Diesel particulate matter (DPM) has adverse health effects. Examining the underlying pathophysiological mechanisms would be facilitated by the introduction of an exposure method that is safe, portable, and cost-effective. The purpose of this study was to establish a novel method to study DPM exposure via nebulization and an inhalation dose that was safe, yet capable of eliciting an inflammatory response. Ten participants enrolled in this nonblinded, nonrandomized study. Subjects inhaled nebulized 0.9% saline and increasing doses of DPM suspended in 0.9% saline (75, 150, and 300
In a variety of epidemiological and experimental studies, researchers have demonstrated that respiratory exposure to airborne pollutants is associated with systemic sequelae that can not only have effects on the respiratory tract itself but also create fertile conditions for the development of atherosclerosis, plaque destabilization, atherothrombosis, and consequent cardiovascular events [
There are a few existing models for human experimental exposures to DPMs. The most common protocol is to direct exhaust from a diesel engine into an exposure chamber. Although very effective, the disadvantage of this system is the elaborate, fixed setup, which is expensive to build and operate. Nasal instillation of DPM is a less expensive method which induces local inflammation in the nasal lavage fluid [
Nebulization of particles has not been described in the literature for use in human subjects, although it is frequently used in animal studies. If established as safe and effective, inhalation of nebulized DPM would be an inexpensive and simple method to facilitate health research on the effects of particulates in the lungs and systemically. In this safety trial, we examined whether DPM exposure via nebulization is safe for experimental study, aiming to identify the lowest effective dose of DPMs capable of reliably eliciting inflammatory responses in the airways and blood.
Twelve healthy subjects were enrolled in the study (female: 10; male: 2). Prior to study entry subjects were examined by a physician to confirm their overall good health. This included a physical exam and blood work, assessing C-reactive protein (CRP) levels, kidney and liver function, and (for female subjects) a pregnancy test. Subjects were excluded from the study if their baseline CRP levels were above 3 mg/L, they were pregnant, or for any other health consideration identified by the examining physician. Subjects were nonsmokers, were not using any medication other than birth control, and had not experienced respiratory infection within the previous six weeks. Two subjects (1 male; 1 female) were excluded during screening due to high levels of serum CRP. All subjects were instructed to refrain from taking over-the-counter nonsteroidal anti-inflammatories for two weeks prior to and during the course of the study. See Table
Subject characteristics.
Subject | FEV1 | FVC | Gender | Age |
---|---|---|---|---|
1 | 3.1 | 3.8 | F | 35 |
2 | 3.9 | 5.6 | M | 38 |
3 | 3.2 | 4.1 | F | 32 |
4 | 3.2 | 3.8 | F | 34 |
5 | 3.7 | 4.5 | F | 38 |
6 | 3.2 | 3.5 | F | 19 |
7 | 3.4 | 4.1 | F | 19 |
8 | 3.7 | 4.1 | F | 21 |
9 | 2.4 | 3.2 | F | 38 |
10 | 3.9 | 4.5 | F | 23 |
As it was primarily a safety trial, this study was a nonrandom, nonblinded design consisting of four arms. Subjects were required to come into the laboratory on thirteen separate sessions. Visit one was the screening visit, with the following twelve sessions comprising the four arms of the study. For each arm, subjects visited the laboratory for three consecutive days. Each arm was separated by a minimum of 7 days and all visits occurred in the morning within 1 hour of each other to minimize circadian variations. A physician was onsite during all study visits and examined patient results prior to continuing with the next inhalation challenge.
For each arm of the study, on study day 1 (baseline), subjects were asked if they had any medical issues since the last visit and it was confirmed that they had not taken any medications prior to their sessions. Baseline spirometric measurements (FEV1, FVC), pulse rate, and pulse oximetry were taken followed by sputum induction and blood sampling. On day 2, baseline spirometry was repeated, followed by the inhalation of the nebulized saline or DPM. After inhalation challenge, pulse rate, pulse oximetry, spirometric measurements, and a symptoms questionnaire were measured for 2 h, at which time blood sampling and sputum induction were performed. On day 3, all the measurements taken on day 1 were repeated.
This study protocol was approved by the Laurentian University Ethics Board, and all subjects provided written, informed consent prior to participation in the study.
Standardized DPM (SRM2975) was obtained from the National Institute of Standards and Technology (Gaithersberg, MD, USA). The standardized DPM was collected from the exhaust of a diesel forklift and hot bag filter system, as described in the certificate of analysis for this material [
The AeroEclipse II Breath Actuated Nebulizer (Monaghan Medical Corporation, Plattsburgh, NY, USA) was used for inhalation challenge. 3 mL of either 0.9% saline or a mixture of 0.9% saline with DPM doses of 75
Oximetry and pulse rate were measured using a SuperSpiro Spirometer that was equipped with a Nonin SpO2 probe (Micro Medical Ltd., Kent, UK). Oximetry and pulse rate were measured concurrently on day 1 and at 2 min, 30 min, 2 hours, and 24 hours after inhalation.
Spirometry was performed with a SuperSpiro Spirometer V1.05 (Micro Medical Ltd., Kent, UK) according to the American Thoracic Society standards [
An eight-question symptom score questionnaire was administered at 2 h after inhalation, querying the subjects’ experience of: headache, nausea, dizziness, difficulty concentrating, fatigue, weakness, heart rate, and dyspnea.
Sputum was induced by inhalation of a hypertonic saline mist and processed according to Pin et al. [
Venous blood samples were obtained at baseline and 2 and 24 hours after inhalation challenge. Complete blood counts (CBC), erythrocyte sedimentation rate (ESR), international normalized ratio (INR), and C-reactive protein (CRP) measurements were performed by LifeLabs Medical Laboratory Services. Blood serum was separated by centrifugation and stored at −80°C. IL-6, GM-CSF, and IL-8 protein levels were quantified using commercially available ELISAs (eBioscience, USA, and BD OptEIA, Canada, resp.). The limits of detection for IL-6 and GM-CSF were approximately 2 pg/mL. The limit of detection for IL-8 was approximately 3.1 pg/mL. Values below the limit of detection were assumed to be 0 pg/mL for statistical analysis.
Summary statistics were expressed as mean ± SEM. Data were analyzed using repeated measures (rm) ANOVA (between group analysis: saline versus DPM75 versus DPM150 versus DPM300; within group analysis: before versus 2 hr after versus 24 hr after inhalation). Statistical significance was accepted as
Baseline FEV1 values were similar on all study days. During the two hours after inhalation challenge, there was a significant decrease in the mean maximal fall in FEV1 from baseline after all challenges (
Change in forced expiratory volume per second (FEV1) following inhalation of saline (a), 75
Saline
75
150
300
Overlay
Total cell counts following inhalation of saline, DPM75, DPM150, and DPM300 (×106 cells/g).
Baseline | 2 h | 24 h | |
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Saline |
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DPM 75 |
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DPM 150 |
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DPM 300 |
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Values given as mean ± SEM. No significant differences between time or groups was found.
Following inhalation challenge, there were no significant differences in the total number of or in the percent change in neutrophils, macrophages, or eosinophils at 2 or 24 hours compared to baseline for all of the challenges (data not shown). There were no significant differences in the number or percentage of these cells between groups.
The majority of sputum macrophages (80–90%) had no visible particle inclusions in all groups at all time points (data not shown). The majority of sputum macrophages with visible particle inclusions fell into the “low-positive” category (i.e., fewer than 20 inclusions) with the proportion of “high-positive” (i.e., more than 20 inclusions) sputum macrophages under 0.5% of total sputum macrophages (data not shown). Thus, for the purposes of analysis, the “low-positive” and “high-positive” macrophages were grouped together (Figure
Percent of sputum macrophages with particle inclusions at baseline (BL), 2 h, and 24 h after exposure to saline or varying doses of DPM. Data are shown as mean
At baseline, approximately 10–13% of sputum macrophages had particle inclusions (Figure
Following inhalation challenge, there were no significant differences in the total number of neutrophils, monocytes, eosinophils, or platelets at 2 or 24 hours compared to baseline for all of the challenges and no significant differences between groups (data not shown).
Baseline values for erythrocyte sedimentation rate, prothrombin time, and C-reactive protein were similar on all study days. Following inhalation challenge, there were no significant changes in either measure at 2 or 24 hours compared to baseline for all of the challenges and no significant differences between groups (data not shown).
Serum granulocyte macrophage colony-stimulating factor (GM-CSF) levels measured by ELISA at baseline (BL), 2 h, and 24 h after exposure to saline or varying doses of DPM. Data are shown as mean ± SEM. *Significant difference from baseline and †significant difference from saline at a given timepoint, as determined by repeated measures ANOVA.
Baseline values were similar on all study days. Following inhalation challenge, there were no significant changes in blood oxygen saturation or pulse rate at 2 min, 30 min, 2 h, or 24 h compared to baseline for all of the challenges (data not shown).
None of the subjects reported any serious adverse symptoms during the saline inhalation challenge. Following inhalation challenge of DPM75, 3 subjects answered positively to the symptoms questionnaire: 1 subject said that they felt both mild nausea and had a mild headache, 1 subject said they felt mild dizziness, and 1 subject said they experienced mild weakness. At DPM150 1 subject answered positively to the symptoms questionnaire, saying they experienced mild fatigue. At DPM300, 4 subjects answered positively to the symptoms questionnaire: 1 experienced mild headache and mild fatigue; 1 experienced mild headache, mild weakness, and mild difficulty breathing; 1 experienced mild fatigue and mild difficulty concentrating; and 1 respondent experienced mild fatigue. None of these symptoms for any of the 4 arms were statistically significant. One symptom that was not on the questionnaire that was spontaneously reported by subjects was a scratchy throat: 6 out of 10 subjects reported a mild scratchy throat at DPM300.
Our findings establish that inhalation of nebulized DPM mixed in saline via a nebulizer is a safe and effective method for research on the local and systemic effects of particulate matter in healthy human subjects. Inhalation of DPM using this method induced declines in FEV1 and some mild symptoms such as nausea, dizziness, fatigue, headache, and a scratchy throat. We also saw evidence that the DPM reached the lower airways through an increase in the positive macrophage particle inclusions and evidence of systemic effects with an increase in GM-CSF in the blood serum. To our knowledge, this is the first published study of the isolated effects of DPM using nebulization as a delivery method. As a pilot study, with safety being the paramount concern, we used a nonrandom, nonblinded design, so that we could establish the tolerability of each dose in each subject before escalating the dose further.
We observed small but significant decreases in FEV1 following all inhalation challenges, which were transient and returned to baseline by the 24 h time point. We were somewhat surprised to see a small decrement in FEV1 after inhalation of saline, as we would expect that in healthy individuals, we would not see any change in FEV1 after isotonic, hypotonic, or hypertonic saline inhalations [
Our findings indicate that DPM exposure has acute effects on lung function of healthy individuals, in contrast with what has been reported by others [
The doses used in this study did not elicit a lung inflammatory response as measured in induced sputum, in contrast with other human exposure studies [
The lack of inflammation we observed cannot be attributed to a failure of the nebulized DPM to reach the lower airways, as we measured a significant increase in particle-containing macrophages in the sputum after DPM exposure. This not only demonstrates that the particles reached the alveoli but is also significant because alveolar macrophages phagocytosing particulate matter can release cellular mediators, which can stimulate the bone marrow, indirectly signaling an inflammatory response [
Not surprisingly in the absence of notable local inflammation, inhalation of nebulized DPM did not induce discernable systemic cellular inflammation after inhalation of DPM. However, we did see a significant increase in serum levels of GM-CSF at 2 h following inhalation of DPM150, and serum GM-CSF levels were significantly elevated 24 h after exposure to DPM150 and DPM300 (Figure
This increase in serum GM-CSF, however, was not accompanied by an increase in sputum supernatant GM-CSF or in granulocyte levels in the sputum or blood. Increased levels of GM-CSF generally have not been detected in the supernatant following sputum induction [
The increased level of GM-CSF present at baseline in the DPM300 might suggest that the increase in serum GM-CSF persisted for at least one week after inhalation and that our wash-out period needed to be longer. However, given that there were no other indicators of a persistent inflammatory response, we suspect that the more likely explanation is that some of our subjects were exposed to an unidentified stimulus outside the laboratory that increased GM-CSF levels.
The presence of GM-CSF is of interest to us because GM-CSF is a cytokine that stimulates bone marrow hematopoietic stem cells to produce increased numbers of neutrophils, eosinophils, basophils, and monocytes, in addition to promoting dendritic cell maturation and antigen presentation. Thus, GM-CSF-rich airway environments have been hypothesized to promote the development of an immune response by creating conditions conducive to
Finally, this study showed that DPM may act as an irritant, as some of our subjects complained of mild symptoms immediately after exposure that were resolved by the 2 h time point. Rudell et al. [
Overall, this novel method of inhaling nebulized DPM mixed in saline proved to be a safe and effective way to examine the effects of DPM and our protocol provides a framework for future research. Inhalation of nebulized DPM delivered the particles to the lower airways and elicited transient decrements in FEV1. However, even at the highest dose of DPM administered in this study, we did not observe cellular inflammatory responses in the airways and blood and measured only very small changes in proinflammatory cytokine levels. For studies aimed at examining inflammatory responses, higher doses may be required but should be tested carefully for safety, particularly in subjects who may have airway hyperreactivity. Understanding the mechanisms through which airborne particulates influence inflammation and lung function will help us to understand the correlation between particulate exposure and increases in cardiopulmonary morbidity and mortality that have been demonstrated epidemiologically. It may also help us identify the threshold dose that is able to elicit an inflammatory response in healthy and susceptible populations, which could be a critical consideration in defining emission standards and workplace policies.
The authors declare that there is no conflict of interests regarding the publication of this paper.