Non-invasive analysis of leukotrienes as biomarkers of airway inflammation : Method development and clinical applications

Sammanfattning: Asthma and chronic obstructive pulmonary disease (COPD) are two common diseases characterized by chronic airway inflammation, but the inflammation in asthma is different from the one in COPD. In addition, asthma is a heterogeneous disease presenting with many different phenotypes (e.g. allergic asthma, aspirin-intolerant asthma), and different patterns of airway inflammation. A better characterization of the different phenotypes allows more accurate diagnosis and consequently tailor-made therapies. There is a need to develop new non-invasive methods for diagnosis of airway inflammation in asthma and COPD. The use of exhaled breath condensate (EBC) has gained considerable interest as a new non-invasive technique for measuring markers of airway inflammation. The thesis examined the possibility that saliva could be a new, non-invasive means to measure markers of airway inflammation. The specific focus concerned the analysis of leukotrienes (LTs) in EBC and saliva. Leukotrienes generated by the 5-lipoxygenase (5-LO) pathway are potent inflammatory lipid mediators implicated in asthma, allergy and COPD. The cysteinyl-leukotrienes (CysLTs = LTC4, LTD4 and LTE4) mediate central components of asthmatic airway inflammation, whereas leukotriene B4 (LTB4) is a very potent chemotactic and proinflammatory agent. In paper I, LTB4 and the saliva marker alpha-amylase were measured in EBC and sputum, collected before and after challenges that induced a strong neutrophilic inflammation in healthy subjects. For comparison, LTB4 was measured in saliva from healthy subjects. Results: Only four out of 102 EBC samples had detectable LTB4 (28 100 pg/mL). alpha-amylase activity was detected in the LTB4-positive samples. In contrast, LTB4 was detected in all examined sputum supernatants in the same study (median 1,190 pg/mL). The median LTB4 level in saliva was 469 pg/mL. Conclusion: High levels of LTB4 in saliva and the presence of LTB4 in EBC only when alpha-amylase was detected, indicate that LTB4 found in EBC is the result of saliva contamination. In paper II, LT levels in urine, blood stimulated ex vivo, and saliva were compared, using the 5-LO inhibitor zileuton to assess the sensitivity of different sampling methods to detect inhibition of leukotriene formation. Healthy non-atopic subjects and atopic individuals (with or without asthma) were treated with zileuton. Blood, urine and saliva were collected before and after treatment. To investigate further the effects of zileuton, immunoexpression of 5-LO pathway enzymes was quantified in peripheral blood leukocytes by flow cytometry, and fraction of exhaled nitric oxide (FENO) was monitored. Results: LTB4 and LTE4 concentrations in saliva and blood stimulated ex vivo, were significantly decreased after zileuton treatment (p<0.05), but urinary LTE4 was not significantly altered. Flow cytometry showed high levels of 5-LO, FLAP and LTA4 hydrolase expression in neutrophils and LTC4 synthase in eosinophils, but zileuton had no acute effect on the expression of these enzymes. FENO was also unaffected. Conclusion: Leukotrienes in saliva are sensitive and convenient markers of altered 5-LO activity in vivo, as demonstrated by response to zileuton treatment. In paper III, LT levels in saliva were compared with levels in sputum, blood stimulated ex vivo, and urine from subjects with aspirin-intolerant asthma (AIA) and aspirin-tolerant asthma (ATA), respectively. FENO and urinary 9alpha,11beta-prostaglandin F2 (9alpha,11beta-PGF2) were also measured. Samples were collected before and after aspirin provocation. Results: Subjects with AIA had higher basal levels of FENO and CysLTs in saliva, sputum, blood ex vivo and urine than subjects with ATA. There were no differences in basal levels of LTB4 or 9alpha,11beta-PGF2 between the groups. Levels of urinary LTE4 and 9alpha,11beta-PGF2 increased after aspirin provocation in AIA subjects, whereas LT levels in saliva and ex vivo stimulated blood were not increased post challenge. Conclusion: The findings support a global and specific exaggeration of CysLT production in AIA. Measurement of CysLTs in saliva has the potential to be a new and convenient non-invasive biomarker of AIA. Overall conclusions: 1) Saliva is one likely source of LTB4 in exhaled breath condensate; 2) Leukotrienes in saliva may be used as novel and sensitive biomarkers of altered in vivo 5-LO activity; 3) Cysteinyl-leukotrienes in saliva may be used as a new and non-invasive biomarker of aspirin-intolerant asthma.