Elevated serum interferon-gamma in atopic asthma correlates with increased airways responsiveness and circadian peak expiratory flow variation.

Interleukin (IL)-4, IL-5 and interferon (IFN)-gamma are thought to play an important role in chronic airway inflammation in asthmatic subjects. Increased airways responsiveness and nocturnal airway obstruction are important clinical manifestations of asthma. The aim of this study was to investigate whether IL-4, IL-5 and IFN-gamma values are elevated in atopic asthma and correlate with its clinical manifestations. Serum IL-4, IL-5 and IFN-gamma levels of 17 atopic asthmatics and eight nonatopic healthy subjects were determined at 16:00 and 04:00 h by a chemiluminescence enzyme-linked immunosorbent assay (ELISA) method. The clinical manifestation of asthma was determined by assessment of the degree of airway obstruction, airways responsiveness to methacholine and severity of nocturnal airway obstruction, defined as the mean circadian (16:00-04:00 h) peak expiratory flow (PEF) variation. Serum IL-4, IL-5 and IFN-gamma levels were significantly higher in asthmatic subjects as compared to healthy controls, both at 16:00 and 04:00 h. In asthmatic subjects serum IFN-gamma at both time points correlated significantly with the provocative concentration of methacholine causing a 20% fall in forced expiratory volume in one second (PC20,meth) (rho= - 0.55) and with the mean 16:00-04:00 h PEF variation (rho = 0.53). In contrast, no relationship was found between the levels of IL-4 and IL-5 and the parameters of clinical manifestation of asthma. The results suggest that the serum interferon-gamma level is a reflection of the severity of airway inflammation in atopic asthma. More studies are needed to detect the cellular sources and to clarify the exact roles of interferon-gamma and other pro-inflammatory cytokines in asthma.

Submucosa 1.0 x 0.1 mm in size is sufficient to count inflammatory cell numbers in human airway biopsy specimens.

Counting of inflammatory cells in human airway biopsy specimens is difficult because immunopositive cells are present in varying density in lung tissue. The goal of our study was to assess the minimal amount of tissue that is necessary for the counting of constant cell numbers. In bronchial biopsy specimens from 5 healthy controls and 5 patients with asthma, we evaluated 20 successive areas of submucosa 0.1 x 0.1 mm in size. We recorded positive and negative changes of more than 10% in the counted numbers of CD4-, CD8-, and EG2-positive cells. We demonstrated that tissue 1.0 x 0.1 mm in size, along 1-mm basement membrane, is sufficient to obtain constant cell numbers.

Eicosanoids and lipocortin-1 in BAL fluid in asthma: effects of smoking and inhaled glucocorticoids.

Both smoking and asthma are associated with inflammatory changes in the lung, which may be suppressed with the help of exogenous anti-inflammatory drugs or by the endogenous defense system. Lipocortin-1 (LC-1; annexin-1) is an anti-inflammatory protein present in respiratory tract secretions. We report an inverse correlation between extracellular LC-1 concentration and the bronchoconstrictor prostaglandin (PG) D2 [n = 15, Spearman rank correlation coefficient (rS) = -0.597, P < 0.05] in bronchoalveolar lavage fluid (BALF) from allergic asthmatic patients, together with positive correlations between extracellular LC-1 per milliliter BALF and the prostacyclin (PGI2) metabolite 6-keto-PGF1 alpha (n = 15, rS = 0.480, P < 0.05) and between LC-1 per milliliter BALF and concentration of histamine causing a 20% decrease in forced expired volume in 1 s (n = 15, rS = 0.720, P < 0.01) in these subjects. We found no significant difference between the LC-1 concentration in BALF from nonsmoking asthmatic patients who were receiving inhaled glucocorticoid therapy (2 x 100 micrograms beclomethasone 4 times/day for 2.5 yr; median 186 ng LC-1/mg albumin; n = 6) and those who were not (median 126 ng LC-1/mg albumin; n = 12), perhaps because inhaled drugs deposit predominantly in central airways, which are poorly represented in bronchoalveolar lavage. Both asthmatic and healthy volunteers who smoked had higher levels of LC-1 in their BALF than did their nonsmoking counterparts (e.g., asthmatic smokers, median 317 ng LC-1/mg albumin, n = 10; asthmatic nonsmokers, median 162 ng LC-1/mg albumin, n = 18; P < 0.05), perhaps because smokers' lungs contain more alveolar macrophages, cells that release LC-1. We observed a positive correlation between BALF LC-1 and bronchoalveolar lavage cell number (n = 16, rS = 0.821, P < 0.001). Increased extracellular LC-1 may be part of a protective response of the lung to inflammatory insult. Regulation of prostanoid levels might be one mechanism by which LC-1 suppresses inflammation.

Effects of propranolol inhalation on the diurnal increase in FEV1 and on propranolol airways responsiveness in atopic subjects with asthma.

BACKGROUND: Propranolol inhalation provocation tests are used to measure indirect airways responsiveness in the investigation of asthma. In this study the effects of repeated propranolol inhalation provocation tests within the same day on normal diurnal variation in the forced expiratory volume in one second (FEV1) and subsequent propranolol airways responsiveness were investigated. METHODS: Fifteen atopic asthmatic subjects were challenged with doubling concentrations of propranolol at 08.00 and 16.00 hours on the same study day and at 16.00 hours on a control day to exclude changes related to normal diurnal variation. RESULTS: Mean (SD) baseline FEV1 at 16.00 hours on the study day was 3.38 (0.23) 1, significantly lower than the value at 16.00 hours on the control day of 3.70 (0.24) 1 (p = 0.001). No differences were found between the geometric mean provocative concentration of propranolol causing a 20% fall in FEV1 (PC20) measured on the study day (08.00 hours, 9.3 mg/ml; 16.00 hours, 11.3 mg/ml) and on the control day (16.00 hours 9.3 mg/ml). CONCLUSIONS: The results suggest that propranolol provocation at 08.00 hours has a long lasting effect on FEV1, thereby counteracting the normal diurnal increase in diameter of the airways. This makes propranolol challenge tests less suitable for studying indirect airways responsiveness in the course of one day. Because the FEV1 does not return to control values, it is not possible to determine whether tachyphylaxis to repeated propranolol challenge with a time interval of up to eight hours occurs.

Inflammatory cell number and mediators in bronchoalveolar lavage fluid and peripheral blood in subjects with asthma with increased nocturnal airways narrowing.

BACKGROUND: Increased nocturnal airways narrowing (NAN) in asthma is thought to occur as the result of intensification of inflammatory processes in the airways. In this study we investigated the presence of inflammatory cells and mediators in bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) and assessed their relationship with the occurrence of increased NAN. METHODS: BAL fluid and PB samples were assessed at 16:00 and 04:00 hours, separated by 7 days or more, in eight nonatopic healthy subjects (group 1) and 17 atopic subjects with asthma who were using inhaled bronchodilators only. The latter subjects were prospectively assigned to groups with and without NAN, as defined by a mean circadian peak expiratory flow variation of less than 15% (group 2) and 15% or more (group 3), respectively. RESULTS: Significantly higher eosinophil numbers and inflammatory activation products (eosinophil cationic protein, eosinophil-derived neurotoxin, histamine) were found in BAL fluid and PB from subjects with asthma in comparison with control subjects. However, increased NAN was not generally associated with a circadian fluctuation in cell number and inflammatory mediators in BAL fluid and PB. No differences in inflammatory cell numbers existed that distinguished between groups 2 and 3. However, in group 3 significantly higher BAL prostaglandin D2 levels (70 vs 24 pg/ml; range, 28 to 102 vs 11 to 90 pg/ml; p = 0.04) and serum eosinophil cationic protein levels (17.6 vs 16.1 ng/ml; range, 6.3 to 17.5 vs 6.3 to 60.3 ng/ml; p = 0.03) at 16:00 hours were detected compared with group 2. CONCLUSIONS: Our findings suggest that increased NAN is more likely to occur in subjects with asthma with ongoing increased cellular activation during the day.

Lymphocyte and macrophage activation in bronchoalveolar lavage fluid in nocturnal asthma.

Increased nocturnal airway narrowing is thought to occur as a consequence of an intensification of inflammatory processes at night. Lymphocyte and alveolar macrophage (AM) activation are thought to be associated with the clinical expression of asthma, and may be important in the occurrence of nocturnal asthma as well. The expression of CD25 and HLA-DR receptors on lymphocytes from bronchoalveolar lavage (BAL) and peripheral blood (PB) CD4+, as well as of CD14, IgG Fc, and CD11/CD18 leukocyte adhesion receptors on AM in BAL fluid and monocytes in PB, were determined at 16.00 and 04.00 h by flow cytometry. Their relationship with the occurrence of nocturnal asthma was investigated in eight nonatopic controls (Group 1) and 17 atopic asthmatic subjects, prospectively assigned to groups with a mean circadian peak expiratory flow (PEF) variation < 15% (Group 2) and > or = 15% (Group 3). The occurrence of an increased circadian variation in PEF in asthmatic subjects was on the whole not associated with a day-night fluctuation in lymphocyte numbers and subsets in PB or BAL fluid, nor with day-night changes in receptor expression on AM from BAL or monocytes from PB. The only exception was the presence of a greater day-night change in the proportion of HLA-DR-expressing CD4+ lymphocytes in the BAL fluid along with an increasing circadian PEF rhythm in asthmatic subjects (r = 0.68, p = 0.03). A further finding was that a lower number of BAL CD4+ lymphocytes at daytime was significantly related to a higher circadian PEF variation in asthmatic subjects (r = -0.66, p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Lower leukotriene C(4) levels in bronchoalveolar lavage fluid of asthmatic subjects after 2.5 years of inhaled corticosteroid therapy.

Long-term treatment with inhaled corticosteroids has been shown to result in improvement of symptoms and lung function in subjects with asthma. Arachidonic acid (AA) metabolites are thought to play a role in the pathophysiology of asthma. It was assessed whether differences could be found in bronchoalveolar lavage (BAL) AA metabolite levels between subjects with asthma who were treated for 2.5 years with inhaled bronchodilators alone or in combination with inhaled corticosteroids. Prostaglandin (PG)D(2), PGF(2alpha), 6-keto-PGF(1alpha), thromboxane B(2), leukotriene (LT)C(4) and LTB(4) levels and cell numbers were assessed in BAL fluid from 22 non-smoking asthmatic subjects. They were participating in a randomized, double-blind multicentre drug trial over a period of 2.5 years. Results of the group treated with inhaled corticosteroids (CS(+): beclomethasone 200 mug four times daily) were compared with the other group (CS(-)) which was treated with either ipratropium bromide (40 mug four times daily) or placebo. BAL LTC(4) levels of asthmatic subjects were significantly lower after 2.5 years inhaled corticosteroid therapy (CS(+), 9(1-17) pg/ml vs. CS(-), 16(6-53) pg/ml; p = 0.01). The same trend was observed for the PGD(2) levels. The results suggest that inhaled corticosteroids may exert their beneficial effect on lung function via a mechanism in which inhibition of LTC(4) synthesis in the airways is involved.

Inflammation in nocturnal asthma?

At present there is some indirect evidence for increased nocturnal inflammation in patients suffering from nocturnal asthma: 1. Circulating eosinophil numbers and activation, as reflected by increased levels of ECP and EDN and low-density eosinophils, are increased at night. 2. Circulating histamine levels are increased at night. 3. Hyperresponsiveness to AMP at night is increased compared with hyperresponsiveness to methacholine. However, most results of various studies point to nocturnal asthma's being an expression of more severe asthma: 1. Both AMP and propranolol responsiveness, indirect measures of airway hyperresponsiveness, are lower both at 4:00 A.M. and 4:00 P.M. in asthmatics with nocturnal asthma than those without nocturnal asthma. 2. Patients with nocturnal asthma have higher circulating numbers of eosinophils at both 4:00 A.M. and 4:00 P.M. than those without nocturnal asthma, and eosinophil survival is not different at these times. 3. Patients with nocturnal asthma have higher PGD2 levels in BAL both at 4:00 A.M. and 4:00 P.M. than those without nocturnal asthma, but show no significant difference between levels at these two times. 4. Two studies have shown no difference in BAL eosinophil numbers and activation parameters at night in nocturnal asthma. 5. Histamine levels in BAL fluid are comparable day and night in patients with and without nocturnal asthma. 6. Inflammatory mediators in BAL are higher in asthmatic patients than in normal subjects, but are not different between patients with and without nocturnal asthma. Thus, patients with nocturnal asthmatic symptoms show an overall increased burden of mediators released from mast cells and other inflammatory cells. In conclusion, we feel that the term "nocturnal asthma" is misleading, in that it does not describe a unique entity in certain patients with asthma. We prefer, in view of the previous arguments, to consider nocturnal asthma a mere expression of more severe asthma. Thus we suggest the term "nocturnal asthma" be changed to "asthma with nocturnal symptoms."

Epidemiology and the concept of underlying mechanisms of nocturnal asthma.

Nocturnal symptoms are common in asthma, even when patients are regularly seen at an outpatient clinic. Inflammation is generally accepted as a general feature of asthma and the severity of this basic inflammatory process can be increased by exogenous triggers such as exposure to allergens and nonallergic stimuli. Superimposed endogenous circadian rhythms may play a more important and intricate role in the circadian modulation of the inflammatory process by changing the number of cells, their release of mediators and/or the susceptibility of airway smooth muscle and vasculature. For instance, an increase in vagal tone may induce nocturnal bronchoconstriction which is further enhanced by falling catecholamine levels. Together, the reduced nocturnal catecholamine levels and the diminished bronchodilating capacity of the NANC system and the low cortisol levels oppose possible protection against inflammatory processes leading to nocturnal airflow obstruction.

Circadian variation in airway responsiveness to methacholine, propranolol, and AMP in atopic asthmatic subjects.

Increased airway hyperresponsiveness is thought to be one of the phenomena underlying nocturnal airway obstruction in asthma. To investigate the mechanisms that influence and modulate this phenomenon, we compared circadian variations in airway responsiveness with AMP and propranolol with the circadian variation in airway responsiveness to methacholine. Inhalation provocation tests were performed at 16.00 and 04.00 h in 16 nonsmoking atopic asthmatic subjects (18 to 42 yr of age), prospectively assigned to Group 1 (mean circadian peak expiratory flow rate [PEFR] variation > or = 15%) and Group 2 (< 15%). The circadian change in airway responsiveness to AMP, in contrast to methacholine, was significantly related to the circadian PEFR-variation of the 16 subjects (r = 0.81, p < 0.001). In Group 1 (n = 7) geometric mean PC20 AMP decreased more than PC20 methacholine during the night (2.3 and 0.9 doubling concentrations respectively, p < 0.05), whereas no difference in baseline FEV1 was found at the same time points during the different study days. Geometric mean PC20 propranolol did not change during the night. Daytime PC20 propranolol and PC20 AMP, in contrast to PC20 methacholine, were significantly lower in Group 1 as compared with Group 2. Together, the results show a higher susceptibility to stimulation of "indirect" airway responsiveness in the subjects with increased circadian PEFR amplitude. This suggests that mast cell activation rather than primary changes in smooth muscle cell contraction may play a role in the development of nocturnal airway obstruction.

Airway responsiveness to adenosine 5'-monophosphate in chronic obstructive pulmonary disease is determined by smoking.

In contrast to methacholine, a stimulus that induces airway constriction mainly by "direct" stimulation of airway smooth muscle cells, AMP airway responsiveness reflects "indirectly" induced airway narrowing via inflammatory or neural reflex mechanisms. In order to determine inflammatory contribution to airway narrowing in COPD, we performed AMP and methacholine inhalation provocation tests in nonatopic subjects with COPD and compared the results with those obtained from atopic nonsmoking asthmatics and from healthy smoking volunteers. AMP caused airway narrowing in all but two subjects with COPD and in only three of the 12 healthy smoking subjects. Patients with COPD were significantly more responsive to AMP and methacholine than were healthy smoking volunteers. Geometric mean PC20 AMP was significantly lower in the smokers with COPD (7.2 mg/ml) than in the nonsmokers with COPD (58.5 mg/ml), whereas PC20 methacholine values and baseline FEV1 were comparable. In the nonatopic nonsmoking subjects with COPD, PC20 AMP was significantly higher than in the atopic nonsmoking asthmatics (3.8 mg/ml), whereas they responded similar to methacholine provocation. These results indicate that most subjects with COPD respond to AMP provocation and that smoking determines the degree of airway responsiveness to AMP in COPD. We suggest that increased susceptibility to mediator release by mast cells or neural reflex mechanisms are involved in AMP-induced airway constriction in asthma and in COPD.

There is no activation of O2- production by alveolar macrophages and neutrophil polymorphonuclear leukocytes in rat lung transplants during the reimplantation response and acute rejection.

Activation of phagocytes (alveolar macrophages [AM] and neutrophil polymorphonuclear leukocytes [PMN]) can cause tissue damage in inflammatory lung diseases. In this study we investigated whether phagocytes contribute to the development of tissue damage in lung grafts histologically observed during two different processes: the reimplantation response and the acute rejection. Therefore, the number and profile of bronchoalveolar lavage (BAL) and blood phagocytes and their in vitro spontaneous and serum-treated-zymosan (STZ)-stimulated O2- production were assessed after allogeneic (BN to LEW) and syngeneic (LEW to LEW) transplantation of the left lung in rats. BAL PMN numbers increased during the reimplantation response, whereas during the late phase of the rejection process BAL AM and PMN numbers were increased. The O2- production by the BAL phagocytes and blood PMN were not increased at any stage. Strikingly, the STZ-stimulated O2- production by the BAL phagocytes was significantly impaired during acute rejection. Our data suggest that activation of the O2- production by bronchoalveolar phagocytes does not play an important role in the development of tissue damage in lung transplants during the reimplantation response and acute rejection. The impaired O2- production by alveolar phagocytes during acute lung rejection may contribute to the increased susceptibility for pulmonary infections after lung transplantation.