Howard Kehrl

Lung function and inflammatory responses in healthy young adults exposed to 0.06 ppm ozone for 6.6 hours.

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 FEV(1) (± 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 FEV(1), 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 FEV(1) and an increase in neutrophilic inflammation in the airways. GSTM1 genotype alone appears to have no significant role in modifying the effects.

Increased specific airway reactivity of persons with mild allergic asthma after 7.6 hours of exposure to 0.16 ppm ozone

BACKGROUND Exposure to ozone causes decrements in lung function, increased airway reactivity to nonspecific bronchoconstrictors, and lung inflammation. Epidemiology studies show an association between ambient oxidant levels and increased asthma attacks and hospital admissions. OBJECTIVE The purpose of our study was to evaluate the response of persons with mild asthma to inhaled allergen after ozone exposure conditions similar to those observed in urban areas of the United States. METHODS Using a double-blind, counter-balanced design, we exposed 9 (5 women and 4 men) subjects with mild atopic asthma (house dust mite sensitive) to clean air and to 0.16 ppm ozone for 7.6 hours; exposures were separated by a minimum of 4 weeks. During exposure, subjects performed light exercise (ventilation = 24 L/min) for 50 minutes of each hour, and pulmonary function was evaluated before and after exposures. The morning after exposure, subjects underwent bronchial challenge with inhaled house dust mite allergen (Dermatophagoides farinae). Using a series of doubling allergen concentrations, subjects inhaled 5 breaths of nebulized allergen (0.06 to 500 AU/mL) at 10-minute intervals until a minimum of a 20% decrement in FEV(1) was elicited. RESULTS Compared with the change in FEV(1) during air exposure, there was a mean 9.1% +/- 2.5% (SEM) decrement in FEV(1) observed because of ozone (P <.01). Seven of the 9 subjects required less allergen after ozone exposure than after air exposure; there was a 0.58 mean dose shift in the doubling concentration of allergen attributable to the ozone exposure (P =.03). CONCLUSION These findings indicate that exposure of subjects with mild atopic asthma to ozone at levels sufficient to cause modest decrements in lung function also increases the reactivity to allergen. To the extent that this effect occurs in response to ambient exposures, ozone may be contributing to the aggravation of asthma.

Allergen bronchoprovocation of patients with mild allergic asthma after ozone exposure

BACKGROUND Clinical and epidemiologic studies suggest that ambient ozone exposure may increase the response of patients with asthma to inhaled allergen. OBJECTIVES The study was designed to evaluate whether a resting 1-hour exposure to 0.12 ppm ozone increases the sensitivity of patients with atopic asthma to inhaled allergen. METHODS Outside of their allergen season, 15 patients with mild atopic asthma (5 women and 10 men) were exposed, on separate occasions, for 1 hour at rest to clean air and 0.12 ppm ozone. Exposures were separated by a minimum of 4 weeks in a counterbalanced, double-blind design. After exposure, subjects underwent inhalation challenge with doubling doses of aerosolized allergen (0.05 to a maximum of 1600 protein nitrogen units/ml) until we elicited a 20% FEV1 decrement (PC20). RESULTS Baseline symptoms, spirometry, and histamine bronchoreactivity were similar for the two exposures. Neither spirometry results nor symptoms were significantly changed after either exposure. The mean difference in response to allergen challenge on the air and ozone days, for the 12 subjects who attained a PC20 was not significant (p = 0.124). Three subjects required the same allergen dose to reach PC20 for both exposures, five required less allergen after ozone exposure, and four required more. There was no order effect for the acute response to allergen challenges (p = 0.325). However, 20 hours after allergen challenge, histamine bronchoreactivity was increased (p < 0.05) to a similar degree for both air and ozone. CONCLUSIONS A resting exposure for 1 hour to 0.12 ppm ozone did not potentiate an immediate bronchoconstrictive response to grass allergen in this group of patients with mild atopic asthma.

Low-level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans

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.

Patients with Asthma Demonstrate Airway Inflammation after Exposure to Concentrated Ambient Particulate Matter

To the Editor: Of the three major particulate matter (PM) size fractions (ultrafine, fine, and coarse), coarse PM (PM2.5–10) has been the least examined in terms of its health effects on susceptible populations, this despite having characteristics that make it particularly likely to affect those with airway diseases such as asthma. For example, PM2.5–10 preferentially deposits in the bronchial airways, a site proximal to asthma pathology (1), and contains biological agents such as endotoxin and allergens that are primary triggers associated with asthma exacerbation (2). We have reported that endotoxin inhalation challenge in subjects with allergic asthma enhances airway inflammation, a key underlying pathophysiological feature of asthma, and modifies airway phagocyte function 4–6 hours after exposure (3). We have also shown that subjects with late-phase allergen-responsive asthma demonstrate enhanced bronchial airway deposition of inhaled particles and slowed clearance of those particles from the central airways 4 hours after particle inhalation, a time coinciding with enhanced inflammation from endotoxin inhalation (4). Hence, specific characteristics of PM2.5–10, together with the fact that individuals with asthma compared with those without asthma have greater sensitivity to air pollutants in general (5), make it likely that individuals with asthma will demonstrate deleterious pulmonary responses after exposure to PM2.5–10. These responses, however, remain largely speculative because they have been described only in healthy individuals. Indeed, we have previously shown that healthy individuals exposed to coarse size (PM2.5–10) concentrated ambient particles (CAPs) demonstrated only modest increases in pulmonary neutrophil levels with no increase in inflammatory mediators (6). The assumption that individuals with asthma will demonstrate a comparatively more robust inflammatory response than those without asthma must be verified by a proof-of-concept study. We undertook a proof-of-concept study to determine whether exposure to coarse size (PM2.5–10) CAPs, at a concentration previously shown to induce only mild changes in healthy subjects (6), would induce robust pulmonary inflammatory and innate immune alterations in subjects with allergic asthma. The experimental design and details of the study replicate those of our previously published coarse CAP study in healthy subjects without asthma (Graff and colleagues, 2009 [6]). Specifically, the urban PM exposure source, exposure months, mechanism of PM concentration, concentrator type, and exposure chamber used in this study were identical to those used in the Graff and colleagues study (6). Furthermore, the PM concentration and calculated dose compared closely between the two studies. This study was approved by the institutional review board at the University of North Carolina (Chapel Hill, NC). In brief, this study was a single-blind crossover study of 10 subjects with mild to moderate allergic asthma, in which each subject was studied on two occasions (2-h exposure to CAPs or filtered air [FA] from ambient Chapel Hill, NC) at least 4 weeks apart. The concentration of coarse particles suspended in the particulate exposure chamber at the U.S. Environmental Protection Agency (EPA) Human Studies facility in North Carolina was measured on a continuous scale and varied from subject to subject depending on the outdoor particle concentration that day. There was a mean overall total particle concentration of 101.8 ± 18.0 μg/m3 and a PM2.5–10 concentration of 86.9 ± 17.4 μg/m3 on CAP days (FA days had a mean total particle concentration of 1.2 μg/m3). The mean coarse PM concentration (101.8 ± 18.0 μg/m3) measured in this study was not unrealistically high and can be found in many areas throughout the world, including locations in the U.S. Southwest (7). Individual and overall coarse PM exposure data are shown in Table E1 (see the online supplement). Lung cells and fluid-phase components were obtained by bronchoalveolar lavage (BAL) and bronchial wash (BW, the first 30 ml of BAL recovered) 24 hours postexposure. Differential leukocytes and fluid-phase components were examined as previously described (6). Flow cytometry was performed on BAL leukocytes for assessment of cell surface phenotypes associated with innate host defense (CD11b/CR3, mCD14, CD64/FcγRI [Fc γ receptor type I]), antigen presentation/T-cell interaction (CD23/low-affinity IgE, HLA-DR, CD86/B7.2, CD80/B7.1, CD40), and inflammation (CD16/FcγRIII). The detailed flow methodology appears in the online supplement and in our review (8). Informed consent was obtained before study and all volunteers with asthma (n = 10; age, 18–45 yr) were nonsmokers with mild to moderate disease severity. Inclusion criteria, baseline medical assessment, and study procedures are detailed in the online supplement. Parametric and nonparametric paired analyses were used to compare all study end points 20 hours after filtered air and CAP exposures, and a linear mixed-effects model was used to compare with the data of Graff and colleagues (6). Significance was set at α = 0.05. As shown in Table 1, we observed a robust increase in BW polymorphonuclear neutrophils after CAP exposure (8 vs. 13%), an effect significantly (P < 0.05) different when compared with our earlier study in subjects without asthma (6). Furthermore, we demonstrated significantly elevated levels of IL-1β and IL-8 in both BW and BAL. Although BAL IL-6 was not significantly different after exposure to CAPs, it was significantly negatively associated with PM dose (R = –0.65) and PM concentration (R = –0.62). These negative IL-6 associations were unexpected because IL-6 is typically positively associated with increased ambient PM levels (9). However, one explanation may be that IL-6–producing alveolar macrophages have become tolerant from preexisting ambient PM exposure and elevated airway inflammation, an underlying feature of asthma, thereby producing the negative association observed here after an acute exposure to PM2.5–10. No change in lung function (FEV1, FVC) was reported after CAP exposure (Table E2), and with the exception of decreased blood IL-6 after CAP exposure (3.255 ± 1.068 vs. 1.740 ± 0.2914 pg/ml), no markers of systemic inflammation were modified by CAPs (IL-8, tumor necrosis factor-α [TNF-α], CD40 ligand [CD40L], E-selectin, soluble vascular cell adhesion molecule-1 [sVCAM1], plasminogen, fibrin, C-reactive protein [CRP], fibrinogen, soluble intercellular adhesion molecule-1 [sICAM-1], myeloperoxidase [MPO]). Immunophenotyping of immature and mature macrophages revealed decreased cell surface expression (mean fluorescence intensity [MFI]) of innate immune receptors (CD11b/CR3, CD64/FcγRI) (Figures 1A and 1B) and antigen presentation receptors (CD40, CD86/B7.2) (Figures 1E and 1F); with increased expression of inflammatory receptors CD16/FcγRIII and the low-affinity IgE receptor (CD23) (Figures 1C and 1D) after CAP exposure. It is intriguing that we have found similar inflammatory responses and cell surface phenotype changes in subjects with asthma exposed to ozone (e.g., elevated IL-1β and IL-8 and increased expression of low-affinity IgE receptor/CD23) (10), and that subjects with asthma have enhanced response to allergen after challenge with both ozone and PM component endotoxin (11). Like endotoxin and ozone, coarse PM induced a neutrophil response albeit at a comparatively reduced magnitude. This component of the overall PM response may be nonspecific and in common with xenobiotics in general. However, downstream effects of coarse PM appear to differ from those of ozone and, depending on the dose, may be similar or different from those of endotoxin. Because CAP endotoxin levels were not measured in this study or in the study by Graff and colleagues (6), we were unable to assess or compare the impact of endotoxin as a driver of observed cell responses. However, our previous mechanistic coarse PM studies (2, 12) clearly point to endotoxin as an important driver of immune cell responses after coarse PM exposure. We also note that the up-regulation of the CD23/IgE receptor reported here suggests an asthma-specific pathway induced by coarse PM not typically observed with other xenobiotics, such as ozone or endotoxin. The observations reported here, namely significant CAP-induced pulmonary inflammation, altered innate host defense response, and potentially enhanced IgE signaling, lead us to hypothesize that coarse-mode CAP exposure increases the responsiveness of individuals with allergic asthma to inhaled allergens and therefore enhances the risk of exacerbation. Table 1. Airway Neutrophil Proportion and Inflammatory Cytokine Levels Figure 1. Cell surface marker expression (MFI) on BAL inflammatory cells after filtered air (FA) and particulate matter (PM) exposure. BAL = bronchoalveolar lavage; IgE R = IgE receptor; Imm Mac = immature macrophages; Mac = macrophages; MFI = mean fluorescence ... This proof-of-concept study confirms the assumption that coarse-size PM, like other pollutants, can initiate deleterious responses in individuals with asthma at concentrations not observed in healthy individuals without asthma. These responses include increased airway inflammation and alterations in immune cell phenotype expression. Our data suggest that individuals with asthma have increased susceptibility to coarse-size PM exposure compared with healthy individuals without asthma, and interventions focused on these responses may be useful approaches to mitigate the impact of PM air pollution in those with asthma.

The glutathione-S-transferase mu 1 (GSTM1) null genotype and increased neutrophil response to low-level ozone (0.06 ppm)

associated with IFN-g therapy at doses up to 200 mg/m; she shows slow clinical improvement characterized by weight gain and absence of fever and diarrhea. We have described a case of composite heterozygousmutations of IL12RB1 gene encoding for the b1 subunit of IL-12 receptor that is associated with disseminated M genavense infection. In a recent survey of 141 patients with IL-12Rb1 deficiency, isolated BCG infection has been observed in most patients (43 subjects of 102 index cases), environmental mycobacteria were observed in 6 patients, and the remaining patients had infections caused byMycobacterium tuberculosis (2 patients), other intracellular pathogens (mostly Salmonella, but also Klebsiella and Nocardia species), or combinations of the above-described pathogens. Infection by M genavense was reported in a single patient with IL-12Rb1 deficit, suggesting that this pathogen is a rare cause of MSMD. In addition, IL-12Rb1 deficiency has incomplete clinical penetrance because 8 of 29 known genetically affected siblings investigated in the survey did not develop MSMD-related infections. This observation suggests that environmental factors, including the route of infection (eg, subcutaneous injection of BCG), or the use of immunosuppressant drugs can facilitate the development of disseminated infections as observed in our patient who was receiving an immunosuppressive drug for the treatment of autoimmune hepatitis. Likewise, M genavense infection was reported in patients receiving immunosuppressive therapy after transplantation or in patients with AIDS. Development of lymphopenia is not usually observed in patients with IL-12Rb1 deficiency, although the patient that we describe has shown a profound lymphopenia that might be related to a defect of lymphocyte generation in bone marrow or to intestinal lymphangiectasia. In conclusion, our observation suggests that development of disseminated mycobacterial infection in patients receiving immunosuppressive treatment could constitute a presenting phenotype of MSMD. Laura Tassone, PhD Anna Cristina C. Carvalho, MD, PhD Alessandra Calabresi, MD Enrico Tortoli, MD Alessandra Apostoli, MD Omar Scomodon, PhD Cecilia Spina, PhD Donatella Vairo, PhD Vincenzo Villanacci, MD Alberto Matteelli, MD* Raffaele Badolato, MD, PhD*