Inflammatory mediators in acute severe asthma
This thesis is concerned with the measurement of inflammatory mediators in acute severe asthma with particular reference to the high molecular weight neutrophil chemotactic activity (HMW-NCA). Measurements of eosinophil chemotactic activity (ECA), histamine and leukotriene C4 (LTC4) are also documented. The latter half of the study addresses the cellular origin of HMW-NCA and the possible role of monocyte and lymphocyte-derived mediators in acute severe asthma. Serum neutrophil and eosinophil directed chemotactic activities, and plasma histamine and LTC4 were measured in patients with acute severe asthma and compared with control groups (mild asthma, stable chronic irreversible airflow obstruction ie. chronic bronchitis and emphysema, allergic rhinitis, non-infective lung conditions and normal asymptomatic individuals). Statistically significant elevations in serum NCA serum ECA and plasma histamine were detected in acute severe asthma, when compared with control groups. In contrast no differences in plasma LTC4 were observed in acute severe asthma when compared to controls. Serial measurements of serum NCA, ECA and plasma histamine were undertaken on patients with acute asthma during the course of their admission at three time points: 1) day 0 at the time of admission to hospital; 2) after 72 hours during the convalescent phase; and 3) approximately a week later (day 7) at the time of discharge from hospital. Highly significant reductions in serum NCA, ECA and plasma histamine were obtained on discharge (day 7) as compared with admission (day 0), and these changes correlated inversely with the improvement in lung function (PEFR). Gel filtration by fast protein liquid chromatography (FPLC) using Superose 6 prep grade (6PG) indicated that serum NCA in acute severe asthma was heterogeneous, consisting of at least four peaks of activity associated with proteins having molecular weights of approximately 800 kD, 600 kD, and < 20 kD. The 800kD and 150 kD peaks were also observed, but to a lesser degree, in control subjects. The 600 kD and < 20 kD activities were confined to the patients with acute asthma. FPLC chromatofocusing of the 600 MW peak from the acute asthmatics, using a Mono-P column with a pH gradient from 8.3 to 5.0, revealed considerable activity in fractions eluting between pH 6.0 and 7.0 which was not observed in normal controls. A second peak, associated with pI of > 7.0, was observed not only in acute asthma but also in mild asthma and normal subjects. Activity at less than pH 6.0 was seen in most asthmatics as well as controls. Eosinophil chemotactic activity was also significantly elevated in acute severe asthma compared to controls but was confined to a low molecular weight fraction of less than 5 kD. Further characterisation is required for final identification of ECA. No ECA activity was seen in the control group. These findings indicate that eosinophil and neutrophil directed chemotactic factors and histamine are elevated in acute severe asthma, are temporally related to bronchoconstriction and decrease significantly with treatment. The NCA of acute severe asthma and some control groups was heterogeneous but a major peak of chemotactic activity was partially characterised as having a molecular weight of 600 kD and slightly acidic isoelectric point between pI 6.0 and 7.0. The eosinophil chemotactic activity resided in a low molecular weight region as assessed by Superose 6 prep grade gel filtration. These eosinophil and neutrophil chemotactic factors identified in the serum of patients with acute severe asthma resemble the activities previously described in association with allergen- and exercise-induced early- and late-phase asthmatic reactions. The precise cellular source of HMW-NCA remains speculative although circumstantial evidence implicates the mast cell. Recent attention has focussed on the role of the alveolar macrophage, T lymphocyte and cell-mediated immunity in the pathogenesis of asthma. To address these points and to test the hypothesis that lymphocyte and/or monocyte-derived products contribute to the enhanced neutrophil and eosinophil chemotactic activity seen in acute severe asthma, NCA was measured in the supernatants of unstimulated peripheral blood mononuclear cells cultured from patients with acute severe asthma. A dose dependent increase in neutrophil chemotactic activity in the 24 and 48 hour supernatant cultures as compared to mild asthmatics and normal non-asthmatic controls was observed. Chemotactic activity decreased during the recovery phase. Time course studies indicated that mononuclear cell derived neutrophil chemotactic activity was detectable after 12 hours of culture, increased up to 24 hours and was maintained at 48 hours of culture. These observations are in keeping with the properties of a cytokine rather than a preformed mediator. By FPLC gel filtration this mononuclear cell derived-neutrophil chemotactic activity was heterogeneous, consisting of three peaks of activity, associated with molecular weights of 600 kD, 40 kD and less than 15 kD. Eosinophil chemotactic activity and histamine in the unstimulated mononuclear cell supernatants from patients with acute severe asthma is the subject of ongoing investigation. The conclusions derived from this study are that, in addition to mast cell dependent mediators, lymphocyte- and/or monocyte-derived chemotactic factors are involved and acting either singly or in unison, activate or amplify the inflammatory response in acute asthma. They may also account for the local recruitment and activation of eosinophils, neutrophils and mononuclear cells during an asthmatic attack.