basic proteins (A, H ), mucus-containing goblet cells (B, I ), CGRP (C-F, J-L), and the pan neuronal marker PGP9.5 (G, M, N) (arrowheads). In sensitized but not control mice, ovalbumin challenge induced characteristic airway eosinophilic inflammation (H ) and goblet cell hyperplasia (I ) and resulted in a considerable depletion of CGRP from the epithelium ( J, K ) and submucosal nerve fibers (L) without alteration in the overall density of airway nerve fibers (G, M, N ). Scale bars represent 50 µm.

To quantitatively analyze the expression of CGRP in airway tissue, we divided the mouse airways into central and peripheral airways based on our preliminary data showing distinct airway morphometric characteristics (Table 1). The morphometric analyses further revealed significant neuroanatomical differences between central and peripheral airways. The overall density of nerve fibers and CGRP immunoreactivity, in both the airway epithelium and submucosal nerve fibers, were remarkably decreased from central to peripheral intrapulmonary airways (Table 2). Most importantly, the results demonstrated considerable depletion of CGRP in the airways of sensitized/challenged mice, and both sensitization and allergen challenge were required for the depletion of CGRP. Indeed, the expression of CGRP remained unchanged in the lungs of nonsensitized mice that were exposed to aerosolized ovalbumin or saline and in mice that were sensitized to ovalbumin but subsequently exposed to saline.

Timing of CGRP Depletion

To further define the time frame in which CGRP depletion occurs during the development of the allergic airway inflammatory response, the expression of CGRP was measured morphometrically in the airways of mice at 24 and 48 hours following one, two, or three ovalbumin challenges. The results, shown in Table 3, demonstrate that CGRP depletion occurred in the airways of sensitized mice only after the third ovalbumin challenge and was more pronounced at 48 hours after this challenge. Forty-eight hours and three challenges resulted in the most robust airway and tissue inflammatory cellular infiltration (Figure 2). These data indicated that CGRP depletion coincides with the development of the allergic airway response, typically characterized by an eosinophilic inflammation.

View larger version (130K): [in this window] [in a new window]   Figure 2.   Association of CGRP depletion with ovalbumin-induced airway inflammation at 48 hours after the last allergen challenge. Lung tissue sections from control (A-D) and sensitized mice, exposed to a single (E-H ), two (I- L), or three ovalbumin challenges (M-P), were stained for CGRP, mucus (PAS), and routine histology (H&E). CGRP depletion was more pronounced in the epithelium (M ) and submucosal nerve fibers (N ) after three ovalbumin challenges, which resulted in the most robust airway tissue inflammatory cell infiltration (O, P ). Scale bar represents 50 µm.

CGRP Depletion Is Associated with Eosinophilic Inflammation

To determine if the depletion of CGRP is associated with the development of airway eosinophilic inflammation, we examined the effects of treatment with anti-VLA-4 or anti-IL-5 antibodies that selectively interfere with airway tissue accumulation. Treatment with anti-VLA-4 or anti-IL-5 before allergen challenge markedly reduced the number of eosinophils in the bronchoalveolar lavage (BAL) and abolished the development of AHR, measured as increases in lung resistance and decreases in dynamic compliance in response to inhaled methacholine (MCh) in sensitized/challenged mice (Figure 3). Both treatments eliminated tissue-infiltrating eosinophils and prevented the allergen-mediated depletion of CGRP in these animals (Table 4).

View larger version (40K): [in this window] [in a new window]   Figure 3.   Effects of anti-VLA-4 (PS/2) and anti-IL-5 (TRFK-5) on ovalbumin-induced airway inflammation (A) and airway responsiveness to inhaled MCh (B). Mice were nonsensitized/challenged (PBS); sensitized/challenged (OVA); or sensitized/challenged but treated with rat IgG (OVA/IgG), anti-VLA-4 (OVA/anti-VLA-4), or anti-IL-5 (OVA/anti- IL-5) before the first ovalbumin challenge. Treatment with anti-VLA-4 or anti-IL-5 markedly reduced the numbers of eosinophils in the BAL (A) and prevented the development of AHR (B), measured as increases in lung resistance (RL) and decreases in dynamic compliance (Cdyn). Values labeled with * are significantly different from control (PBS) values (p < 0.05).

Replacement of CGRP Restores Normal Airway Responsiveness

To determine if CGRP can modulate allergen-induced AHR, we evaluated two pharmacologic approaches: administration of the highly selective CGRP receptor antagonist (CGRP, peptide fragment 8-37) to antagonize endogenous CGRP; and administration of exogenous -CGRP to compensate for the in vivo depletion.

Treatment of naive mice by intraperitoneal administration of the receptor antagonist CGRP(8-37), before each (of three) allergen challenges, did not alter their normal airway responsiveness to inhaled MCh, suggesting that under these basal conditions CGRP is not required for maintaining such normal airway responsiveness (Figure 4). Similar treatment of sensitized mice with CGRP(8-37) did not produce significant changes in airway responsiveness, further indicating that endogenous CGRP, if released, is not mediating AHR in these animals (Figure 5A). By contrast, administration of exogenous CGRP to sensitized mice, before each allergen challenge to overcome the in vivo depletion, fully restored normal airway responsiveness to MCh.

View larger version (23K): [in this window] [in a new window]   Figure 4.   Effect of CGRP receptor antagonist on basal airway responsiveness to methacholine. Naive mice were administered (intraveneously) varying doses of CGRP (8-37) and subjected to airway function measurement. No significant increases were observed in lung resistance to methacholine following the treatments with CGRP (8-37).

View larger version (33K): [in this window] [in a new window]   Figure 5.   Effects of CGRP and CGRP receptor antagonist on ovalbumin-induced AHR. CGRP and CGRP(8-37) were administered intraperitoneally to sensitized mice before each ovalbumin challenge (A) or after the last (of three) ovalbumin challenge (B). Administration of exogenous CGRP but not its receptor antagonist, before each ovalbumin challenge, prevented the development of ovalbumin-induced AHR (A). Treatment of mice with exogenous CGRP after the last (of three) allergen challenges restored normal airway responsiveness (lung resistance and dynamic compliance) to inhaled MCh (B). This effect of CGRP was neutralized by prior treatment of mice with the highly selective CGRP receptor antagonist. Values labeled with * are significantly different from control (PBS) values (p < 0.05).

Interestingly, airway responsiveness was also normalized in sensitized/challenged mice administered exogenous CGRP after the last (of the three) allergen challenge, 2 hours before the assessment of airway responsiveness (Figure 5B). Importantly, these inhibitory effects of CGRP were totally abolished by pretreatment of the animals with the receptor antagonist CGRP(8-37), clearly indicating a receptor-mediated effect. No significant effects on BAL or tissue-infiltrating eosinophils were seen following these treatments (Table 5).

To circumvent any potential effect of systemic administration of CGRP that might influence airway function, we evaluated the effects of CGRP delivered via inhalation. Sensitized/ challenged mice were exposed to aerosolized CGRP (10⁶ M) for 15 minutes, and their airway function was assessed after 2 hours. The results clearly show that airway function was also normalized after local administration of CGRP to the airways of sensitized/challenged mice (Figure 6).

View larger version (20K): [in this window] [in a new window]   Figure 6.   Effect of inhaled CGRP on ovalbumin-induced AHR. Mice were nonsensitized and challenged (PBS), sensitized/challenged but not treated (OVA), or sensitized/challenged and treated after the last allergen challenge by 15-minute inhalation of CGRP (OVA+CGRP[inhaled]). Administration of CGRP locally to the airways of sensitized/ challenged mice also restored normal airway responsiveness to inhaled MCh. Values labeled with * are significantly different from control (PBS) values (p < 0.05).

Although CGRP exists in  (dominant) and  form, no significant differences in physiologic effects have been reported to date. To determine if -CGRP can also modulate airway responsiveness to MCh, we administered mice -CGRP, either alone or following pretreatment with the receptor antagonist CGRP(8-37), 2 hours before the assessment of airway function. The results demonstrated that administration of -CGRP, at the same dose shown to be effective for -CGRP (4 nmol/kg), produced significant inhibition of AHR (~ 75% decrease in lung resistance at the highest dose of MCh). This effect was antagonized by the receptor antagonist CGRP (8-37) (Figure 7).

View larger version (22K): [in this window] [in a new window]   Figure 7.   Effect of -CGRP on ovalbumin-induced AHR. Administration of -CGRP to sensitized mice either before (-CGRP, pre-OVA) or after the ovalbumin challenge (-CGRP, post-OVA) resulted in significant decreases in lung resistance. These effects were antagonized by prior treatment with CGRP(8-37) (-CGRP+CGRP[8-37] post-OVA). * = significant difference, compared with control (PBS); # = significant difference, compared with (OVA) sensitized/challenged.

Effect of CGRP on Airway Smooth Muscle Responsiveness to Electric Field Stimulation

We have previously demonstrated that sensitized mice have an increased tracheal smooth muscle (TSM) responsiveness to electric field stimulation, which is due to increased acetylcholine release resulting from altered M2 receptor function (35). In this study, treatment of TSM segments from sensitized mice for 2 hours with CGRP (10⁶ M) did not alter their responsiveness to electric field stimulation and did not affect acetylcholine release following electrical stimulation (mean ± SEM, n = 8: 134 ± 17 versus 151 ± 12 pmol · g tissue¹ · minute stimulation¹ for CGRP-treated versus untreated TSM, respectively).

Effect of CGRP on Postjunctional Cholinergic Airway Smooth Muscle Responsiveness

To further determine if the observed in vivo effects of CGRP were transduced at the postjunctional levels on airway smooth muscle, we investigated the effects of this neuropeptide on the contractile responsiveness of mouse TSM segments to the cholinergic agonist carbachol. TSM segments isolated, 48 hours after the last challenge, from sensitized/challenged mice or normal control (nonsensitized but challenged) mice were equally responsive to carbachol and developed similar dose-dependent contraction to this agonist in vitro (Figure 8). Incubation of TSM for 2 hours with CGRP (10⁶ M) did not inhibit the carbachol-induced contraction and did not alter the responsiveness to carbachol in both groups of mice. When CGRP was added to TSM segments precontracted to half maximal tension, no relaxation could be detected for sensitized/challenged or control groups (not shown). Thus, the in vivo suppression of AHR by CGRP is likely not mediated through a direct action on airway smooth muscle.

View larger version (24K): [in this window] [in a new window]   Figure 8.   Effect of exogenous CGRP on postjunctional cholinergic airway responsiveness. Tracheal smooth muscle (TSM) segments were isolated from nonsensitized/challenged (upper figure) or sensitized/ challenged mice (bottom figure), at 48 hours following the last ovalbumin challenge, and were incubated or not with CGRP (106 M) for 2 hours in Eagle's Minimal Essential Medium supplemented with 1% naive mouse serum. Tracheas were rinsed with Krebs-Henselheit medium and were tested for their contractile responsiveness to the cholinergic agonist carbachol in the presence or absence of exogenous CGRP. CGRP did not inhibit the in vitro cholinergic contraction of TSM isolated from either control or sensitized/challenged mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The overall objective of this study was to define the effects of sensitization and allergen airway exposure on CGRP expression and to determine if CGRP can modulate airway function in a mouse model of allergic airway inflammation and AHR. The morphometric analyses revealed distinct morphological (airway caliber, amount of smooth muscle, and mucus production) and neuroanatomic (distribution of CGRP and density of nerve fibers) features for central and peripheral airways that may have potential implication in the pathophysiology of these airway compartments. The results also demonstrated that allergen challenge of sensitized mice induces significant depletion of CGRP in neuroepithelial bodies and submucosal nerve fibers without altering the overall density of nerve fibers in the airways. This depletion only followed the combination of sensitization and allergen challenge and was subsequent to the development of characteristic airway eosinophilia. Further, maximum depletion coincided with the peak of eosinophil infiltration and AHR. Treatment with anti-IL-5 or anti-VLA-4, which inhibited the accumulation of eosinophils in the airways and normalized airway responsiveness, also prevented the depletion of CGRP. IL-5 is known to be critical for eosinophil differentiation, release from bone marrow, and activation but does not affect the accumulation of lymphocytes in airway tissue, whereas anti-VLA-4 inhibits the recruitment of both eosinophils and VLA-4-expressing T lymphocytes (36, 37). Our observations thus provide a potential link between eosinophils or factors responsible for their recruitment and activation (i.e., IL-5) or their secreted products in the depletion of CGRP following allergen challenge. Whether this eosinophil-associated depletion of CGRP is also accompanied by a loss of other neuropeptides, with potential roles in the development of AHR, remains a possibility to be investigated.

A link has been previously established between neural control of airway function and major basic proteins released from activated eosinophils (38, 39). These studies, performed in the guinea pig model, have demonstrated that major basic proteins can bind to and alter the function of acetylcholine muscarinic M2 receptors present on cholinergic airway nerve fibers, resulting in increased acetylcholine release and enhanced smooth muscle contractility in response to electrical stimulation of the vagus nerve. We have previously demonstrated, pharmacologically, that this enhanced response to electrical field stimulation in sensitized mice is due to increased acetylcholine release caused by an altered M2 receptor function (35). However, this alteration may not be mediated by eosinophil major basic proteins in the mouse system, because recent studies have provided evidence that mouse eosinophils do not obviously degranulate after stimulation in vitro or in vivo following allergic airway challenge (40, 41). This may suggest that other mechanisms, possibly including soluble mediators and/or cell-to-cell contact-mediated interactions, could be involved in eosinophil-mediated effects in the mouse system. In the present study neither electric field stimulation responses nor acetylcholine release were altered by CGRP treatment, suggesting that the effects of CGRP are likely not transduced at the prejunctional level and that M2 receptor function is not restored by this neuropeptide. Our in vitro studies of carbachol-induced TSM contractility further excluded a postjunctional effect of CGRP directly acting on isolated airway smooth muscle. Thus, the in vivo effects of CGRP are likely indirect, possibly requiring the involvement of an integrated network of neurons and/ or mediators that are not recovered along with isolated TSM segments. It must be stressed that the increased lung resistance to MCh measured in vivo in several animal models of allergic AHR, which is contributed to by airway smooth muscle contraction, cannot be differentiated in vitro when using isolated airways.

Some studies have shown that CGRP can be degraded in vitro by purified lung mast cell tryptase (42, 43), suggesting that this enzyme may lower the activity of CGRP in vivo. This may explain why CGRP was ineffective in protecting against substance P-induced constriction of sensitized guinea pig airways (25). However, the effectiveness of CGRP administration in allergic mice reported in this study suggests that such CGRP-degrading enzymes may not be fully active in vivo in this animal model. The finding that exogenous CGRP could normalize airway function, even when administered after the allergen challenge, emphasizes its potential effect downstream of the inflammatory cascade. The lack of effect of CGRP administration on naive mouse airways in this study suggests that CGRP may act on some aspects of the inflammatory response without necessarily modifying the inflammatory cell tissue infiltration. In addition, the antagonistic action of CGRP(8-37) further confirmed the receptor-mediated effect of the administered CGRP and demonstrated its specificity. It is noteworthy that CGRP(8-37) did not alter the responsiveness of either normal or allergic mouse airways to MCh, which demonstrates that endogenous CGRP does not mediate nor does it enhance AHR in this model. These results also provide additional support to previous findings that CGRP is not a mediator of airway constriction (16, 25) and suggest rather a protective role for this neuropeptide.

The -(dominant) and -CGRP forms differ from each other by a single amino acid in the rat and three amino acids in humans, but both isoforms have been shown to produce similar biologic effects (44-46). In the present study, AHR was also inhibited by -CGRP in a CGRP(8-37)-sensitive manner, suggesting that both CGRP isoforms are similarly active and share identical receptors in the mouse lung. Based on the differential potencies of CGRP peptide analogs, including CGRP(8-37), two subtypes of CGRP receptors have been proposed by previous pharmacologic studies (47, 48). At the molecular level, the functional (type 1-like) CGRP receptor has been recently characterized and shown to consist of a complex formed by the G-coupled 7-transmembrane domain protein calcitonin receptor-like receptor and the receptor activity modifying protein family member RAMP-1 (49). When coexpressed with RAMP-2 or RAMP-3, calcitonin receptor-like receptor serves as a receptor for adrenomedullin, the most recently identified peptide of the CGRP family (50). Adrenomedullin has previously been shown to protect against bronchoconstrictive responses to acetylcholine and histamine when administered in a guinea pig model of ovalbumin-induced allergic airway responses (51). Further studies are necessary to define the exact role of these neuropeptides and their family-related receptors in allergic airway responses.

In summary, the present study demonstrates that allergen exposure depletes CGRP from the airways of sensitized/challenged mice. This depletion is associated with allergen-induced airway eosinophilic inflammation that contributes to the development of AHR in this model. When administered, CGRP can restore normal airway responsiveness despite this inflammatory response. These beneficial effects of CGRP may suggest its potential use in the treatment and/or prevention of AHR.