Maria Kumlin

The ENFUMOSA cross-sectional European multicentre study of the clinical phenotype of chronic severe asthma

Since severe asthma is a poorly understood, major health problem, 12 clinical specialist centres in nine European countries formed a European Network For Understanding Mechanisms Of Severe Asthma (ENFUMOSA). In a cross-sectional observational study, a total of 163 subjects with severe asthma were compared with 158 subjects whose asthma was controlled by low doses of inhaled corticosteroids (median dose of beclomethasone equivalents 666 µg). Despite being treated with higher doses of inhaled corticosteroids (median dose 1773 µg) and for a third of the severe asthmatics also being treated with regular, oral-steroid therapy (median daily dose 19 mg), the subjects with severe asthma met the inclusion criteria. The criteria required subjects to have undergone at least one asthma exacerbation in the past year requiring oral steroid treatment. Females dominated the severe asthma group (female/male ratio 4.4:1 versus 1.6:1 in the controlled asthmatics), and compared with controlled asthmatics, they had a predominantly neutrophilic inflammation (sputum neutrophils, 36 versus 28%) and evidence of ongoing mediator release but less atopy. From these findings and other physiological and clinical data reported in this paper, it is suggested that severe asthma might be a different form of asthma rather than an increase in asthma symptoms. The findings prompt for longitudinal studies and interventions to define the mechanisms in severe asthma.

Evidence of mast cell activation and leukotriene release after mannitol inhalation

The aim of this study was to investigate if mannitol inhalation, as a model of exercise-induced bronchoconstriction (EIB), causes mast cell activation and release of mediators of bronchoconstriction. Urinary excretion of previously identified mediators of EIB was investigated in association with mannitol-induced bronchoconstriction. Twelve asthmatic and nine nonasthmatic subjects inhaled mannitol and urine was collected 60 min before andfor90 min after challenge. The urinary concentrations of leukotriene (LT)E4, the prostaglandin (PG)D2 metabolite and the mast cell marker 9α,11β‐PGF2 weremeasured by enzyme immunoassay. Nτ‐methylhistamine was measured by radioimmunoassay. In asthmatic subjects, inhalation of a mean±sem dose of 272±56 mg mannitol induced a reduction in forced expiratory volume in one second (FEV1) of 34.5±2.1%. This was associated with increases in urinary 9α,11β‐PGF2 (91.9±8.2 versus 66.9±6.6 ng·mmol creatinine−1, peak versus baseline) and LTE4 (51.3±7.5 versus 32.9±4.7). In nonasthmatic subjects, the reduction in FEV1 was 1.0±0.5% after inhaling 635 mg of mannitol. Although smaller than in the asthmatics, significant increases of urinary 9α,11β‐PGF2 (68.4±6.9 versus 56.0±5.8 ng·mmol creatinine−1) and LTE4 (58.5±5.3 versus 43.0±3.3 ng·mmol creatinine−1) were observed in the nonasthmatic subjects. There was also a small increase in urinary excretion of Nτ‐methylhistamine in the nonasthmatics, but not in the asthmatics. The increased urinary levels of 9α,11β‐prostaglandin F2 support mast cell activation with release of mediators following inhalation of mannitol. Increased bronchial responsiveness to the released mediators could explain the exclusive bronchoconstriction in asthmatic subjects.

Increased urinary excretion of the prostaglandin D2 metabolite 9α,11β-prostaglandin F2 after aspirin challenge supports mast cell activation in aspirin-induced airway obstruction

Prostaglandin (PG)D2 is a major product of arachidonic acid metabolism in pulmonary mast cells. We therefore attempted to determine whether measurement of the stable urinary metabolite of PGD2, 9 alpha, 11 beta-PGF2, could serve as a marker of mast cell activation in the lungs. A commercially available enzyme immunoassay was validated and found to be specific and sensitive when applied to unpurified urine. There was no diurnal variation in the levels of 9 alpha, 11 beta-PGF2 in healthy volunteers. Morning baseline values of urinary 9 alpha, 11 beta-PGF2 were measured in three groups--healthy volunteers (n = 9), patients with atopic asthma (n = 14), and aspirin-intolerant patients with asthma (n = 12)--and found to be very similar, 54 +/- 9, 62 +/- 6, and 71 +/- 15 ng/mmol creatinine, respectively (means +/- SEM). Urinary excretion of 9 alpha, 11 beta-PGF2 was increased threefold immediately after allergen-induced bronchoconstriction in nine patients with atopic asthma. Bronchial challenge with inhaled lysine aspirin in eight aspirin-intolerant patients with asthma produced bronchoconstriction without extrapulmonary symptoms and was also followed by a significant increase in the urinary excretion of 9 alpha, 11 beta-PGF2. In addition, challenge with a higher dose of aspirin produced an even greater increase in urinary 9 alpha, 11 beta-PGF2, supporting dose-dependent release of PGD2 during aspirin-induced bronchoconstriction. In contrast, the postchallenge levels of urinary 9 alpha, 11 beta-PGF2 were not increased when bronchoconstriction was induced by histamine challenge in the aspirin-intolerant patients with asthma. The study confirms mast cell involvement in allergen-induced bronchoconstriction and provides novel data, which strongly support the hypothesis that pulmonary mast cells are activated during aspirin-induced airway obstruction. It is finally suggested that measurement of urinary 9 alpha, 11 beta-PGF2 with enzyme immunoassay may be used as a new noninvasive strategy to monitor mast cell activation in vivo.

Inhibition of mast cell PGD2 release protects against mannitol-induced airway narrowing

Mannitol inhalation increases urinary excretion of 9α,11β-prostaglandin F2 (a metabolite of prostaglandin D2 and marker of mast cell activation) and leukotriene E4. The present study tested the hypothesis that β2-adrenoreceptor agonists and disodium cromoglycate (SCG) protect against mannitol-induced bronchoconstriction by inhibition of mast cell mediator release. Fourteen asthmatic subjects inhaled mannitol (mean dose 252±213 mg) in order to induce a fall in forced expiratory volume in one second (FEV1) of ≥25%. The same dose was given 15 min after inhalation of formoterol fumarate (24 µg), SCG (40 mg) or placebo. Pre- and post-challenge urine samples were analysed by enzyme immunoassay for 9α,11β-prostaglandin F2 and leukotriene E4. The maximum fall in FEV1 of 32±10% on placebo was reduced by 95% following formoterol and 63% following SCG. Following placebo, there was an increase in median urinary 9α,11β-prostaglandin F2 concentration from 61 to 92 ng·mmol creatinine−1, but no significant increase in 9α,11β-prostaglandin F2 concentration in the presence of either formoterol (69 versus 67 ng·mmol creatinine−1) or SCG (66 versus 60 ng·mmol creatinine−1). The increase in urinary leukotriene E4 following placebo (from 19 to 31 ng·mmol creatinine−1) was unaffected by the drugs. These results support the hypothesis that the drug effect on airway response to mannitol is due to inhibition of mast cell prostaglandin D2 release.

Increased levels of cysteinyl-leukotrienes in saliva, induced sputum, urine and blood from patients with aspirin-intolerant asthma

Background: A diagnosis of aspirin-intolerant asthma requires aspirin provocation in specialist clinics. Urinary leukotriene E4 (LTE4) is increased in aspirin-intolerant asthma. A study was undertaken to investigate new biomarkers of aspirin intolerance by comparing basal levels of cysteinyl-leukotrienes (CysLTs) and leukotriene B4 (LTB4) in saliva, sputum and ex vivo stimulated blood in subjects with aspirin-intolerant and aspirin-tolerant asthma. The effects of aspirin- and allergen-induced bronchoconstriction on leukotriene levels in saliva and ex vivo stimulated blood were also compared with the effects of the provocations on urinary mediators. Methods: Induced sputum, saliva, urine and blood were obtained at baseline from 21 subjects with asthma. At a separate visit, 11 subjects showed a positive response to lysine-aspirin inhalation and 10 were aspirin tolerant. Saliva, blood and urine were also collected on the provocation day. Analyses of CysLTs and LTB4 and the prostaglandin D2 metabolite 9α,11β-prostaglandin F2 were performed and the fraction of exhaled nitric oxide was measured. Results: Subjects with aspirin-intolerant asthma had higher exhaled nitric oxide levels and higher baseline levels of CysLTs in saliva, sputum, blood ex vivo and urine than subjects with aspirin-tolerant asthma. There were no differences in LTB4 levels between the groups. Levels of urinary LTE4 and 9α,11β-prostaglandin F2 increased after aspirin provocation whereas leukotriene levels in saliva and ex vivo stimulated blood did not increase. Conclusion: These findings support a global and specific increase in CysLT production in aspirin-intolerant asthma. Measurement of CysLTs in saliva has the potential to be a new and convenient non-invasive biomarker of aspirin-intolerant asthma.

Monitoring mast cell activation by prostaglandin D2 in vivo

Prostaglandin D2 is a useful in vivo marker of mast cell activation in humans While the pro-inflammatory role of eosinophilic granulocytes in asthma is currently under debate, an increasing body of evidence suggests that mast cells may indeed orchestrate many of the characteristic pathophysiological changes in asthma.1 There are also indications that the mast cell may be an effector cell in other lung diseases such as chronic obstructive pulmonary disease2–4 and lung fibrosis.5 Given the location of mast cells at multiple sites within the airways,1 they clearly have the potential to function as sensors of alterations in the microenvironment—be it to inhaled or bloodborne substances, microbes, or other insults that require a prompt host defence reaction. Their versatility is demonstrated by the great number of stimuli that trigger mast cell activation (fig 1). In addition to classical IgE dependent degranulation of mast cells, transduction pathways resulting in mast cell activation may be triggered by, for example, adenosine,6 hyperosmolarity,7 and lipopolysaccharide.8 Figure 1 Mast cells may produce a large number of mediators, enzymes, cytokines and other factors in response to allergic (IgE dependent) or non-allergic activation (adenosine, exercise, endotoxin, mannitol, non-steroidal anti-inflammatory drugs (NSAIDs) in NSAID intolerant subjects, etc). However, only tryptase and prostaglandin (PG) D2 (boxed) are specific markers of mast cell activation. As reported by Bochenek et al in this issue, measurement of PGD2 and its metabolites is currently the most sensitive strategy to monitor mast cell activation in human subjects. LTC4 = leukotriene C4. Although many mast cell mediators or products serve as useful markers of mast cell activation in vitro, it has been notoriously difficult conclusively to establish mast cell activation in human studies. For example, it is difficult to catch the short lived increase in …

Lipoxin formation in human nasal polyps and bronchial tissue

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15‐hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15‐hydroxyeicosatetraenoic acid was 3–5‐times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co‐incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15‐lipoxygenase in lipoxin formation in human airways.

Identification of 11-dehydro-TXB2 as a suitable parameter for monitoring thromboxane production in the human

In order to identify suitable parameters for measurement of thromboxane production in vivo, the metabolism of TXB2 was studied in the human. [3H8]-TXB2 was given intravenously to a healthy human volunteer. Blood samples were collected for 50 min after the injection, and urine was collected for 24 hours. The urinary and blood metabolic profiles were visualized by the use of two-dimensional TLC and autoradiography. Identification of metabolites was achieved with GC/MS and in some cases by cochromatography with reference compounds in TLC and GC. In blood, unmetabolized TXB2 was the dominating compound during the first 30 min. Three less polar metabolites appeared, two of which were identified as 11-dehydro-TXB2 and 11,15-didehydro-13,14-dihydro-TXB2, respectively. The third compound was tentatively identified as 15-dehydro-13,14-dihydro-TXB2. Since 11-dehydro-TXB2 was one of the major metabolites in blood as well as urine, it was deemed suitable as target for measurement of thromboxane production in vivo. The advantages of 11-dehydro-TXB2 over its parent compound, TXB2, were demonstrated in experiments where unlabeled TXB2 was injected i.v. to a human volunteer, and the blood and urinary levels of both compounds were then followed by radioimmunoassay. Measured levels of 11-dehydro-TXB2 were found to give a more reliable picture of metabolic events than TXB2, the latter compound to a large extent reflecting technical difficulties during blood sample collection.

Saliva is one likely source of leukotriene B4 in exhaled breath condensate

Leukotriene (LT)B4 in exhaled breath condensate (EBC) has been reported to be elevated in airway inflammation. The origin of leukotrienes in EBC is, however, not established. The aims of this study are to measure LTB4 levels in EBC collected in two challenges characterised by a strong neutrophilic airway inflammation and to compare LTB4 levels in EBC with levels in sputum and saliva. LTB4 and α-amylase were measured in EBC from 34 healthy subjects exposed in a pig confinement building or to a lipopolysaccharide provocation. These markers were also measured in induced sputum in 11 of the subjects. For comparison, LTB4 and α-amylase were measured in saliva from healthy subjects. Only four out of 102 EBC samples had detectable LTB4 (28–100 pg·mL-1). α-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-1). The median LTB4 level in saliva was 469 pg·mL-1. High levels of leukotriene B4 in saliva and the presence of leukotriene B4 in exhaled breath condensate only when α-amylase was detected, indicate that leukotriene B4 found in exhaled breath condensate is the result of saliva contamination. As leukotriene B4 was consistently present in sputum supernatants, exhaled breath condensate may be inappropriate for monitoring airway leukotriene B4.

15(S)-Hydroxyeicosatetraenoic acid is the major arachidonic acid metabolite in human bronchi: Association with airway epithelium☆

15(S)-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) was by far the most abundant metabolite of arachidonic acid in chopped human bronchi, as identified by reverse phase HPLC, uv spectrometry, and GC/MS. The quantitation of monohydroxyeicosatetraenoic acids (mono-HETEs) was performed by the use of 16(S)-hydroxy-9(Z),12(Z),14(E)-heneicosatrienoic acid as internal standard. Thus, significant amounts of 15-HETE were obtained in incubations of bronchi in buffer alone, but the addition of exogenous arachidonic acid (3-100 microM), dose-dependently increased the formation, with maximal levels reached at around 10 min. In contrast, challenge with ionophore A23187 or anti-human IgE did not stimulate the production of 15-HETE in the bronchi. Nordihydroguaiaretic acid inhibited the production of 15-HETE, whereas indomethacin did not. Small amounts of 8,15-diHETEs were detected in incubations with exogenous 15H(P)ETE. Lipoxins were however not detected under any of the incubation conditions used. Furthermore, removal of the airway epithelium substantially diminished the production of 15-HETE in the bronchi. Finally, bronchi were obtained from three patients with asthma, and the amounts of 15-HETE in these specimens were significantly higher than those found in tissues from nonasthmatics. Also, in peripheral lung parenchyma and pulmonary blood vessels 15-HETE was the major mono-HETE after stimulation with arachidonic acid but the levels were about 10 times lower than in the bronchi. As another difference, challenge of the parenchyma with the ionophore A23187 made 5-HETE the predominant mono-HETE. Taken together, airway epithelium appears to be the major source of 15-HETE in the human lung and the findings in specimens of asthmatics raise the possibility that 15-HETE somehow is involved in airway inflammation.