Extracellular purines are biomarkers of neutrophilic airway inflammation

Abstract

Purinergic signalling regulates airway defence mechanisms, suggesting that extracellular purines could serve as airway inflammation biomarkers in cystic fibrosis (CF). The purines adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and adenosine were measured in sputum from 21 adults (spontaneously expectorated from seven CF patients, induced from 14 healthy controls) to assess normal values and CF-associated changes. Subsequently, purine levels were measured in bronchoalveolar lavage fluid (BALF) from 37 children (25 CF patients, 12 disease controls) and compared with neutrophil counts, presence of airway infection and lung function. To noninvasively assess airway purines, ATP levels were measured using luminometry in exhaled breath condensate (EBC) from 14 children with CF and 14 healthy controls, then 14 CF children during a pulmonary exacerbation. Both ATP and AMP were elevated in sputum and BALF from CF subjects compared with controls. In BALF, ATP and AMP levels were inversely related to lung function and strongly correlated with neutrophil counts. In EBC, ATP levels were increased in CF relative to controls and decreased after treatment of CF pulmonary exacerbation. The purines adenosine triphosphate and adenosine monophosphate are candidate biomarkers of neutrophilic airways inflammation. Measurement of purines in sputum or exhaled breath condensate may provide a relatively simple and noninvasive method to track this inflammation.

FIGURE 1

Airway purines were elevated in cystic fibrosis (CF). a) Sputum was obtained by spontaneous expectoration from adults with CF (n=7; ●) and after induction in healthy controls (n=14; ○), and purines measured in isolated supernatants. Both adenosine triphosphate (ATP) and adenosine monophosphate (AMP) were significantly elevated in CF compared with control, while adenosine diphosphate (ADP) and adenosine (Ado) levels were similar between groups. b) Supernatant of mucopurulent material (SMM) was obtained from the explanted lungs of adults with end-stage CF at time of lung transplantation (n=13). The pattern of purine levels in SMM was similar to that observed in CF sputum. #: p<0.0001.

FIGURE 2

Purines in bronchoalveolar lavage fluid (BALF) from children were elevated in cystic fibrosis (CF) and correlated with neutrophilic airway inflammation. a) BALF was obtained from children with CF (n=25; ●) and from disease controls (n=12; ○) with other respiratory diseases. Measurement of purines in the supernatant of this BALF revealed elevated levels of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) in the CF group. b) Statistically significant correlations were observed between polymorphonuclear neutrophil (PMN) counts and levels of both ATP (□) and AMP (■) in BALF. ADP: adenosine diphosphate; Ado: adenosine. ^#: p50.0164; ^¶: p50.0300. b) ATP: r=0.81, p<0.0001; AMP: r=0.74, p<0.0001.

FIGURE 3

Purine ratios correlated with neutrophilic airway inflammation and lung function. Ratios of adenosine triphosphate (ATP) to adenosine (Ado; □) and adenosine monophosphate (AMP) to Ado;■ were calculated as markers of purine levels corrected for variable dilution of airway secretions in bronchoalveolar lavage fluid. a) Both ATP/Ado and AMP/Ado ratios were strongly correlated with percentage of polymorphonuclear neutrophils (PMNs) as a dilution-independent marker of inflammation. b) Purine ratios were compared with spirometry data available from a subset of subjects (n=28, 20 cystic fibrosis and eight disease controls). Both ATP/Ado and AMP/Ado ratios were negatively correlated with forced expiratory volume in one second (FEV₁ % predicted). a) ATP/Ado: r=0.76, p<0.0001; AMP/Ado: r=0.67, p<0.0001. b) ATP/Ado: r= −0.41, p=0.0335; AMP/Ado: r= −0.43, p=0.0240.

FIGURE 4

Adenosine triphosphate (ATP) in exhaled breath condensate (EBC) was elevated in cystic fibrosis (CF) and decreased with treatment of a CF exacerbation. ATP levels in EBC were measured by luminometry. a) Pilot EBC collections were obtained from four healthy controls (subjects 1–4) and four CF subjects (5–8). A luminescent signal was detected in all samples that was stable to incubation at 37°C for 30 min (), compared with samples kept at 0°C on ice (■), and disappeared after incubation with 2 units·mL⁻¹ of the ATP degrading enzyme apyrase (□). b) ATP levels were measured in EBC from CF (n=14) and healthy controls (n=14). ATP was elevated in the CF group. c) ATP levels in EBC were measured at the start and end of a course of antibiotics to treat a pulmonary exacerbation in children with CF (n=14). ATP levels were lower at the end of treatment. ALU: arbitrary light units. ^#: p=0.0441;^¶: p=0.0175.