Capsule Summary

Deficits in inflammasomes, a key element of innate immunity, confer increased susceptibility to infection. We report that sputum cells from asthmatics have decreased expression of inflammasome factors, consistent with reports of increased infection risk in asthmatics.

Rationale: Exposure to ozone causes a decrease in spirometric lung function and an increase in airway inflammation in healthy young adults at concentrations as low as 0.08 ppm, close to the National Ambient Air Quality Standard for ground level ozone.

Objectives: To test whether airway effects occur below the current ozone standard and if they are more pronounced in potentially susceptible individuals, such as those deficient in the antioxidant gene glutathione S-transferase mu 1 (GSTM1).

Methods: Pulmonary function and subjective symptoms were measured in 59 healthy young adults (19–35 yr) immediately before and after exposure to 0.0 (clean air, CA) and 0.06 ppm ozone for 6.6 hours in a chamber while undergoing intermittent moderate exercise. The polymorphonuclear neutrophil (PMN) influx was measured in 24 subjects 16 to 18 hours postexposure.

Measurements and Main Results: Subjects experienced a significantly greater (P = 0.008) change in FEV1 (± SE) immediately after exposure to 0.06 ppm ozone compared with CA (−1.71 ± 0.50% vs. −0.002 ± 0.46%). The decrement in FVC was also greater (P = 0.02) after ozone versus CA (−2.32 ± 0.41% vs. −1.13 ± 0.34%). Similarly, changes in %PMN were greater after ozone (54.0 ± 4.6%) than CA (38.3 ± 3.7%) exposure (P < 0.001). Symptom scores were not different between ozone versus CA. There were no significant differences in changes in FEV1, FVC, and %PMN between subjects with GSTM1-positive and GSTM1-null genotypes.

Conclusions: Exposure of healthy young adults to 0.06 ppm ozone for 6.6 hours causes a significant decrement of FEV1 and an increase in neutrophilic inflammation in the airways. GSTM1 genotype alone appears to have no significant role in modifying the effects.

Context

Ozone exposure triggers airway inflammatory responses that may be influenced by biologically active purine metabolites.

Objective

Examine the relationships between airway purine metabolites and established inflammatory markers of ozone exposure, and determine if these relationships are altered in individuals with atopy or asthma.

Materials and Methods

Mass spectrometry was utilized to measure concentrations of purine metabolites (AMP, adenosine, hypoxanthine, uric acid) and non-purine metabolites (taurine, urea, phenylalanine, tyrosine) in induced sputum obtained from 31 subjects with normal lung function (13 healthy controls, 8 atopic non-asthmatics, and 10 atopic asthmatic) before and four hours after ozone exposure.

Results

At baseline, the purines AMP and hypoxanthine correlated with multiple inflammatory markers including neutrophil counts and the cytokines IL-6, IL-8, TNF-α, and IL-1β (r ranged from 0.41–0.66, all p<0.05). Following ozone exposure, these purines remained correlated with IL-6, IL-8, and TNF-α (r=0.37-0.68). However, AMP and hypoxanthine increased significantly post ozone exposure in atopic nonasthmatics but not atopic asthmatics. In contrast, the non-purine metabolite taurine correlated with baseline neutrophil counts (r=0.56) and IL-6 (r=0.53) and was elevated post exposure in both atopic cohorts.

Discussion and Conclusions

The purine metabolites AMP and hypoxanthine are correlated with multiple inflammatory markers at baseline and after ozone exposure. However, changes in these purine metabolites after ozone appear to differ from other inflammatory markers, with less response in atopic asthmatics relative to atopic nonasthmatics. Purine metabolites may play a role in the signaling responses to ozone, but these responses may be altered in subjects with asthma.

CD14, a co-receptor for endotoxin, plays a significant role in the inflammatory response to this environmentally important pollutant. The C-159T single nucleotide polymorphism (SNP) in the CD14 gene promoter is reported to affect expression of CD14, with TT homozygous persons having higher CD14 expression. This SNP has been linked to pathogenesis of asthma and with cardiovascular diseases in smokers. We hypothesize that CD14 also plays a role in development of COPD in smokers who are exposed to inhaled endotoxin by cigarette smoking and to endotoxin released from Gram-negative microbes colonizing their airways. To assess the effect of the C-159T SNP of the CD14 gene promoter on lung function and GOLD score in smokers with COPD, we recruited 246 smokers with COPD with a range of 10–156 pack-year smoking exposures. We found that the C-159T single gene polymorphism of the CD14 gene promoter may play a role in modulating severity of obstructive impairment in smokers with COPD: The TT genotype was associated with lower lung function in smokers with a moderate smoking history. However, the CC genotype was associated with decreased lung function in heavy smokers (>56 pack-years). The result on CC genotype in risk for COPD is analogous with the effect of this genotype in risk for asthma. CD14 may be a factor in the pathophysiology of COPD, as it is in asthma and smoking-related cardiovascular diseases.

Background

Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone (O3) causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of O3-induced inflammation has not been determined.

Objective

To determine if atopic status modulates O3 response phenotypes in humans.

Methods

Fifty volunteers (25 normal volunteers, 14 atopic non-asthmatics, 11 atopic asthmatics not requiring maintenance therapy) underwent a 0.4 ppm O3 exposure protocol. Ozone response was determined by changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell surface markers, cytokine and hyaluronic acid concentration.

Results

All cohorts experienced similar decreases in lung function post O3. Atopics and atopic asthmatics had increased sputum neutrophils and IL-8 after O3 exposure; levels did not significantly change in normal volunteers. Following O3 exposure, atopic asthmatics had significantly increased sputum IL-6 and IL-1 β, and airway macrophage TLR4, FceRI, and CD23 expression; levels in normal volunteers and atopic non-asthmatics showed no significant change. Atopic asthmatics had significantly decreased IL-10 at baseline compared to normal volunteers: IL-10 did not significantly change in any group with O3. All groups had similar levels of hyaluronic acid at baseline, with increased levels after O3 exposure in atopics and atopic asthmatics.

Conclusion

Atopic asthmatics have increased airway inflammatory responses to O3. Elevated TLR4 expression suggests a potential pathway through which O3 generates the inflammatory response in allergic asthmatics but not in atopics without asthma.

Clinical Implications

These observations suggest that mild atopic asthma confers increased risk for exacerbation of O3-induced lung disease through promoting an enhanced innate immune inflammatory response to O3.

The effects of low-level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known; however, much less is known about the inflammatory and immunomodulatory effects of low-level ozone in the airways. Techniques such as induced sputum and flow cytometry make it possible to examine airways inflammatory responses and changes in immune cell surface phenotypes following low-level ozone exposure. The purpose of this study was to determine if exposure to 0.08 parts per million ozone for 6.6 h induces inflammation and modifies immune cell surface phenotypes in the airways of healthy adult subjects. Fifteen normal volunteers underwent an established 0.08 part per million ozone exposure protocol to characterize the effect of ozone on airways inflammation and immune cell surface phenotypes. Induced sputum and flow cytometry were used to assess these endpoints 24 h before and 18 h after exposure. The results showed that exposure to 0.08 ppm ozone for 6.6 h induced increased airway neutrophils, monocytes, and dendritic cells and modified the expression of CD14, HLA-DR, CD80, and CD86 on monocytes 18 h following exposure. Exposure to 0.08 parts per million ozone is associated with increased airways inflammation and promotion of antigen-presenting cell phenotypes 18 hours following exposure. These findings need to be replicated in a similar experiment that includes a control air exposure.

Ozone and lipopolysaccharide (LPS) are environmental pollutants with adverse health effects noted in both healthy and asthmatic individuals. The authors and others have shown that inhalation of ozone and LPS both induce airway neutrophilia. Based on these similarities, the authors tested the hypothesis that common biological factors determine response to these two different agents. Fifteen healthy, nonasthmatic volunteers underwent a 0.4 part per million ozone exposure for 2 h while performing intermittent moderate exercise. These same subjects underwent an inhaled LPS challenge with 20,000 LPS units of Clinical Center Reference LPS, with a minimum of 1 month separating these two challenge sessions. Induced sputum was obtained 24 h before and 4–6 h after each exposure session. Sputum was assessed for total and differential cell counts and expression of cell surface proteins as measured by flow cytometry. Sputum supernatants were assayed for cytokine concentration. Both ozone and LPS challenge augmented sputum neutrophils and subjects’ responses were significantly correlated (R = .73) with each other. Ozone had greater overall influence on cell surface proteins by modifying both monocytes (CD14, human leukocyte antigen [HLA]-DR, CD11b) and macrophages (CD11b, HLA-DR) versus LPS where CD14 and HLA-DR were modified only on monocytes. However, LPS significantly increased interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, with no significant increases seen after ozone challenge. Ozone and LPS exposure in healthy volunteers induce similar neutrophil responses in the airways; however, downstream activation of innate immune responses differ, suggesting that oxidant versus bacterial air pollutants may be mediated by different mechanisms.

Background

Exposure of humans to air pollutants such as ozone and particulate matter (PM) may result in airway and systemic inflammation and altered immune function. One putative mechanism may be through modification of cell-surface costimulatory molecules.

Objectives

We examined whether changes in expression of costimulatory molecules on circulating cells are associated with ambient levels of fine PM [aerodynamic diameter ≤ 2.5 μm (PM2.5)] in a susceptible population of diabetic individuals.

Methods

Twenty subjects were studied for 4 consecutive days. Daily measurements of PM2.5 and meteorologic data were acquired on the rooftop of the exam site. Circulating cell-surface markers that mediate innate immune and inflammatory responses were assessed by flow cytometry on each day. Sensitivity analysis was conducted on glutathione S-transferase M1 (GSTM1) genotype, body mass index, and glycosylated hemoglobin A1c (HbA1c) levels to determine their role as effect modifiers. Data were analyzed using random effects models adjusting for season, weekday, and meteorology.

Results

We found significantly increased monocyte expression (mean fluorescent intensity) of CD80, CD40, CD86, HLA-DR, and CD23 per 10-μg/m3 increase in PM2.5 at 2- to 4-day lag times after exposure. These findings were significantly higher in obese individuals, in individuals with HbA1c > 7%, and in participants who were GSTM1 null.

Conclusions

Exposure to PM2.5 can enhance antigen-presenting cell phenotypes on circulating cells, which may have consequences in the development of allergic or autoimmune diseases. These effects are amplified in diabetic individuals with characteristics that are associated with insulin resistance or with oxidative stress.

Background

The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. However, it is not known if GSTM1 modulates these ozone responses in vivo in humans

Objective

The purpose of this study was to determine if the GSTM1 null genotype modulates ozone responses in humans.

Methods

Thirty-five normal volunteers were genotyped for the GSTM1 null mutation and underwent a standard ozone exposure protocol to determine if lung function and inflammatory responses to ozone were different between the 19 GSTM1 normal and 16 GSTM1 null volunteers.

Results

GSTM1 did not modulate lung function responses to acute ozone. Granulocyte influx 4 hours after challenge was similar between GSTM1 normal and null volunteers. However, GSTM1 null volunteers had significantly increased airway neutrophils 24 hours after challenge, as well as increased expression of HLA-DR on airway macrophages and dendritic cells.

Conclusion

The GSTM1 null genotype is associated with increased airways inflammation 24 hours following ozone exposure, consistent with the lag time observed between increased ambient air ozone exposure and exacerbations of lung disease.

Clinical Implications

These observations suggest that the GSTM1 null genotype likely confers increased risk for exacerbation of ozone-induced lung disease through promoting an enhanced neutrophilic and monocytic inflammatory response to ozone.

Capsule summary

The GSTM1 null genotype is associated with increased risk for ozone-induced lung disease. We report the GSTM1 genotype modulates ozone-induced inflammation but not lung function, and may predict persons at risk for environmental lung disease.

CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IκBα degradation. Inhibition of NFκB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon γ (IFNγ) levels in the medium of PBMCs. Neutralization of IFNγ and IFNγ receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS induced CD40 expression on human PBMCs through activation of NFκB and JNK, and partially through the induction of IFNγproduction.

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces “mucus” with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

Background

Organic dust exposure in the agricultural industry results in significant airway disease and lung function decline. Mononuclear phagocytes are key cells that mediate the inflammatory and innate immune response following dust exposure.

Objective

To investigate the effect of organic dust extract (ODE) from modern swine operations on monocyte-derived macrophage (MDM) phenotype and function.

Methods

Peripheral blood monocytes were obtained by elutriation methodology (>99% mCD14+) and differentiated into macrophages in the presence of GM-CSF (1 week) with and without ODE (0.1%). At one week, cells were analyzed by flow cytometry for cell surface marker expression (HLA-DR, CD80, CD86, TLR2, TLR4, mCD14, CD16), phagocytosis (IgG-opsonized zymosan particles), and intracellular killing of Streptococcus pneumoniae. At one week, MDMs were re-challenged with high dose ODE (1%), lipopolysaccharide (LPS), and peptidoglycan (PGN), and cytokines (TNFα, IL-6, IL-10, CXCL8/IL-8) were measured. To elucidate ODE-associated factors, comparisons were made to MDMs conditioned with heat-inactivated dust, endotoxin-depleted dust, LPS, and PGN.

Results

Expression of HLA-DR, CD80, CD86, phagocytosis and intracellular bacterial killing were significantly decreased with ODE- versus control-MDMs. Responses were retained after marked depletion of endotoxin. PGN, LPS and PGN + LPS significantly reduced MDM surface marker expression, and except for LPS alone, also reduced phagocytosis. ODE-MDMs had significantly diminished cytokine responses (TNFα, IL-6, IL-10) following repeat challenge with high dose ODE. Cross-tolerant cytokine responses were also observed.

Conclusion

Repetitive organic dust exposure significantly decreases markers of antigen presentation and host defense function in monocyte-derived macrophages. Bacterial cell components appear to be driving these impaired responses.

Key Messages

Repetitive organic dust exposure impairs monocyte-derived macrophage host defense functions.

Gram positive bacterial cell components may be driving this impaired response.

Clinical implications: Organic dust-induced macrophage dysfunction may be important in respiratory disease development.

Capsule Summary

Repetitive organic dust exposure in vitro impairs host defense function in monocyte-derived macrophages, which appear to be driven by gram positive bacterial cell components. Organic dust-induced macrophage dysfunction may be important in respiratory disease development.

We have recently reported that gamma tocopherol (γT) reduces allergen and zymosan-induced inflammation using rodent models. As an initial step in extending these observations to humans, we conducted an open-label, Phase I dosing study of two doses (one or two capsules/daily for one week) of a gamma tocopherol rich preparation containing 623mg of γ tocopherol, 61.1mg of d-α-tocopherol, 11.1 mg of d-β-tocopherol (11.1mg), and 231 mg of d-σ-tocopherol per capsule. Endpoints for this study include serum levels of 5-nitro-gamma tocopherol, as a marker of oxidative stress, and changes in serum gamma, alpha and delta tocopherol and γ-2′-carboxyethyl-6-hydroxychroman (CEHC) six and 24 hours after the first dose and after 1 week of treatment. To assess biological activity of this treatment, we obtained peripheral blood mononuclear cells at baseline and after 1 week of treatment with 2 capsules of a gamma tocopherol rich preparation/day, and examined the inflammatory cytokine response of these cells in culture to ex-vivo endotoxin/LPS (0.01 ng/ml) challenge. We also monitored a number of safety endpoints to examine how well this preparation is tolerated in 8 normal volunteers (4 allergic and 4 non-allergic) and 8 allergic asthmatics. We further obtained human monocytes from a subset of these volunteers and treated them ex vivo with γT, αT,γ-CEHC and α-CEHC and assessed their actions on LPS induced degradation of IkBα, and JNK signaling and ROS generation. As detailed herein, this open label study demonstrates that gamma tocopherol enriched supplementation decreased systemic oxidative stress, increased serum levels of gamma tocopherol, and inhibited monocyte responses to LPS without any adverse health effects. Further,in vitro treatment of human monocytes with γ-CEHC and α-CEHC inhibits ROS generation and LPS-induced degradation of IκB and JNK activation.

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB.

Calgranulins are a family of powerful chemoattractants, which have been implicated as biomarkers in inflammatory diseases. To determine how different respiratory diseases affect the expression of calgranulins, we measured the expression of S100A8/A9 and S100A12 in bronchoalveolar lavage fluid (BALF) of ARDS patients and healthy volunteers by ELISA. Analysis of calgranulin expression revealed a high level of S100A12 in the lavages of patients suffering from ARDS compared to controls (p< 0.001). Based on the hypothesis that the increased expression of S100A12 relative to the S100A8/A9 heterodimer was a characteristic of respiratory diseases with neutrophilic inflammation, we measured calgranulin expression in BALF of cystic fibrosis (CF) patients. Despite similarly elevated levels of S100A8/A9, S100A12 was significantly higher in ARDS compared to CF BALF (p<0.001). The differential expression of calgranulins was unique for inflammatory markers, as an array of cytokines did not differ between CF and ARDS patients.

Since ARDS is an acute event and CF a chronic inflammation with acute exacerbations, we compared calgranulin expression in sputum obtained from CF and patients with chronic obstructive lung disease (COPD). Levels of S100A12 and S100A8/9 were elevated in CF sputum compared to COPD sputum, but the ratio of S100A12 to S100A8/A9 was similar in COPD and CF and reflected more closely that seen in healthy controls. The results indicate that the regulation of human calgranulin expression and the ratio of S100A8/A9 to S100A12 may provide important insights in the mechanism of respiratory inflammation.

Background

Ozone exposure induces airway neutrophilia and modifies innate immune monocytic cell-surface phenotypes in healthy individuals. High-dose inhaled corticosteroids can reduce O3-induced airway inflammation, but their effect on innate immune activation is unknown.

Objectives

We used a human O3 inhalation challenge model to examine the effectiveness of clinically relevant doses of inhaled corticosteroids on airway inflammation and markers of innate immune activation in healthy volunteers.

Methods

Seventeen O3-responsive subjects [> 10% increase in the percentage of polymorphonuclear leukocytes (PMNs) in sputum, PMNs per milligram vs. baseline sputum] received placebo, or either a single therapeutic dose (0.5 mg) or a high dose (2 mg) of inhaled fluticasone proprionate (FP) 1 hr before a 3-hr O3 challenge (0.25 ppm) on three separate occasions at least 2 weeks apart. Lung function, exhaled nitric oxide, sputum, and systemic biomarkers were assessed 1–5 hr after the O3 challenge. To determine the effect of FP on cellular function, we assessed sputum cells from seven subjects by flow cytometry for cell-surface marker activation.

Results

FP had no effect on O3-induced lung function decline. Compared with placebo, 0.5 mg and 2 mg FP reduced O3-induced sputum neutrophilia by 18% and 35%, respectively. A similar effect was observed on the airway-specific serum biomarker Clara cell protein 16 (CCP16). Furthermore, FP pretreatment significantly reduced O3-induced modification of CD11b, mCD14, CD64, CD16, HLA-DR, and CD86 on sputum monocytes in a dose-dependent manner.

Conclusions

This study confirmed and extended data demonstrating the protective effect of FP against O3-induced airway inflammation and immune cell activation.

Background

Recent reports suggest that endotoxin exposure can blunt phagocyte functions. The aim of this study was to examine whether lung phagocytic cells have altered host defense function in young cystic fibrosis (CF) patients, and to explore the contribution of neutrophil elastase (NE) and surfactant proteins to these effects.

Methods

BALF cells from CF children (N=12) and disease controls (N=12) were analyzed by flow cytometry for mCD14 and HLA-DR expression and phagocytosis. The effects of exogenous surfactant protein A and D (SP-A,D) and proteases on BALF cells in short-term culture were assessed experimentally.

Results

Expression of the surface markers mCD14 and HLA-DR, and phagocytosis, were all blunted on CF phagocytes compared to disease controls (p<0.05). In CF phagocytes, SP-A enhanced both phagocytosis and mCD14 expression (p<0.05). Both CF BALF and NE reduced phagocytosis and expression of mCD14 and HLA-DR (p<0.05) by non-CF phagocytes; the latter effect was attenuated by protease inhibitor.

Conclusion

CF airway phagocytes appear to have altered host defense functions that could contribute to poor bacterial clearance. These impairments can be reproduced by incubation of non-CF cells with NE, while SP-A can partially reverse them. Decreasing protease activity and increasing collectin activity may be beneficial in early CF.

We combined two techniques, radiolabeled aerosol inhalation delivery and induced sputum, to examine in vivo the time course of particle uptake by airway macrophages in 10 healthy volunteers. On three separate visits, induced sputum was obtained 40, 100, and 160 min after inhalation of radiolabeled sulfur colloid (SC) aerosol (Tc99 m-SC, 0.2 μm colloid size delivered in 6-μm droplets). On a fourth visit (control) with no SC inhalation, induced sputum was obtained and SC particles were incubated (37°C) in vitro with sputum cells for 40, 100, and 160 min (matching the times associated with in vivo sampling). Total and differential cell counts were recorded for each sputum sample. Compared with 40 min (6 ± 3%), uptake in vivo was significantly elevated at 100 (31 ± 5%) and 160 min (27 ± 4%); both were strongly associated with the number of airway macrophages (R = 0.8 and 0.7, respectively); and the number and proportion of macrophages at 40 min were significantly (P < 0.05) elevated compared with control (1,248 ± 256 versus 555 ± 114 cells/mg; 76 ± 6% versus 60 ± 5%). Uptake in vitro increased in a linear fashion over time and was maximal at 160 min (40 min, 12 ± 2%; 100 min, 16 ± 4%; 160 min, 24 ± 6%). These data suggest that airway surface macrophages in healthy subjects rapidly engulf insoluble particles. Further, macrophage recruitment and phagocytosis-modifying agents are factors in vivo that likely affect particle uptake and its time course.

Oxidative stress plays a significant role in allergic airway inflammation. Supplementation with alpha-tocopherol (alone or combined with ascorbate/vitamin C) has been assessed as an intervention for allergic airway diseases with conflicting results. Enhancing levels of airway antioxidants with oral supplements has been suggested as an intervention to protect individuals from the effect of inhaled oxidants, although it is unclear whether supplementation changes tocopherol or vitamin C levels in both serum and airway fluids. Our objective was to obtain pilot safety and dosing data from 14 allergic asthmatic volunteers examining the effect of daily combination oral therapy with 500 mg alpha-tocopherol (αT) and 2 g vitamin C for 12 wk. We examined serum and airway fluid and cellular levels of alpha- and gamma-tocopherol (γT) and vitamin C to plan for future studies of these agents in asthma and allergic rhinitis. Six volunteers completed 12 wk of active treatment with αT and vitamin C and 8 completed placebo. Blood and sputum samples were obtained at baseline and at 6 wk and 12 wk of therapy and were analyzed for αT, γT, and vitamin C levels in the serum, sputum supernatant, and sputum cells. Combination treatment increased serum vitamin C and significantly decreased sputum αT and serum γT levels. No changes were found in sputum supernatant or sputum cell vitamin C or serum αT levels in the active treatment group. In conclusion, supplementation with αT and high-dose vitamin C does not augment vitamin C levels in the respiratory-tract lining fluid.