Activation of Calcitonin Gene-Related Peptide Receptor during Ozone Inhalation Contributes to Airway Epithelial Injury and Repair

Abstract

The authors investigated the importance of the neuropeptide, calcitonin gene-related peptide (CGRP), in epithelial injury, repair, and neutrophil emigration after ozone exposure. Wistar rats were administered either a CGRP-receptor antagonist (CGRP_8–37) or saline and exposed to 8 hours of 1-ppm ozone or filtered air with an 8-hour postexposure period. Immediately after exposure, ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, airway dissected lung lobes were stained for 5′-bromo-2′-deoxyuridine, a marker of epithelial proliferation. Positive epithelial cells were quantified in specific airway generations. Rats treated with CGRP_8–37 had significantly reduced epithelial injury in terminal bronchioles and reduced epithelial proliferation in proximal airways and terminal bronchioles. Bronchoalveolar lavage and sections of terminal bronchioles showed no significant difference in the number of neutrophils emigrating into airways in CGRP_8–37-treated rats. The airway epithelial cell line, HBE-1, showed no difference in the number of oxidant stress positive cells during exposure to hydrogen peroxide and a range of CGRP_8–37 doses, demonstrating no antioxidant effect of CGRP_8–37. We conclude that activation of CGRP receptors during ozone inhalation contributes to airway epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Introduction

Ozone, a major component of photochemical air pollution, continues to be a significant health concern. In the lung, ozone first reacts with components of the fluid lining airway epithelial cells, resulting in reaction products that cause oxidant stress to the underlying cells. This ultimately results in death of ciliated cells in the large conducting airways and terminal bronchioles and type I pneumocytes in the distal terminal bronchioles and proximal alveolar ducts (Plopper, Dungworth, and Tyler 1973). The oxidant-stressed epithelial cells release numerous mediators, including chemotactic compounds, which leads to a predominately neutrophilic influx into the airways that peaks 8–12 hours after exposure (Pino, Levin, et al. 1992; Vesely, Hyde et al. 1999). The neutrophilic influx is important in removing oxidant injured airway epithelial cells and also likely contributes to resulting epithelial proliferation and subsequent repair in both acute and episodic ozone exposures (Hyde et al. 1999; Schelegle et al. 2003). Epithelial repair proceeds with airway basal cells and Clara cells replacing lost ciliated cells in proximal conducting airways and terminal bronchioles, respectively (Evans et al. 1976; Puchelle et al. 2006). Type II pneumocytes replace lost type I pneumocytes in distal terminal bronchioles and proximal alveolar ducts (Evans et al. 1976).

Acute ozone exposure also stimulates C fibers, small non-myelinated sensory nerves originating from the vagus nerve and terminating beneath and within the airway epithelium and around submucosal bronchial glands and blood vessels (Solway and Leff 1991). C fiber activation induces central nervous-system-mediated reflexes and the release of neuropeptides, including substance P, neurokinin (NK) A, and calcitonin gene-related peptide (CGRP) (Maggi et al. 1995). In rats, CGRP containing C fibers and endocrine cells is widely distributed throughout the respiratory epithelium (Shimosegawa and Said 1991). CGRP levels have been shown to be increased in both guinea pig and rabbit lungs following ozone exposure (Wu et al. 2007; Ren et al. 2004).

Two CGRP receptors have been identified based on their affinities for the competitive peptide antagonist CGRP_8–37, with CGRP₁ receptor having a higher affinity. CGRP receptors are widely expressed on blood vessels in human airways with fewer present on airway epithelium and smooth muscle (Mark and Barnes 1988). One study has demonstrated an increase in mRNA levels of CGRP receptor with increasing exposure to ozone in rabbits (Ren et al. 2004).

Functionally, neuropeptides, including CGRP, play a role in regulating airway blood flow, airway smooth muscle responses, airway inflammation (Groneberg et al. 2004), and epithelial migration and proliferation (White et al. 1993; Sanghavi et al. 1994; Mikulec and Tanelian 1996; Kim et al. 1995). CGRP induces degranulation in human neutrophils; promotes neutrophil adherence to endothelial cells (Richter et al. 1992; Zimmerman, Anderson, and Granger 1992); and modulates migration and proliferation of cells, including tracheal epithelial cells (White et al. 1993; Mikulec and Tanelian 1996; Kim et al. 1995).

Using neonatal capsaicin treatment, a nonselective method of C fiber ablation and neuropeptide (including CGRP) depletion, we have previously shown that airway C fibers modulate ozone-induced injury and repair of the distal airways. In a series of experiments, we found that rats depleted of C fibers using neonatal capsaicin treatment did not develop rapid shallow breathing, epithelial injury of terminal bronchioles was greater, and there were reductions in epithelial cell turnover and proliferative repair (Sterner-Kock et al. 1996; Vesely, Schelegle et al. 1999).

To overcome the limitations of the nonselective neuropeptide depletion of the capsaicin studies, we have determined the specific role that NK-1 and CGRP receptors play in airway epithelial injury and the proliferative phase of repair after acute ozone inhalation. A recent study demonstrated that substance P, acting via NK-1 receptors, contributes to airway epithelial injury and subsequent epithelial proliferation but does not affect neutrophil emigration after acute ozone exposure (Oslund et al. 2008).

In the present study, we extend our observations to the role of CGRP in acute ozone injury by administering a selective CGRP receptor antagonist, CGRP_8–37, to rats and exposing them to 1 ppm ozone for 8 hours. The competitive CGRP receptor antagonist, CGRP_8–37, has been used previously in rats to study the in vivo effects of CGRP in the lungs (Fox, Peterson, and McCormack 1996). Previous work in our laboratory with this model of ozone exposure in the rat has demonstrated that an 8-hour exposure to 1 ppm ozone, with an 8-hour postexposure time in filtered air, is ideal to study epithelial injury, repair, and neutrophil influx (Pino, Levin, et al. 1992; Pino, Stovall, et al. 1992). We labeled airway epithelium with ethidium homodimer (a marker of epithelial necrosis) and 5-bromo-2′-deoxyuridine (BrdU; a marker of epithelial proliferation) and sampled sites along short and long airway paths using previously described techniques (Schelegle et al. 2001). Ethidium homodimer readily crosses cells with disrupted cell membranes and is a marker of necrotic cells. BrdU, a thymidine analog, has been used reliably and extensively in our laboratory and is an excellent cumulative and quantitative marker of epithelial proliferation. In addition, we examined the effect of CGRP receptor antagonism on ozone-induced airway neutrophilic influx by quantifying the number of neutrophils present in bronchoalveolar lavage (BAL) fluid and airway tissue. Our hypothesis was that blocking CGRP receptors would inhibit epithelial proliferation after ozone exposure without affecting epithelial injury or neutrophil influx.

Materials and Methods

Animals and Exposure

Male Wistar rats (200–250 grams) were obtained from a specific pathogen-free colony (Charles River Laboratories, Kingston, NY). Rats were immediately housed in clean stainless steel chambers upon arrival to our facility and allowed to acclimate in the chambers for at least 1 week prior to the start of the exposure. All protocols described were approved by the University of California, Davis, Office of Environmental Health and Safety, which is responsible for the proper care and use of experimental animals.

Role of CGRP in Epithelial Injury and Repair

Rats were randomly divided into one of three groups (n = 8 per group): (1) ozone + CGRP_8–37, (2) ozone + saline control, or (3) filtered air (FA) + CGRP_8–37. On the morning of exposure, two miniosmotic pumps (ALZET pump Model 2001D, nominal pumping rate 8.0 μl/h; ALZA Corp., Palo Alto, CA) were utilized for this experiment. One pump contained BrdU (30 mg/ml BrdU dissolved in 0.01 N NaOH) and was primed in 0.9% saline for 16 hours at 27°C prior to implantation. The second pump contained either the CGRP receptor antagonist (Cat #: C-2806 8–37 peptide fragment, Sigma Chemicals, St. Louis, MO) or sterile saline and was primed for 3 hours at 27°C prior to implantation. The antagonist (or saline) pump was outfitted with a polyethylene tubing catheter (I.D. 0.76 mm, O.D. 1.22 mm Intramedic, Clay Adams, Franklin Lakes, NJ) that delivered the compound intravenously into the right external jugular vein. The CGRP receptor antagonist (1.0 mg/ml) was delivered to the animal at 200 nM/kg diluted in sterile saline.

Approximately 12 hours prior to the beginning of the exposure period, rats were anesthetized with a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) intraperitoneally (IP). Using aseptic surgical techniques, two incisions were made: one between the shoulder blades and one ventral to the right jugular vein, which was then isolated for cannulation. Using blunt dissection, the skin was separated from the underlying subcutaneous tissue on the dorsal aspect of the torso and also circumferentially joining the ventral incision made ventral to the right external jugular vein. First, the miniosmotic pump containing the antagonist or saline was placed subcutaneously between the shoulder blades and the cannula was fed subcutaneously around the right side of the neck and then inserted into the right external jugular vein. The cannula was advanced just proximal to the superior/inferior vena cava junction and held in place with silk sutures. Then, the BrdU minipump was also packed subcutaneously between the shoulder blades adjacent to the antagonist/saline pump. BrdU was administered subcutaneously continuously throughout the course of the experiment. Incisions were closed with 7.5 mm stainless steel wound clips (Roboz Surgical Instrument Co., Inc. Gaithersburg, MD).

Rats were allowed to recover in the exposure chambers until the beginning of exposure. One hour prior to the beginning of exposure, rats received a booster of 200 nM/kg of the antagonist delivered in 200 μl saline SC. Control rats received 200 μl saline SC. The rats were exposed to either 1 ppm ozone or filtered air for 8 hours followed by an 8-hour post-exposure in filtered air as previously described (Vesely, Hyde et al. 1999) and remained conscious throughout the exposure. Water and food were available during the entire length of the exposure.

Prior to the start of the study in a separate experiment, groups of rats were exposed to the same ozone and treatment regimens with continual monitoring of breathing pattern to determine if the antagonist altered either respiratory frequency (f) or tidal volume (VT), which could lead to an altered distribution of ozone uptake within the airway (Alfaro et al. 2004). In brief, whole-body plethysmography was used to measure respiratory frequency and estimate tidal volume as previously described (Schelegle et al. 2001).

Immediately following the ozone exposure, rats were deeply anesthetized with 4% sodium pentobarbital and the trachea was cannulated. After the chest was opened, 0.6 mM ethidium homodimer (Invitrogen Molecular Probes, Eugene, OR) was instilled in the lungs with gentle pressure until the lungs were just inflated. After a 15-minute incubation time in the dark, the remaining ethidium homodimer was aspirated out and the lungs were lavaged once with warm 0.9% NaCl in water via the tracheal cannula. A cytospin sample was stained for a cellular differential of the bronchoalveolar lavage fluid. Lungs were then fixed in situ with a buffered zinc-formalin fixative (Z-fix, Anatech, Battle Creek, MI) via intratracheal instillation at 30 cm water pressure. Lungs and duodenums were stored in the same fixative for at least 24 hours before processing.

To quantify the number of necrotic and proliferating epithelial cells, the left lung lobes were airway dissected, and whole mounts were stained for BrdU as previously described (Schelegle et al. 2001). Stained whole mounts of duodenum served as positive controls for BrdU. Blinded to the treatment group, an observer counted the number of ethidium homodimer and BrdU positive cells at nine sites along the airway tree using a stereomicroscope at 150X. Ethidium positive cells fluoresced red while BrdU positive cells were counted with bright field (DAB positive nuclei) in the same microscopic field. The positive cells were squamated and lined the airways, consistent with epithelial cells. Their epithelial morphology was subsequently confirmed with light microscopy. Five distinct airway generations were sampled: central airway (CA), short path proximal airway (SP PA), short path terminal bronchiole (SP TB), long path proximal airway (LP PA), and long path terminal bronchiole (LP TB), as shown in the schematic diagram of Figure 1. Counts were expressed as number of positive cells per average surface area of counting field (#/mm²). Surface area of airways was estimated by measuring heights, widths, and angles of all the airway sites in rats followed by trigometric calculations as described previously (Schelegle et al. 2001).

Neutrophils in Terminal Bronchioles

After cells in the left lung whole mounts were counted, the short path terminal bronchioles (SP TB) and long path terminal bronchioles (LP TB) from each rat were paraffin-embedded. Sections 7-μm thick were cut and stained with a FITC conjugate of a polyclonal rat neutrophil antibody (Accurate Chemical Inc., Buffalo, NY) as previously described (Oslund et al. 2008). The SP TB and LP TB were then viewed on an epifluorescent microscope (Leica MZ FLIII Stereo-fluorescent microscope, San Jose, CA). A semiquantitative grading scheme was used to estimate the number of neutrophils within the epithelium and surrounding interstitium. Each section was given a score quantifying the number of neutrophils. Scores were based on a 1 to 5 grading scheme, where 1 = no neutrophils, 2 = 1–20 neutrophils, 3 = 21–40 neutrophils, 4 = 41–60 neutrophils, and 5 = more than 60 neutrophils.

Active Caspase-3 Immunohistochemistry of Terminal Bronchioles

Using the paraffin-embedded blocks prepared for the neutrophil staining, additional 7-μm sections were stained with polyclonal active caspase 3 antibody (Cell Signaling, Danvers, MA) as previously described (Oslund et al. 2008). Negative control sections were incubated in PBS only and without polyclonal active caspase 3 antibody. Rat duodenum served as a positive control tissue. The slides were developed with AEC (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol and counterstained with Meyer’s hematoxylin (Sigma, St. Louis, MO).

Oxidant Stress in HBE Cells after CGRP_8–37 Treatment

An immortalized human airway epithelial cell line, HBE-1, was maintained in serum-free Dulbecco’s modified Eagle’s medium/Ham’s F12 with insulin (5 μg/ml), transferrin (5 μg/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 μM), cholera toxin (10 ng/ml), and bovine hypothalamus extract (15 μg/ml). HBE cells were plated on chamber cover-slips (Fisher Scientific Pittsburgh, PA) and grown to confluence. Prior to the start of the experiment, monolayers of cells were loaded with 1 μM of the oxidant stress sensitive dye, 5-(and 6)-chloromethyl-2,′7′-dicholorodihydrofluorescein diacetate (Invitrogen Molecular Probes, Eugene, OR) for 20 minutes at 37°C in the dark. Subsequently, the monolayers were treated with 0, 1 μg/ml, 4 μg/ml, or 7 μg/ml of CGRP_8–37 for 30 minutes prior to oxidant exposure. Hydrogen peroxide was used as the oxidant, and each concentration of CGRP_8–37 was treated with either 0, 400 μM, or 800 μM H₂O₂. The cells were imaged during oxidant stress in the presence of the inhibitor while kept at 37°C using a DeltaVision microscope. Using stratified random sampling, 10 fields at 40X were imaged for each condition. Oxidant stress positive and oxidant stress negative cells were counted using Stereology Toolbox (Davis, CA).

Statistical Analysis

For the receptor antagonist study, separate analysis of variance (ANOVA) tests were performed for the ethidium homodimer and BrdU data at each airway generation (CA, SP PA, SP TB, LP PA, LP TB) followed by Fisher’s least-significant-difference test to compare individual groups (Systat^® 10.0, SPSS Inc., Chicago, IL). Before determining if there were significant differences in the numbers of neutrophils in lavage between the groups, an ANOVA followed by a Bonferroni comparison test was performed between the ozone exposed groups. Because there were no significant differences in these tests, the number of neutrophils in lavage was compared between the ozone exposed groups and filtered air exposed group using Student’s T-test (Systat^® 10.0, SPSS Inc., Chicago, IL). Kruskal-Wallis one-way ANOVA was performed to determine if there were significant differences in neutrophil scores between rats exposed to ozone and treated with CGRP_8–37 or saline. For the oxidant stress experiment, an overall ANOVA test was followed by a Fisher’s least-significant-difference test to compare individual groups. Results were considered significant if p < .05.

Results

Breathing Pattern Response to the CGRP Receptor Antagonist

Ozone induced a significant decrease in tidal volume and increase in breathing frequency compared to preexposure baseline and filtered air controls (p < .05). There were no significant differences in tidal volume (Figure 2A), breathing frequency (Figure 2B), or minute ventilation (data not shown) between groups exposed to ozone. Although CGRP_8–37-treated rats had a trend toward an elevated breathing frequency, this was considered biologically irrelevant. As a result of these findings, we exposed the majority of the rats studied in large exposure chambers capable of housing several rats at one time without further monitoring of the breathing pattern.

Diminished Necrosis and Epithelial Proliferation in Rats Treated with the CGRP-Receptor Antagonist

As shown in the results of Figure 1, in the terminal bronchioles of rats exposed to ozone, there were significantly fewer necrotic (ethidium positive) cells in the terminal bronchioles of rats that were also treated with the CGRP receptor antagonist (p = .028).

As shown in the results of Figure 1, epithelial proliferation was significantly less in ozone-exposed rats treated with CGRP_8–37 compared to rats treated with the saline vehicle in the proximal airway (SP PA + LP PA; p = .00015) and terminal bronchioles (SP TB + LP TB; p < .0001).

CGRP Receptor Antagonist Does Not Significantly Alter Neutrophil Emigration

To determine if CGRP influenced neutrophil migration into the airways after ozone exposure, the percentage of neutrophils in bronchoalveolar lavage and the numbers of neutrophils in tissue sections were compared between the groups. The only significant difference in neutrophil numbers in BAL fluid was between ozone exposed rats and filter air exposed rats. There was no significant difference in the number of neutrophils between the rats treated with CGRP_8–37 or saline and exposed to ozone either in the BAL fluid or in the terminal bronchioles of the tissue sections (Figure 3). We conclude that CGRP_8–37 did not significantly affect neutrophil emigration into airways after ozone exposure.

Ozone Does Not Induce Epithelial Cell Apoptosis in Terminal Bronchioles

To determine if a decrease in epithelial cell necrosis was associated with a change in apoptotic epithelial cells in CGRP_8–37-treated rats, terminal bronchioles were stained with active caspase 3, a marker of apoptotic cells. Only extremely rare positive airway epithelial cells were found in rats exposed to ozone and treated with CGRP receptor antagonist. These cells were interpreted as incidental and there was no significant difference in the number of apoptotic cells between the treatment groups. These results indicate that ozone induces a non-caspase-mediated form of cell death. Additionally, the decrease in epithelial cell necrosis was not associated with a change in the mode of cell death to a caspase-mediated form of apoptosis in CGRP_8–37-treated rats.

CGRP Receptor Antagonist, CGRP_8–37, Does Not Function as an Antioxidant

One possible explanation for the diminished epithelial injury when rats were treated with CGRP_8–37 is that the antagonist is an antioxidant and effectively protects the airway epithelium from ozone exposure. We tested this hypothesis by exposing an immortalized normal human airway epithelial cell line (HBE-1) to 0, 400 μM, or 800 μM H₂O₂ in the presence of either 0, 1 μg/ml, 4 μg/ml, or 7 μg/ml CGRP_8–37 and the oxidant stress sensitive dye, 5-(and 6)-chloromethyl-2,′7′-dicholorodi-hydrofluorescein diacetate. The fraction of oxidant stress positive cells to the total cell number was determined for each condition. As shown in Figure 4, no significant correlation was found between the oxidant stress positive cells and the dose of CGRP_8–37. We conclude that CGRP_8–37 did not diminish epithelial injury and subsequent proliferation by reducing the oxidant injury after ozone exposure.

Discussion

In this article, we examine the role of CGRP receptors in airway epithelial injury and proliferation during repair and whether they influence neutrophil emigration into airways after acute ozone inhalation. First, we demonstrate that the CGRP receptor antagonist, CGRP_8–37, did not significantly affect the reflex rapid shallow breathing induced by acute ozone exposure in Wistar rats. Second, we demonstrate that treatment with CGRP_8–37 significantly attenuated epithelial injury in terminal bronchioles but not proximal airways, providing further evidence for an airway-generation-specific response to ozone-induced cell death in terminal bronchioles similar to our previous findings with blockade of NK-1 receptors (Oslund et al. 2008). Supplementing this observation, we show that CGRP_8–37 does not attenuate cell injury by functioning as an antioxidant. Third, we show that treatment with CGRP_8–37 significantly attenuated epithelial cell proliferation after acute ozone exposure in both proximal and distal airways. Fourth, CGRP_8–37 did not influence the emigration of neutrophils into airways after ozone injury. Taken together, these data indicate that CGRP is an important mediator in regional airway epithelial cell death and proliferation after acute ozone exposure with no impact on neutrophil emigration.

Our laboratory recently reported that substance P, acting via NK-1 receptors, is also an important mediator in airway epithelial cell death and subsequent proliferation after ozone exposure (Oslund et al. 2008). In our prior report and this one, we have observed airway regional differences in the amount of epithelial cell death and proliferation after ozone exposure. Consistently, the highest density of ozone-induced epithelial injury and proliferation is in terminal bronchioles, a finding supported by prior studies of acute ozone exposure (Castleman, Tyler, and Dungworth 1977; Schelegle et al. 2003). Schelegle and coworkers have shown that the distribution of epithelial injury after ozone exposure is in part dependent on the development of rapid shallow breathing, which tends to spare the large conducting airways but induce a more evenly distributed injury in short path and long path terminal bronchioles (Schelegle et al. 2001). The CGRP receptor antagonist, CGRP_8–37, did not affect ozone-induced rapid shallow breathing. Therefore, the observed attenuation in epithelial injury and proliferation are due to the direct effect of CGRP_8–37 and not secondary to alterations in ozone distribution. These results, combined with our previous similar findings that substance P affects airway epithelial injury and proliferation most significantly at the terminal bronchioles, strongly implicate neuropeptides as important mediators in the injury and reparative phases of acute ozone exposure particularly in the terminal bronchioles.

Our observation that CGRP_8–37 attenuated epithelial necrosis in the terminal bronchioles is a novel finding, but the underlying mechanism is uncertain. We investigated the possibility that CGRP_8–37 could have direct antioxidant effects thus protecting the epithelium from ozone-induced injury. We found no evidence of antioxidant effects when HBE-1 cells were treated with 400 μM and 800 μM H₂O₂ as an oxidant in the presence of a range of antagonist levels including that used in the rats in this study. Nevertheless, it is theoretically possible that the CGRP_8–37 antagonist could be metabolized in vivo to an active antioxidant.

The potential protective effect of CGRP_8–37 may be related to its ability to block CGRP-induced vasodilation and increased microvascular permeability (Ay and Tuncer 2004; Grant, Gerard, and Brain 2002; Amann et al. 1995). Acute ozone inhalation in humans elevates several macromolecules, including complement 3a, in bronchoalveolar lavage fluid, which are normally restricted to the vascular compartment and that may contribute to cellular injury and necrosis (Koren et al. 1989). Blocking increased microvascular permeability could limit the influx and up-regulation of many mediators to the site of ozone-induced oxidant stress reducing the subsequent epithelial cell necrosis.

Acute ozone exposure induced epithelial cell death but did not induce caspase-mediated apoptosis as assessed by active caspase 3 immunohistochemistry. Therefore, the decrease in epithelial necrosis in CGRP_8–37-treated rats was not associated with a change in the mode of cell death to caspase-mediated apoptosis.

The mode of cell death that is induced by acute ozone exposure warrants further study. We have previously shown that ethidium homodimer positive cells colocalize with expression of the orphan nuclear receptor Nur77 after ozone exposure (Oslund et al. 2008). Nur77 has been associated with non-caspase-mediated cell death in other experimental models of acute pulmonary injury (Thompson and Burcham 2008; Dolinay et al. 2006; Shin et al. 2004). The neuropeptide substance P induces phosphorylation of Nur77 via arrestin 2 in a neuronal cell line after activation of the NK-1 receptor (Castro-Obregon et al. 2004). While it is unknown if the CGRP₁ receptor induces a similar non-caspase-mediated form of programmed cell death, there is evidence that CGRP up-regulates the expression of NK-1 receptors in the lung and could thus indirectly be associated with NK-1 receptor–mediated airway epithelial cell injury (Wu et al. 2007).

In addition to attenuating epithelial cell necrosis, CGRP_8–37 reduced epithelial cell proliferation in all the airways studied, but significantly in the proximal airways and terminal bronchioles. It is likely that the inhibition of epithelial proliferation in the terminal bronchioles was, at least in part, a direct consequence of the attenuated epithelial injury. CGRP is, however, established as an important mediator in cellular proliferation and migration, critical events for epithelial repair. CGRP stimulates proliferation and migration of primary guinea pig tracheal epithelial cells and re-epithelialization of the cornea; and acting indirectly, CGRP has been shown to be important in intestinal epithelial repair (White et al. 1993; Sanghavi et al. 1994; Mikulec and Tanelian 1996; Bulut et al. 2008). Vesely, Schelegle and colleagues (1999) found less BrdU incorporation in terminal bronchiolar epithelium of capsaicin-treated rats exposed to filtered air and ozone, suggesting neuropeptides released by C fibers may modulate basal and reparative airway epithelial cell proliferation. The present results and those of Oslund and coworkers (2008) support these findings, showing that two C-fiber-derived neuropeptides, substance P and CGRP, are important in epithelial proliferation after acute ozone exposure.

Our results indicate that CGRP_8–37 did not significantly affect neutrophil emigration into airways after ozone exposure. These results are consistent with our previous observation that rats treated neonatally with capsaicin, causing a decrease in C-fiber-produced CGRP, had no differences in the number of neutrophils in lavage fluid after ozone exposure (Sterner-Kock et al. 1996). Taken together, these results suggest that CGRP is not a critical mediator for neutrophil emigration into airways following ozone injury in the rat.

Within the literature there is a discrepancy as to the importance of CGRP in neutrophil function and recruitment in different experimental models. While CGRP can activate human neutrophils, other studies are conflicting as to its importance in neutrophil recruitment (Richter et al. 1992). A study of IL-1β-induced inflammation in a mouse air pouch model concluded that exogenous and endogenous CGRP potentiates neutrophil recruitment, while other studies have concluded that exogenously administered CGRP reduced neutrophil accumulation in experimental skin flaps of rats (Ahluwalia and Perretti 1994; Gherardini et al. 1998; Jansen et al. 1999).

In conclusion, these observations illustrate the pivotal role that the activation of C fibers, with the subsequent release of CGRP, plays in the complex cascade of events that is initiated by the acute inhalation of ozone. This role is not limited to reflex responses, such as rapid shallow breathing, but includes the modulation of cellular injury and subsequent proliferation of epithelial cells during repair. Further studies need to be conducted to examine the direct and/or indirect mechanisms that contribute to this modulation of cellular injury and repair by the CGRP₁ receptor.