Abstract
Airway concentrations of many reactive nitrogen and oxygen species are high in asthma. The stability and bioactivities of these species are pH-dependent; however, the pH of the airway during acute asthma has not previously been studied. As with gastric and urinary acidification, asthmatic airway acidification could be expected dramatically to alter the concentrations and bioactivities/cytotoxicities of endogenous nitrogen oxides. Here, we demonstrate that the pH of deaerated exhaled airway vapor condensate is over two log orders lower in patients with acute asthma (5.23 ± 0.21, n = 22) than in control subjects (7.65 ± 0.20, n = 19, p < 0.001) and normalizes with corticosteroid therapy. Values are highly reproducible, unaffected by salivary or therapeutic artifact, and identical to samples taken directly from the lower airway. Further, at these low pH values, the endogenous airway compound, nitrite, is converted to nitric oxide (NO) in quantities sufficient largely to account for the concentrations of NO in asthmatic expired air, and eosinophils undergo accelerated necrosis. We speculate that airway pH may be an important determinant of expired NO concentration and airway inflammation, and suggest that regulation of airway pH has a previously unsuspected role in asthma pathophysiology. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TAE, Gaston B. Endogenous airway acidification: implications for asthma pathophysiology.
Subglottic air is saturated with water that may be condensed during exhalation (1). In asthma, exhaled airway vapor condensate contains high concentrations of nitrogen oxides (NOx) and reactive oxygen species, reflecting changes in lower respiratory tract lining fluid during inflammation (2, 3). Of note, the interactions and toxicity of many of these NOx and reactive oxygen species are critically pH-dependent. We hypothesized that exhaled airway vapor condensate pH might be low in asthma, and that this acidification might contribute to asthma pathophysiology. We report that (1) airway vapor condensate from patients with acute asthma has a pH substantially lower than normal; (2) airway acidity appears to be relevant to asthma in that it both accelerates human eosinophil necrosis and causes the conversion of endogenous nitrite (NO2 −) to nitric oxide (NO); and (3) the acidic airway vapor condensate pH in asthma normalizes with anti-inflammatory therapy. These observations present asthmatic airway inflammation in an entirely new light, and may have critical diagnostic and therapeutic implications.
We studied patients with asthma, defined as a history of three or more episodes of β2-agonist–reversible airway obstruction, who were admitted to the hospital for dyspnea and demonstrated both tachypnea and an inspiratory:expiratory ratio less than 0.5. We excluded subjects who smoked, had clinical evidence of pneumonia, or had a chronic disease other than asthma. Selected subjects were followed longitudinally with repeated sample collection during and after their hospitalization. Control patients were recruited from hospital staff and inpatients admitted for acute, nonrespiratory diseases. Additionally, three patients undergoing direct, undiluted tracheal suctioning were studied, and three subjects were studied before and after three consecutive jet nebulization treatments with albuterol (total of 7.5 mg albuterol in 9 ml of normal saline). This study was approved by the Institutional Human Investigation Committee.
Subjects performed quiet tidal breathing through inert one-way valves and a 0.3-μm particle filter (Marquest Respirgard II, Englewood, CO) into an aluminum condensing conduit surrounded by coolant. The conduit had been rinsed with distilled water, dried with forced air, and frozen at −40° C. One milliliter of exhaled airway vapor condensate was obtained during 10 min of breathing. Samples were excluded if gastric air was expelled during collection. Stable pH was achieved in all cases after deaeration of the condensate with argon (350 ml/min) for 10 min. pH was measured using a Cardy Twin pH meter (Horiba, Japan), or Corning pH microelectrode (Corning, New York).
Isolation. Fresh human peripheral blood was applied to a density gradient consisting of one part Mono-Poly Resolving Medium (ICN Biomedicals, Aurora, OH), two parts Polymorph (Accurate Science, Westbury, CT), and one part neutrophil isolation media (NIM) (Cardinal, Santa Fe, NM). After centrifugation (25 min; 300 × g; 25° C), the polymorphonuclear (PMN) fraction was washed in Hank's balanced salt solution (HBSS), counted, and incubated with anti-CD16 immunomagnetic microbeads (Milteny Biotech, Sunnyvale, CA) (30 min; 6° C) before being applied to a magnetic column to remove neutrophils (4). An aliquot of eosinophil cell suspension was mixed 1:1 with a 0.4% solution of trypan blue for light microscopic analysis of viability. Eosinophil isolates that were less than 95% pure and viable were discarded.
Determination of apoptosis and necrosis. Mechanism of cell death was assessed by two methods. First, cells underwent Hansel staining and light microscopy for morphologic assessment of apoptosis as evidenced by cytosolic vacuolization, nuclear and cytoplasmic condensation, and presence of apoptotic bodies (5). Additionally, cells underwent DNA quantification using flow cytometry as previously described (6). Briefly, purified eosinophils were fixed in 70% ethanol, pelleted, resuspended in 40 μl of phosphate–citrate buffer for 30 min, washed in HBSS, and stained with propidium iodide (50 μg/ml) for 30 min. Cells were counted by flow cytometry on a FACScan (Immunocytometry Systems, Becton Dickinson, San Jose, CA) for DNA content, gating on forward and side scatter, and (FL-2) area versus FL-2 width to exclude debris. Apoptotic ratios were calculated (ratio of counts in apoptotic range to counts in diploid range).
Nitrite was assayed as NO by anaerobic chemiluminescence after reduction in 1% potassium iodide (KI; glacial acetic acid; 50° C) according to the manufacturer's recommendations (NOA 280; Sievers, Boulder, CO). NO evolution from airway vapor condensate was measured by chemiluminescence in the headspace of 2-ml sealed glass tubes after incubation of 200-μl samples with NO2 − (100 μM) (7).
Data are presented as median and range or, when parametrically distributed, as arithmetic or geometric (pH) mean ± SEM. Differences were analyzed by analysis of variance (ANOVA) or ANOVA on ranks with appropriate pairwise comparisons. Linear regression and correlation coefficients were used to assess relationships. Differences were considered significant at p values < 0.05. Statistical calculations were performed using SigmaStat 2.0 (Jandel Corporation, San Rafael, CA).
Twenty-two subjects with acute asthma, including 16 subjects treated for less than 48 h with glucocorticoids (mean age 19.5 ± 2.1 yr, 10 males), 19 control subjects (20.5 ± 3.2 yr, 10 males), and 12 subjects with stable asthma (age 21.5 ± 2.0 yr, 6 males) were enrolled. The mean pH of exhaled airway vapor condensate samples from patients with early acute asthma was 5.23 ± 0.21 compared with a mean in the control group of 7.65 ± 0.20 (p < 0.001) (Figure 1). The mean condensate pH of subjects with stable asthma was 7.8 ± 0.1 (p = 0.95 compared with normal subjects). Hospitalized patients who had received systemic glucocorticoid therapy for longer than 48 h had higher condensate pH values than acutely ill subjects, in fact approaching normal (7.4 ± 0.23, n = 11, p < 0.001 versus acute asthmatic subjects on systemic steroids less than 48 h) (Figure 1). Patients followed longitudinally showed steady increases in condensate pH to normal values during anti-inflammatory therapy (Figure 2). Measurements were highly stable and reproducible (average coefficient of variation = 3.3%, 2 to 16 samples each from six normal subjects and three subjects with acute asthma).
Fig. 1. Airway vapor condensate is acidified in acute asthma. Asthmatic subjects were studied on admission to the hospital for acute bronchospasm. Control subjects had no history of asthma and were free of respiratory symptoms. All subjects breathed orally through one-way valves and a 0.3-μm filter into a clean, dry condensing chamber. Condensate was deaerated for 10 min in argon and the pH was measured under anaerobic conditions. This test was highly reproducible (average coefficient of variation 3.3%). Mean acute asthmatic pH (5.23 ± 0.21) was over two logs lower than controls (7.65 ± 0.20) on admission, and approached normal after treatment with systemic glucocorticoid therapy (7.4 ± 0.23, p < 0.001).
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Fig. 2. Airway vapor condensate pH normalizes during corticosteroid treatment for acute asthma. Serial measurements were made on five patients during treatment. Mean pH after treatment (7.5 ± 0.23) was over two logs higher than at the initiation of treatment (5.3 ± 0.38, p = 0.001).
[More] [Minimize]Nonpulmonary causes for airway vapor acidification were extensively considered. First, direct comparison was made between the pH of condensate and undiluted tracheal secretions obtained from subjects undergoing subsequent bronchoscopy. Deaerated condensate pH values were identical to the pH of unprocessed native lower airway secretions at both low and normal pH (r2 = 1.0; n = 3). Further, there was no association between the pH of condensate and matched salivary samples (n = 20; r2 = 0.17; p = NS) (Figure 3), and the pH decline did not depend on the presence or absence of supplemental oxygen. There was no change in condensate pH after administration of 0.083% albuterol (three unit dose treatments of 3.0 ml each) by nebulizer over 45 min to three subjects (one with stable asthma, and two healthy control subjects), nor by nebulized unbuffered solutions at pH of 3.5 (three subjects) and 9.8 (one subject). In this regard, it should also be noted that airway vapor condensate acidosis in subjects with acute asthma did not appear to be an artifact of airflow obstruction because β2-agonist therapy and methacholine challenge in asthmatic subjects did not change the pH of condensates, and because nonasthmatic subjects with chronic obstructive pulmonary disease, cystic fibrosis, and immotile cilia syndrome—though obstructed—consistently had condensate pH values greater than 7.0 (data not shown).
Fig. 3. Airway vapor condensate pH and salivary pH. Simultaneous specimens were collected both from subjects with asthma and from control subjects. n = 20, r2 = 0.17, p = not significant (NS).
[More] [Minimize]Consistent with our previous report (2), we found that median condensate NO2 − concentrations in asthmatics were substantially higher than in control subjects. However, this effect was more evident in specimens from treated asthmatic subjects with median normal condensate pH values (NO2 − = 1.5 μM [range, 0.45 to 2.74] versus median control values of 0.55 μM [ranage 0.31 to 2.33]; p < 0.001) than in samples from subjects with acute asthma whose pH values were less than 7.0 (median, 0.77 μM [range, 0.18 to 4.85]; p = NS compared with control subjects) (Figure 4A). Lower NO2 − values in the acidified samples could be accounted for simply by loss of NO. Indeed, NO was evolved from exogenous NO2 − added to acute asthmatic, but not control, condensates in a pH-dependent fashion (Figure 4B). Because of the small volumes of condensate samples, and the importance of controlling for airflow when measuring NO concentrations from the human lung (8), we used an in vitro model of airflow through acidified water (pH 5.0) with a physiologically relevant concentration of NaNO2 (100 μM) (7) to further assess NO evolution from mildly acidified NO2 −. This system evolved NO at a rate (2.7 nanomoles/min) adequate fully to account for the high NO concentrations exhaled by asthmatic patients (9), assuming an airway lining fluid volume of 25 ml (10) (Figure 4C).
Fig. 4. Nitrite is converted to NO in the airway vapor condensate of patients with acute asthma. (A) Airway vapor condensate NO2 − concentrations were higher in asthmatic subjects with normal pH than with low pH. Nitrite was measured by chemiluminescence after reduction in acidified KI. Median asthmatic NO2 − values (1.26 μM [range, 0.18 to 4.85]) were higher than control (0.55 μM [range, 0.31 to 2.33]; p < 0.001). Median nitrite values were lower in the acidic condensates of subjects with acute asthma (0.77 μM [range, 0.18 to 4.85]) than the normal pH condensates of subjects with controlled/treated asthma (1.5 μM [range, 0.45 to 2.74]; p < 0.05). (B) NO was evolved from acute asthmatic condensates after exposure to exogenous NO2 −. 200 μl of asthmatic (n = 8) and control (n = 5) condensate samples were incubated for 5 h with 100 μM NO2 − in 2-ml sealed glass chambers. Headspace NO was measured by chemiluminescence. NO was evolved from the acidic asthmatic samples in a pH-dependent fashion, but not from control subjects (p < 0.001). (C) Aliquots of 5 ml of deionized water at pH values characteristic of control and asthmatic subjects were reacted with 100 μM NO2 −, and headspace NO in helium (200 ml/min) was measured by chemiluminescence. The amount of NO produced at pH 5.0 (2.7 nmol/min) in this simplified system is equivalent to that in the exhaled air of subjects with asthma, assuming a total airway lining fluid volume of 25 ml, and minute ventilation of 5 L.
[More] [Minimize]Morphologic and nuclear DNA content studies of isolated human eosinophils from two subjects with asthma revealed that 86 ± 4% of cells had necrosed after incubation for 48 h at pH in the range found in acute asthmatic condensate (pH = 6.2). On the other hand, when incubated for the same time period at pH 8.0, minimal necrosis occurred (13.5 ± 4.5%, p < 0.01), but an additional 26% showed morphologic and DNA content evidence of apoptosis (Figures 5A and 5B). Incubation of the cells with 250 μM of S-nitrosoglutamyl-cysteine (CGSNO)—a compound of the endogenous S-nitrosothiol class—or the peroxynitrite (OONO−) donor SIN-1(250 nM), prevented apoptotic cell death at neutral pH (Figures 5C and 5D).
Fig. 5. Necrotic cell death occurs in human eosinophils exposed to the pH seen in acute asthma. Freshly isolated human eosinophils (5 × 105 per aliquot) were incubated for 48 h in RPMI 1640 plus 10% fetal calf serum (37° C, 5% CO2) at pH 8.0 and pH 6.2. (A) Cell death, assessed by trypan blue exclusion, occurred in 88 ± 2.5% at pH 6.2 (n = 6), compared with 45 ± 1% at pH 8.0 (n = 2, p < 0.001). Flow cytometric analysis of DNA content after staining with propidium iodide revealed that cell death at pH 6.2 was almost entirely due to necrosis (98 ± 0.3%, n = 6), whereas at pH 8.0, apoptosis (black shading) predominated and necrosis (no shading) was minimal (14.2 ± 4.5%, n = 2, p < 0.001), consistent with morphologic assessments (data not shown). (B) Raw flow cytometric counts reveal absence of apoptosis, but profound cellular necrosis when incubated in low pH media. Top: Freshly isolated eosinophils with normal prominent diploid peak. Middle: After 48 h in media at pH 6.2 with loss of diploid peak and extensive debris consistent with necrosis. Bottom: After 48 h in media at pH 8.0— large subdiploid population consistent with apoptosis. (C) Eosinophils were incubated for 96 h at pH 8.0 with addition of 250 nM SIN-1 or 250 μM CGSNO. Cell death occurred in 67.5 ± 5.2% of controls (n = 4), but only 37 ± 7% of cells treated with SIN-1 (n = 2), and 4 ± 0% of cells treated with CGSNO (n = 2, p = 0.001). (D) Flow cytometric analysis of DNA content revealed an apoptotic ratio (ratio of apoptotic to diploid cells) of 2.1 ± 0.3 in controls, 1.2 ± 0.4 in SIN-1-treated cells, and only 0.17 ± 0.005 in CGSNO-treated cells (p = 0.02).
[More] [Minimize]We found that the water derived from the airway was acidified in patients with acute asthma. The pH was sufficient to cause both NO evolution from endogenous NO2 − and necrosis of eosinophils. Further, pH normalized during glucocorticoid treatment. These observations suggest that regulation of airway pH may have a role in the pathophysiology of acute asthma.
Breath condensates have been studied extensively as tools to measure airway inflammation. They are noninvasive, simple to perform, highly reproducible, and reflect abnormalities noted in specimens obtained bronchoscopically (11, 12) and in sputum (13). In this regard, condensate levels of nitrate (NO3 −), nitrite (NO2 −) (2), hydrogen peroxide (H2O2) (3), and certain cytokines (14) are high in asthma. Because the concentrations and bioactivities of many of these asthma markers are critically pH-dependent, we speculated that low airway pH could contribute substantially to airway inflammation in asthma. Several lines of evidence had suggested a possible role for abnormalities of airway pH in this setting. These included the observations that (1) citric acid inhalation causes acute cough and bronchoconstriction in guinea pigs (15); (2) inhalation of the carbonic anhydrase inhibitor, acetazolamide, protects against cold air– and sulfite-induced bronchoconstriction in asthmatic humans (16, 17). Of note, pH may be generally reduced in human airway inflammation when measured bronchoscopically (18), though dilution by saliva and/or sampling medium, use of topical anesthetics, reflex responses to airway instrumentation, and limited sample size have complicated interpretation of direct lower airway studies.
Our evidence suggests that abnormalities in condensate chemistry reflect intrinsic abnormalities of the airway lining fluid. Direct comparison revealed that deaerating condensate—to control for variations in pH ex vivo with exposure to atmosphere—yielded pH values that were identical to those of specimens suctioned directly from the tracheobronchial tree. Although airway pH has not before been studied in acute asthma, data from our control subjects are consistent with prior studies of sputum pH (19), and identical to invasive measurement of tracheobronchial secretion pH in 126 patients with artificial airways (20). We excluded salivary, nasal, and gastric contamination from our samples as previously described (2), and showed that there was no association between salivary and condensate pH. We also demonstrated that condensate acidity was not likely to have been caused either by airflow obstruction itself or by inhaled asthma medications, as (1) three consecutive albuterol nebulizer treatments caused no change in pH, and (2) patients admitted for nonasthmatic obstructive pulmonary disease who were treated similarly to patients with asthma had normal condensate pH. Taken together, these observations suggest strongly that acidification of asthmatic airway vapor condensate reflects an intrinsic abnormality in the regulation of nonvolatile species in the lower airway.
Our data suggest that pH is a determinant of NOx concentrations and bioactivities in the airways of subjects with acute asthma. In particular, NO2 − protonation to form nitrous acid (HNO2, negative logarithm of the acid ionization constant [pK a] = 3.4) (21) resulted in evolution of NO gas. Of note, expired NO concentrations are high in asthmatic patients and decrease with glucocorticoid therapy (22). The observations that (1) pH conditions in the airways of subjects with acute asthma favor protonation of the high μM concentrations of airway NO2 − (7)—liberating NO in quantities consistent with those observed in expired air; (2) endogenous airway fluid acidification depletes NO2 −; and (3) pH normalizes with therapy, raise the possibility that NO2 − may serve as an NOx reservoir, converted to NO by airway acidification during an asthma exacerbation.
From a teleological standpoint, airway acidosis may have antimicrobial effects mediated through protonation reactions involving reactive nitrogen and oxygen species. Nitrite acidification has been proposed as a mammalian host defense mechanism (21). The abundant NO2 − of the airway is present as bacteriotoxic HNO2 in relevant quantities only when the pH is low. Mycobacterium tuberculosis produces a gene product specifically protecting against the 'cidal effects of HNO2 (23). Some of the toxicity of HNO2 occurs because of its reactive decomposition to NO, which is known to inhibit mycobacterial growth (24). Additionally, hydrogen peroxide (H2O2), which is elevated in the condensed breath of asthmatic patients (3), acts synergistically with HNO2 to kill gram-negative organisms (21). A decline in airway pH would also favor protonation of the relatively stable NO-superoxide reaction product, peroxynitrite (OONO−), to peroxynitrous acid (HONOO; pK a = 6.8) (25), an oxidizing and nitrating species involved in macrophage-mediated Mycoplasma killing (26). These observations suggest that mild airway acidification may be a subtle and titratable innate host defense mechanism, one which takes advantage of the pK a's of weak endogenous acids to defend the airway against airborne pathogens.
Several pathogens also have developed mechanisms to protect against the 'cidal effect of S-nitrosoglutathione (GSNO) and other S-nitrosothiols (SNO) (27) that are present endogenously in the human airway (28). Though SNO formation and stability are favored at low pH, SNO concentrations are paradoxically low in the airway of children with near-fatal asthma (29). We speculate that accelerated catabolism of GSNO in the asthmatic airway—ultimately forming NO and glutathione— may serve as a compensatory mechanism, preserving levels of anti-oxidant glutathione in the face of the overwhelming nitrosative stress of a fall in airway pH, thus serving as a nitrosative “relief valve.” The airway may be viewed as maintaining an intrinsic nitrosative defense system that switches effector molecules from SNOs to HNO2 during acute asthma. We suggest that this switch could be advantageous—and might be exploited for therapeutic benefit—in subjects infected with an organism adapted to detoxify SNOs, but not HNO2 (30).
Given the array of cytotoxic reactions associated with mild acidification, it is not surprising that the pH found in asthmatic condensates caused necrosis of eosinophils in vitro. In this regard, release of mediators from eosinophils is central to asthma pathophysiology (31), and, in contrast, apoptosis of these cells is associated with resolution of asthmatic inflammation (32). At normal pH, addition of OONO− directly (data not shown) or from the donor SIN-1 suppressed eosinophil apoptosis—similar to the effect of the cell-permeable, low mass endogenous S-nitrosothiol, CGSNO, and other low-molecular-weight NO donors (33). Recent evidence suggests the possibility that these effects may involve S-nitrosylation reactions, inhibiting proapoptotic proteins such as caspase-3 (34). On the other hand, incubation of eosinophils at asthmatic pH induced rapid necrosis. Therefore, we speculate that while eosinophils are maintained (protected from apoptosis) by high concentrations of NOx in the baseline asthmatic airway—permitting low-grade eosinophil-mediated inflammation—a sudden drop in airway pH before or during an asthma exacerbation would cause extensive eosinophil necrosis with an acute release of inflammatory and bronchoconstricting products.
A decline in airway pH may have additional consequences relevant to asthma. Normal ciliary beating is substantially reduced or eliminated when bronchial epithelium is bathed in medium with a pH less than 6.5 ex vivo (35), and acidic pH can increase airway mucous viscosity (36). At pH less than 6.7, epithelial cell membranes are damaged in the tracheal mucosa of cows, in which the normal tracheal pH is 7.9 (37). Further, exogenous introduction of acids to the airway causes bronchoconstriction, in part through stimulation of ion channels in capsaicin-sensitive neurons (38)—including neurons causing kinin-induced bronchoconstriction in the guinea pig lung (15, 39). Taken together with evidence for accelerated eosinophil necrosis and conversion of inert to cytotoxic NOx, these various lines of evidence suggest that there may be a causal relationship between airway acidification and the airflow limitation observed in acute asthma.
In conclusion, we report that airway vapor condensate pH is over two log orders lower than normal in patients with acute asthma, with neutralization occurring during systemic anti- inflammatory therapy. This observation suggests that it may be critical to consider endogenous pH when interpreting the airway biochemistry and cell biology of asthma. For example, we have shown that acidosis at levels seen in subjects with acute asthma causes both necrosis of human eosinophils and conversion of airway NO2 − to NO—effects relevant to asthma pathophysiology. Because differences among control, acutely ill, and treated subjects are robust, and because these simple assays are noninvasive and reproducible, measurement of pH may prove clinically useful as a mechanism for diagnosing and titrating therapy in acute asthma. Further, we speculate that therapies directed at normalizing airway pH early in the course of an acute exacerbation of asthma will help to prevent the cascade of events that leads to airflow obstruction.
The authors would like to express our appreciation to Mark Conaway, Ph.D., for his careful review of the manuscript; and to William Ross for his assistance with flow cytometry.
Supported by The Virginia Thoracic Society (J.H.), NIH 1RO1BL59337 (B.G.), American Lung Association Grant RG-110-N (B.G.), NIH Asthma Center Grant 1U19-A134607 (B.G., T.A.E.P.M.), and the University of Virginia Children's Medical Center.
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