American Journal of Respiratory and Critical Care Medicine

To the Editor:

Diesel exhaust (DE) is an important component of traffic-related air pollution (1). A landmark field study (2) showed significant acute DE–associated FEV1 reductions in subjects with asthma. Controlled human exposures complement such observations regarding the potential effect of DE in asthma. In healthy volunteers, DE causes increased airway inflammation (3). In individuals with asthma it increases airway responsiveness (4), but not airway inflammation (4, 5). One proposed mechanism for its toxicity is the increase in neurogenic inflammation (NI) (6). NI refers to the release of neuropeptides, like substance P (SP) and calcitonin gene-related peptide (CGRP), at sensory nerves endings that, independent of airway inflammation, can have various physiological effects (7) including bronchoconstriction and increased airway responsiveness.

In this study we aimed to measure changes in NI-associated neuropeptides in the upper airway of subjects with asthma exposed to DE. We hypothesized that exposure to DE would increase neuropeptide concentration. Specifically, we aimed to verify if, in response to DE in a controlled exposure setting, (1) the concentrations of SP and CGRP increase in the airways of subjects with asthma, and (2) exposure-related changes of these two neuropeptides are correlated with airway responsiveness.

Eighteen subjects with asthma, recruited at the Air Pollution Exposure Laboratory in Vancouver, were exposed to filtered air (FA) and DE (300 μg/m3 PM2.5) for 2 hours on two separate occasions, at least 2 weeks apart using a randomized and counterbalanced crossover design. Eighteen hours before and 30 hours after exposure onset, nasal lavage (NL) was collected and airway responsiveness to methacholine was quantified by calculating the dose–response slope (DRS). This metric, as opposed to the more common provocative concentration of methacholine inducing a 20% fall in FEV1 (PC20), allows linear regression analyses.

Concentrations of SP and CGRP were measured in duplicate in the NL fluid supernatant using specific ELISA kits (for SP: Cayman Chemical, Ann Arbor, MI; and for CGRP: Phoenix Pharmaceuticals, Burlingame, CA). For both assays, plate loading and incubation were performed at 4°C to ensure stability of the neuropeptides. IL-8 and tumor necrosis factor α (TNF-α) were similarly measured as markers of inflammation (Quantikine ELISA Kit; R&D Systems, Minneapolis, MN). Total protein concentration was measured with a modified Lowry assay (Thermo Scientific, Waltham, MA).

Comparisons between exposures were performed using repeated-measures analysis of variance and correlation between neuropeptide concentration and airway responsiveness were performed using least-square estimations.

Baseline characteristics of study participants are presented in Table 1. The main results are shown in Figure 1. There were no significant differences in IL-8, TNFα, and total protein concentration between FA and DE.


 Mean (SD)Range
Age, yr28.6 (7.0)19 to 46
Sex, % male47
BMI, kg/m224.8 (3.6)19.5 to 34.7
Atopic status, % positive skin tests72.2
Proportion of subjects with physician-diagnosed rhinitis, %11.1
Proportion of subjects using inhaled corticosteroids, %11.1
Proportion of subjects using long acting β-agonists, %16.7
Proportion of subjects using short-acting beta agonists, %44.4
Proportion of subjects using antihistamines, %0
Baseline PC20, mg/ml2.53 (1.94)0.23 to >16
Proportion of subjects with baseline airway hyperresponsiveness*, %83.3
Baseline FEV1, L3.68 (0.95)2.00 to 5.04
Baseline FEV1, % of predicted value93 (14)66 to 120

*Airway hyperresponsiveness was defined as a provocative concentration causing a 20% fall in FEV1 < 8 mg/ml.

Baseline SP concentrations were similar under both exposure conditions. Postexposure SP concentrations were higher in the DE condition compared with the FA condition (P = 0.004). The pre- to postexposure change in SP (ΔSP) was significantly greater for DE compared with FA (mean absolute ΔSP 1.6 ± 2.1 pg/ml and 0.17 pg/ml ± 0.67 respectively; P = 0.02).

Baseline and postexposure CGRP concentrations were not statistically different between DE and FA. The baseline to post-exposure change in CGRP (ΔCGRP) was significantly greater for DE compared with FA (mean absolute ΔCGRP 0.31 ± 0.51 ng/ml and 0.06 ± 0.34 ng/ml respectively; P = 0.04).

A repeated measures analysis of covariance did not show significant interaction of atopy, physician-diagnosed rhinitis, baseline airway hyperresponsiveness (PC20 < 8 mg/ml) and inhaled corticosteroid on neuropeptides concentration increases. There were no significant differences in neuropeptide levels between atopic and nonatopic subjects.

On a group level, airway responsiveness was not significantly higher after DE compared with FA (mean DRS 1.31 ± 0.84 and 1.04 ± 0.71 for DE and FA respectively, P = 0.21). However, after DE exposure, the individual changes in methacholine DRS (ΔDRS) correlated significantly with ΔSP (simple linear regression, R2 = 0.48, P = 0.002) but not ΔCGRP (R2 = 0.21, P = 0.07). In multiple regression analysis, ΔSP was significantly associated with ΔDRS (P = 0.01) when controlling for CGRP.

These findings suggest that exposure to high concentrations of DE augments local levels of upper airway NI-related neuropeptides. These phenomena, in relation to air pollution, have been proposed in rodent models (8), but never formally in humans. There are two possible mechanisms by which DE can lead to NI. It can either directly trigger airway sensory nerves (9) or indirectly decrease SP degradation by inhibiting neprilysin (6, 10), the primary protease implicated in SP metabolism. CGRP, on the other hand, is released by the same sensory triggers as SP, but its metabolic pathway is different (7, 11). In our experiment, the fact that we observe increases in both neuropeptides suggests sensory nerve activation as the main mechanism. If decreased neuropeptide metabolism by neprilysin was a major contributor, we would expect increases in SP alone, not both SP and CGRP.

The significant correlation between ΔSP and ΔDRS in this crossover study shows that those subjects with the highest increase in DE-related airway responsiveness were also those with the highest increase in SP. Thus, increases in SP concentration in the airways could contribute to the inter-individual variability in the airway response to DE. Moreover, in accordance with the literature (4, 5), we did not observe increases in IL-8 and TNF-α, two proinflammatory cytokines. Although not directly addressed in this study, SP could be the factor through which DE leads to increased airway responsiveness without inducing airway inflammation in individuals with asthma.

One limitation is that it was not possible to determine whether these observations are specific to asthma because of the absence of healthy controls. However, we believe that these findings in a population with asthma are a valuable contribution to the literature and that they should motivate future studies comparing subjects with asthma versus subjects without asthma.

Our findings suggest a role for SP in the pathophysiology of DE-related effects on asthmatic airways. This mechanistic explanation is of clinical interest; SP might contribute to traffic-related air pollution’s effect on bronchoconstriction. To our knowledge, this is the first study showing the potential link between DE, SP, and airway responsiveness in humans. This is a novel and sparsely explored mechanism by which DE may affect asthmatic airways in humans, through the release neuropeptides such as SP.

1. Fujita EM. Concentrations of air toxics in motor vehicle-dominated environments. Boston, MA: Health Effects Institute; 2011.
2. McCreanor J, Cullinan P, Nieuwenhuijsen MJ, Stewart-Evans J, Malliarou E, Jarup L, Harrington R, Svartengren M, Han IK, Ohman-Strickland P, et al. Respiratory effects of exposure to diesel traffic in persons with asthma. N Engl J Med 2007;357:23482358.
3. Behndig AF, Mudway IS, Brown JL, Stenfors N, Helleday R, Duggan ST, Wilson SJ, Boman C, Cassee FR, Frew AJ, et al. Airway antioxidant and inflammatory responses to diesel exhaust exposure in healthy humans. Eur Respir J 2006;27:359365.
4. Nordenhäll C, Pourazar J, Ledin MC, Levin JO, Sandström T, Adelroth E. Diesel exhaust enhances airway responsiveness in asthmatic subjects. Eur Respir J 2001;17:909915.
5. Behndig AF, Larsson N, Brown JL, Stenfors N, Helleday R, Duggan ST, Dove RE, Wilson SJ, Sandstrom T, Kelly FJ, et al. Proinflammatory doses of diesel exhaust in healthy subjects fail to elicit equivalent or augmented airway inflammation in subjects with asthma. Thorax 2011;66:1219.
6. Wong SS, Sun NN, Keith I, Kweon CB, Foster DE, Schauer JJ, Witten ML. Tachykinin substance P signaling involved in diesel exhaust-induced bronchopulmonary neurogenic inflammation in rats. Arch Toxicol 2003;77:638650.
7. Groneberg DA, Quarcoo D, Frossard N, Fischer A. Neurogenic mechanisms in bronchial inflammatory diseases. Allergy 2004;59:11391152.
8. Costa SK, Kumagai Y, Brain SD, Teixeira SA, Varriano AA, Barreto MA, de Lima WT, Antunes E, Muscará MN, Costa SK. Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ). Arch Toxicol 2010;84:109117.
9. Deering-Rice CE, Romero EG, Shapiro D, Hughen RW, Light AR, Yost GS, Veranth JM, Reilly CA. Electrophilic components of diesel exhaust particles (DEP) activate transient receptor potential ankyrin-1 (TRPA1): a probable mechanism of acute pulmonary toxicity for DEP. Chem Res Toxicol 2011;24:950959.
10. Wick MJ, Buesing EJ, Wehling CA, Loomis ZL, Cool CD, Zamora MR, Miller YE, Colgan SP, Hersh LB, Voelkel NF, et al. Decreased neprilysin and pulmonary vascular remodeling in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2011;183:330340.
11. Martling CR, Saria A, Fischer JA, Hökfelt T, Lundberg JM. Calcitonin gene-related peptide and the lung: neuronal coexistence with substance P, release by capsaicin and vasodilatory effect. Regul Pept 1988;20:125139.

Supported by the AllerGen Network Centre of Excellence, the Michael Smith Foundation for Health Research, and WorkSafeBC (RS2011-OG07). F.S.'s salary is provided by a Michael Smith Foundation for Health Research/AllerGen Research Trainee Award.

Author Contributions: C.R.C. is the senior investigator of the laboratory; he participated at each step of this study, including design, data collection and analysis, and manuscript writing. M.J.M. participated in data collection and analysis, and manuscript writing. F.S. is the primary investigator; he participated in data collection and analysis, and manuscript writing.

Author disclosures are available with the text of this letter at


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