Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma

Abstract

Rationale: Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A(4) (LXA(4)) is an arachidonic acid-derived mediator that serves as an agonist for resolution of inflammation.

Objectives: Airway levels of LXA(4), as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma.

Methods: Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA(4) and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA(4) receptors were monitored by flow cytometry.

Measurements and main results: Individuals with severe asthma had significantly less LXA(4) in bronchoalveolar lavage fluids (11.2 +/- 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 +/- 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA(4) receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes.

Conclusions: Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.

Figure 1.

Airway lipoxin A₄ (LXA₄) and 15(S)-hydroxyeicosatetraenoic acid (15-HETE) levels in severe asthma. Samples of bronchoalveolar lavage fluid (BALF) were obtained from healthy individuals and from subjects with asthma, the latter separated into severe asthma and mild to moderate (“nonsevere”) cohorts on the basis of criteria stated by the Severe Asthma Research Program of the National Heart, Lung and Blood Institute. After extraction, materials were analyzed by HPLC and ELISA (see Methods) for (A) LXA₄ and (B) 15-HETE. Results are expressed as means ± SEM (healthy, n = 17; nonsevere, n = 38; severe, n = 14) for immunoreactive materials. *P < 0.05 in comparison with healthy subjects; **P < 0.05 in comparison between severe and nonsevere asthma cohorts.

Figure 2.

Airway generation of cysteinyl leukotrienes (CysLTs) relative to LXA₄ in severe asthma. (A) Levels of immunoreactive CysLTs in bronchoalveolar lavage fluid (BALF) from healthy individuals and subjects with asthma were determined (see Methods). (B) To normalize comparisons of eicosanoid levels between cohorts, individual ratios of LXA₄ to CysLT levels in BALF were measured. Results are expressed as means ± SEM (healthy, n = 17; nonsevere, n = 38; severe, n = 14). *P < 0.05 for subjects with nonsevere and severe asthma compared with healthy individuals; **P < 0.05 for subjects with severe asthma compared with nonsevere asthma.

Figure 3.

Lipoxin biosynthetic gene expression in blood, bronchoalveolar lavage (BAL) cells, and endobronchial lung biopsies (EBBs). Changes in cycle threshold (ΔC_t) values for (A) 5-lipoxygenase (5-LO), (B) 15-lipoxygenase (15-LO)-1, (C) 15-LO-2, and (D) cyclooxygenase (COX)-2 in subjects with severe and nonsevere asthma were obtained from quantitative polymerase chain reaction results, using cyclophilin A as the control gene (see Methods). Results are expressed as means of ΔC_t values (nonsevere [n = 10], severe [n = 24] for blood; nonsevere [n = 6], severe [n = 6] for BAL cells; and nonsevere [n = 17], severe [n = 14] for EBBs). *P < 0.05 for subjects with severe versus nonsevere asthma; **P < 0.01 for subjects with severe versus nonsevere asthma.

Figure 4.

Relative lipoxin biosynthetic gene expression in peripheral blood, bronchoalveolar lavage (BAL) cells, and endobronchial lung biopsies (EBBs). 5-LO (shaded columns), 15-LO-1 (solid columns), 15-LO-2 (hatched columns), and COX-2 (open columns) gene expression in samples from individuals with severe asthma was analyzed by quantitative polymerase chain reaction, using fluorescence TaqMan technology (see Methods), and compared with samples from individuals with nonsevere asthma; samples represented peripheral whole blood (top), BAL cells (middle), and EBBs (bottom). Results are expressed as means of fold change for severe asthma relative to nonsevere asthma (blood: healthy [n = 7], nonsevere [n = 10], severe [n = 24]; BAL cells: nonsevere [n = 6], severe [n = 6]; EBBs: nonsevere [n = 17], severe [n = 14]).

Figure 5.

Expression of LXA₄ receptor (ALX) RNA and protein in peripheral blood leukocytes. (A) ALX RNA in whole blood from subjects with severe asthma, subjects with nonsevere asthma, and healthy individuals was analyzed by quantitative polymerase chain reaction (see Methods). Results are expressed as the fold change in ALX expression (2^−ΔΔC_t), using the healthy group as the calibrator arbitrarily set at 1 (healthy, n = 7; nonsevere, n = 10; severe, n = 24). (B) ALX surface expression on peripheral blood leukocytes was determined by flow cytometry (ALX antibody, 1:50 dilution) (see Methods). Histograms show the isotype control (shaded) overlaid in boldface by ALX expression in neutrophils. Inset: Numbers indicate the median fluorescence intensity (MFI) minus the MFI of the isotype control. (C) Quantitation of ALX surface expression in leukocyte subsets (neutrophils, eosinophils, lymphocytes, and macrophages) in subjects with severe and nonsevere asthma and healthy individuals. Results are expressed as individual values. *P < 0.05 for asthma subjects compared with healthy individuals.