Chemotactic and Phagocytic Activity of Blood Neutrophils in Allergic Asthma

Allergic asthma is a chronic inflammatory airway disease, and has been considered a T helper-2-biased response. Studies suggest that neutrophils may be associated with exacerbation and asthma severity. We sought to evaluate the chemotactic activity and phagocytic capacity by peripheral blood neutrophils from individuals with controlled and uncontrolled allergic asthma, and compare the results with non-asthmatic controls groups. Blood neutrophils were isolated from 95 patients: 24 with controlled asthma, 24 uncontrolled asthma, 24 healthy subjects and 23 patients with IgE-mediated allergies other than asthma. The neutrophil chemotaxis, stimulated with LPS, autologous serum or homologous serum, was determined using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by NBT test. The phagocytic digestion phase and chemotaxis by neutrophils from asthmatic patients was higher than in non-asthmatic controls (p??<?0.05). Autologous serum-induced neutrophil chemotaxis in patients with uncontrolled asthma was greater (p??<?0.05) than in other study groups. The ingestion phase of phagocytosis showed similar values in asthmatics and non-asthmatics. We conclude that the blood neutrophil from controlled and uncontrolled asthmatic patients exhibit activation markers, particularly phagocytic digestion and chemotactic activities.     

Effect of exercise on isoprenaline-induced lymphocyte cAMP production in atopic asthmatics and atopic and non-atopic, non-asthmatic subjects

The effect of exercise on isoprenaline-induced cyclic adenosine monophosphate (cAMP) production was studied in peripheral-blood lymphocytes obtained from ten patients with atopic asthma, seven subjects who were atopic but did not have asthma and eight non-atopic, non-asthmatic control subjects. The asthma in the atopic subjects was mild only requiring intermittent treatment with inhaled β adrenoceptor agonists, none of which were taken in the 48 hr prior to the study. Exercise consisted of a standardized 6-min run on a treadmill sufficient to raise the subject's pulse rate to > 160 bpm and respiratory function was measured before and at 5,10,15,20,30 and 60 min after the test. Blood samples were taken 5 min before and at 10 and 60 min after exercise, lymphocytes were separated by density gradient centrifugation and cAMP measured by a competitive radioimmunoassay. Exercise led to a significant decrease (27%) in the forced expiratory volume in I sec (FEV₁) in the ten atopic asthmatic subjects but no change (< 3%) in the non-atopic and atopic non-asthmatics. There was no significant difference in the unstimulated cAMP levels before exercise in the three groups, but stimulation with isoprenaline caused a significantly greater rise in cAMP in the non-atopic, non-asthmatic subjects when compared to both the atopic asthmatics and the atopic subjects without asthma. Exercise led to a significant elevation of cAMP in all three groups of subjects, but the same differences between the groups remained. These results suggest that there are differences in lymphocyte β receptor function not between patients who are asthmatic or non-asthmatic but between individuals who are atopic as opposed to non-atopic.     

Atopic asthma: differential activation phenotypes among memory T helper cells

Background T cells have been implicated in the pathogenesis of atopic asthma. We have previously shown that memory T helper cells (CD4⁺CD45RO⁺) are preferentially activated relative to naïve T helper cells (CD4⁺CD45RA⁺) after bronchial allergen challenge. However, specific T helper subpopulations that are activated in atopy and/or asthma remain undefined. Objective To determine the T helper subpopulations and activation phenotypes relevant to acute and stable asthma that may be common with or distinct from atopy. Methods Two groups of atopic asthmatics (ten acute and nine stable asthmatics) and two non‐asthmatic groups (14 non‐asthmatic atopics and eight normal non‐atopic controls) were analysed. Ten acute asthmatics were assessed in the emergency room during an acute episode (FEV₁ 43.6% ± 18.4). Nine stable asthmatics were assessed during a symptom‐free period (FEV₁ 85% ± 6). Using multiple colour flow cytometry we analysed T cell subpopulations and the expression of IL‐2‐receptor (IL‐2R) and MHC‐class II antigens (MHC II) on naïve and memory T helper cells in the peripheral blood of asthmatic and non‐asthmatic groups. Results Atopic asthmatics (acute and stable) had an increased percentage of memory T helper cells expressing IL‐2R compared with normal non‐atopics (mean SD 16.1 ± 6%, 12.4 ± 2% and 7.7 ± 1.8%, P < 0.05) but not compared with non‐asthmatic atopics (10 ± 3.5%). Naïve T helper cells had low expression of IL‐2R and MHC II in all four groups. MHC II antigen expression was increased in memory T helper cells of asthmatics (acute and stable) compared with normal non‐atopics (13.9 ± 7.5, 10.6 ± 5 and 4.9 ± 2.5, P < 0.05) but not compared with non‐asthmatic atopics (7.92 4). A novel finding was that IL‐2R and the MHC II molecules were mainly expressed in non‐overlapping populations and coexpression was found predominantly on memory T helper cells. Asthmatics (acute and stable) had higher proportion of double positive memory T helper cells (IL‐2R⁺MHC II⁺) compared with both non‐asthmatic groups (P < 0.05). Conclusions We demonstrate a differential expression of IL‐2R⁺ and MCH II⁺ on CD45RO⁺ T helper cells that would suggest that there are three subsets of activated memory T helper cells in asthmatics. Two non‐overlapping IL‐2R⁺ or MHC II⁺ CD45RO⁺ T helper cells and a third subpopulation of activated cells that coexpress IL‐2R and MHC II (double positives). This latter subpopulation is significantly higher in asthmatics (acute or stable) compared with both non‐asthmatic groups, suggesting a specific T helper activation phenotype distinct to atopic asthmatics as compared with atopic non‐asthmatics.     

Comparison of the effects of salmeterol and salbutamol on clinical activity and eosinophil cationic protein serum levels during the pollen season in atopic asthmatics

Background In atopic asthma there is strong evidence of eosinophils playing an active role in pathogencsis. Some investigations demonstrated that eosinophil cationic protein (ECP) serum levels increased in atopic patients with asthma during pollen season. Objective The aim of the study was to evaluate the effects of short-term (1 week) β2-agonist treatment on lung function and eosinophil activity in asthmatic patients. Methods We used an open, randomized, cross-over design to compare the effects of salbutamol (200μg q.i.d.) and salmeterol (50μg b.i.d.) on peak expiratory flow rate (PEFR), blood eosinophil count and serum levels of ECP as a measure of eosinophil activity in 20 mild atopic asthmatics. Results Morning and evening PEFR values were both significantly higher during salmeterol treatment than during the salbutamol period. Conversely, both morning and evening daily asthma symptom scores were significantly lower during salmeterol treatment compared with those recorded during the salbutamol period. The mean basal eosinophil blood count on salmeterol treatment (601 ± 189mm³) was not higher than the mean count on salbutamol treatment (612 ± 204 mm³). After both treatments the mean eosinophil blood counts were unchanged (619 ± 189mm³ and 576 ±212 mm³, respectively). No significant differences in blood eosinophil counts were observed between or within treatments at any time. No significant difference was observed in baseline mean ECP serum concentration (43.8 ± 263 μg L on salmeterol treatment and 41.7 ± 29.8 μg L on salbutamol treatment, respectively). After salmeterol treatment the mean ECP serum concentration had fallen significantly to 20.9± 18.6μg/L (P < 0.01), whereas after salbutamol treatment it was unchanged (42.0 ± 25.1 μg /L). Salmeterol treatment produced a decrease in ECP serum levels without any changes in blood eosinophil count. Conclusion This study demonstrates that salmeterol affords a significant improvement in asthma control during the pollen season, measured by both subjective and objective parameters, compared with salbutamol. This greater efficacy may be related to inhibition of eosinophil degranulation during the pollen season.     

Inhibition of neutral endopeptidase potentiates bronchoconstriction induced by neurokinin A in asthmatic patients

The endogenous tachykinins exhibit a range of properties which may he relevant in the pathophysiology of asthma. Their effects on the airways seem to be modulated by a variety of lung peptidases, including neutral endopeptidase (NEP). In order to evaluate the potential role of endogenous NEP activity in modulating tachykinins-induced bronchconstriction in man in vivo, six atopic asthmatic patients, with a mean FEV₁ value of 3.38 ± 0.76 1, and a histamine PD20 mean value of 0.024 mg. were studied. The influence of inhaled phosphoramidon (a potent NEP inhibitor) was examined against the NKA-induced bronchospasm in a double-blind, placebo-controlled randomized study. Changes in airway calibre were followed as FEV₁ and agonists responsiveness expressed as PD20 and PD15 for histamine and NKA respectively. Patients received nebulized phospharamidon sodium salt (10^?5 M) or a control solution 10 min prior to the bronchoprovocation test with NKA. No significant difference was noticed between any of the study days and after inhaled phosphoramidon on baseline FEV₁ values (3.29 ± 0.90 1) in comparison with the control solution (3.31 ± 0.79 1). Inhaled NKA produced a dose-dependent fall in FEV₁ values in all the subjects studied with a mean PD15 value of 20.91 × 10^?9 mol. Phosphoramidon administered by inhalation elicited a significant (P < 0.0l vs baseline and control solution) potentiation in the airway responsiveness to inhaled NKA, the NKA PD15 value decreasing to 9.45 × 10^?9 mol. The present study confirms that inhaled NKA induces a dose-related bronchconstriction in asthmatic patients and demonstrates that inhaled phosphoramidon potentiates NKA-induced bronchoconstriction.     

Expression of CCR8 is increased in asthma

Background Chemokines and their receptors could play key roles in the recruitment of T cells to the asthmatic lung. CCR8 is preferentially expressed on T‐helper type 2 cells, and is thought to play a role in the pathogenesis of human asthma. Objective Determine the expression of CCR8 on T cells in blood, bronchoalveolar lavage (BAL) and bronchial mucosa from asthmatics and normal subjects. Methods CCR8 expression in blood and BAL from asthma and normal subjects was studied using flow cytometry. CCR8 expression on IFN‐γ⁺ and IL‐4⁺/IL‐13⁺ blood and BAL T cells was studied following stimulation with Phorbol–Myristate–Acetate and Calcium Ionophore. Paraffin‐embedded bronchial biopsies were used to study CCR8 in bronchial epithelium. Results The percentage of CD3⁺ cells expressing CCR8 in the blood was higher in asthmatics (4.7±0.4%) compared with normal subjects (3.0±0.4%; P<0.01). There was an approximately sixfold enrichment of CCR8 on IL‐4⁺/IL‐13⁺ cells compared with IFN‐γ⁺ T cells (P<0.001) in both asthmatic and normal subjects in both blood and BAL. Significantly more BAL T cells expressed CCR8 in asthmatic (8.6±0.8%) compared with normal subjects (3.9±0.7%) (P<0.01). In paired blood‐BAL samples from asthmatics, significantly more CCR8+CD3⁺ T cells were present in BAL (9.0±0.9%) than in blood (5.6±0.9%; P<0.05). There were more CCR8‐positive cells in bronchial biopsies from asthmatic (93±11 cells/mm²) compared with normal subjects (30±16 cells/mm²) (P<0.05). The ligand CCL1 was increased in the BAL of asthmatics compared with normal subjects (35±6 vs. 12.9±7 pg/mL; P<0.05). Conclusion There may be a role for CCR8 in the recruitment of T cells to the lung in asthmatics. Cite this as: K. Mutalithas, C. Guillen, C. Raport, R. Kolbeck, D. Soler, C. E. Brightling, I. D. Pavord and A. J. Wardlaw, Clinical & Experimental Allergy, 2010 (40) 1175–1185.     

The effect of verapamil on histamine and methacholine-induced bronchoconstriction

Calcium antagonist, verapamil given by inhalation did not alter histamine bronchial hyper-reactivity in ten patients with extrinsic bronchial asthma and similarly did not modify methacholine-induced bronchoconstriction in further five patients. In eight non-asthmatic subjects verapamil reduced histamine sensitivity with increase in PC_20H from 8.07(±2.33) to 12.10(±2.71, P < 0.05) but failed to have an effect on methacholine sensitivity in five controls. The failure of inhaled verapamil to modify histamine and methacholine bronchial hyper-reactivity in asthmatic patients and the beneficial effect of calcium antagonists in exercise asthma suggests that these agents may act predominantly on the mast cell degranulation rather than the bronchial smooth muscle.     

Exhaled nitric oxide of childhood asthma]

Chronic airway inflammation is a central feature of pathology of bronchial asthma. In order to evaluate inflammatory status in asthma, examinations such as bronchoscope or induced sputum test can be done. Because of difficulty of those examinations we need non-invasive and simple measures for childhood asthma. Here we investigated eNO in childhood asthma. Twenty-six of atopic asthma, 13 non-asthmatic atopic children and 12 normal children were enrolled in this study. eNO was measured by chemiluminescence analyzer. eNO was significantly collerated with % FEV 1.0 and blood eosinophil counts (R = -0.494, R = 0.416, respectively). Geometrical mean of eNO in normal, non-asthmatic atopic, asthma without inhaled corticosteroid (ICS) and asthma with ICS was 16.3, 23.7, 71.6, 43.6 ppb, respectively. eNO was significantly higher in asthma than in normals. eNO in patients without ICS were significantly higher than in non-asthmatic atopic. We concluded that eNO might be useful marker for evaluation of airway inflammation in asthmatic children.     

Eosinophils from patients with asthma express higher levels of the pan-leucocyte receptor CD45 and the isoform CD45RO

BACKGROUND: Eosinophils and their secreted mediators are heavily implicated as effector cells in asthma and other allergic diseases. Comparisons were made between expression of CD45, CD45RA, CD45RB and CD45RO by eosinophils from asthmatic patients and non-asthmatic atopic and non-atopic, non-asthmatic control subjects. METHODS: Twenty-seven patients with asthma and 33 control subjects were recruited for the study. Eosinophil expression of CD45, CD45RA, CD45RB and CD45RO was established by immunostaining and flow cytometry was performed on whole leucocyte samples. Eosinophil apoptosis in response to CD45 and CD45 isoform monoclonal antibody (mAb)-dependent receptor ligation was assessed by binding of annexin V and flow cytometry. RESULTS: Eosinophils from patients with asthma expressed significantly (P<0.05) higher levels of pan-CD45 and CD45RO compared with eosinophils from non-asthmatic, non-atopic subjects. No significant correlations were found between expression of either pan-CD45 or CD45RO and the degree of symptoms in the asthmatic patients as defined by lung function (FEV1 and FEF25-75) and methacholine PD20. Increased expression of pan-CD45 or CD45RO did not appear to be a consequence of the atopic phenotype. Higher expression of pan-CD45 or CD45RO by eosinophils from asthmatic patients was not associated with greater sensitivity to CD45 and CD45RO mAb receptor ligation-induced eosinophil apoptosis. CONCLUSION: Higher expression of CD45 and CD45RO by eosinophils from asthmatic patients appeared to be a consequence of asthma rather than atopy and further supports a role for activated eosinophils in asthma.     

American cockroach Cr-PI allergen induces lymphocyte proliferation and cytokine production in atopic patients

Background Previous studies have shown cockroach-induced antigen-specific IgF-mediated asthma. In cockroach-Infested areas, more then 50% of asthmatic subjects may have positive skin reactions to this allergen. Partial purified Cr-PI allergen from American cockroaches contains allergens with molecular weights of 72 and 78k Da; however, little is known about its eifect on the lymphoeyte proliferation and cytokine production. Objective IgE synthesis is known to be regulated by interleukin-4 (IL-4) and interferon gamma (IFN-γ). Therefore, we studied Cr-PI allergen-induced cytokine production in atopic patients and healthy normal controls to understand each factors’ role in the disease. Methods Peripheral blood mononuclear cells (PBMC) from cockroach skin-sensitive patients and controls were stimulated with mitogen and Cr-PI for proliferative response and cytokine production. Cr-PI antigen-specific T-cell cultures of atopic patients and healthy normal controls were used to test C r-PI-lnduced proliferation and cytokine mKNA expression. Results PMBC ot atopic subjects showed a significantly (P < 0.01) higher stimulation index for Cr-PI induced proliferation (SI = l l.8.3.7) when compared with that of non-atopic subjects (SI =4.1 ± 0.8) and cord bloods (SI = 2.1 ± 0.4). Cr-Pl-induced IL-4 was observed only in the PBMC of atopic patients, whereas Cr-PI-induced IFNγ was detected in both atopic patients and normal controls. Likewise, Cr-PI-induced IL-4 mRNA expression in T-cell cultures was detected in all atopies but only one of nine controls. Conclusion IL-4 mKNA expression and IL-4 production in PBMC and T-cell cultures of atopic patients showed good correlation with clinical symptoms, skin-reactivity, specific IgE and proliferative response to Cr-PL These results suggests that cockroach allergen may be a hidden cause of asthma and other atopie diseases.     

Evidence of a strong, positive association between atopy and the HLA class II alleles DR4 and DR7

Background Atopy, with or without associated asthma, provides a useful model for evaluating the genetic factors that control human immune responsiveness. HLA class II gene products are involved in the control of immune responses. Objectives We investigated whether susceptibility or resistance to the disease was associated with HLA class II genes. Methods Blood samples were obtained from two groups of unrelated European-born white adults: 56 atopic patients (52 of them with asthma) and 39 healthy controls with no personal or familial history of asthma or atopy. Genomic DNA was extracted from peripheral blood lymphocytes. The exons of DQA1, DQB1, DRB and DPBl genes were selectively amplified by the polymerase chain reaction (PCR) method. Geno-typing was carried out by digestion of the amplified DNA products with allele-specific endonucleases (PCR-RFLP), which can recognize allelic variations in the polymorphic exon. Results We found no significant difTerences in the frequency of DPBl alleles between patients and controls. HLA class II DR4 and DR7 alleles were present in 39.2% of the patients and in 2.5% of the healthy subjects (Pc*²± 3.9 10^?3). Conversely, DQA1*0103 and DQB1*0502 alleles were more frequent in the control subjects. These results confirm a previous study of an extended pedigree, which showed that DR4 and DR7 alleles were absent in all healthy members of the family and were frequently observed in atopic and/or in asthmatic subjects. Conclusion We observed that HLA-DR 4 and DR7 alleles are significantly implicated in their susceptibility to the disease and suggest that this susceptibility is more related to atopy than to specific responses to allergens. According to previous studies, we could also submit that in atopic patients with asthma, DR4 alleles at the least, could be more closely associated with atopy than with asthma per se. Conversely, we suggest that some allelic DQA1 and DQB1 sequences might confer protection against the disease.     

Lymphocyte glucocorticoid receptors in asthmatic and control subjects

Glucocorticoid hormones, which are widely used in the treatment of asthma, have been shown to potentiate physiological and biochemical beta-adrenergic responsiveness in asthmatics. These effects are presumably mediated through glucocorticoid receptors. In order to better understand glucocorticoid pharmacology in asthmatics, we assayed glucocorticoid receptors by directly binding a radioactively labelled glucocorticoid hormone, dexamethasone, to intact lymphocytes prepared from the peripheral blood of asthmatics and control subjects. Binding studies were performed with dexamethasone at 100 nm and 5 nm concentrations. At 100 nm dexamethasone, the mean number of lymphocyte glucocorticoid receptors (per cell) in control subjects (7191 ± 385. n= 9) was not significantly different from that in asthmatic subjects (7772 ± 437, n = 9). At 5 nm dexamethasone, the mean number of glucocorticoid receptors in control subjects (1177 ± 194, n= 5) was not significantly different from that in asthmatic subjects (1215 ± 108. n= 8). At 100 nm dexamethasone, males had significantly more receptors (7939 ± 360. n= 11) than females (6764 ± 72, n= 7). Our results suggest that the number of lymphocyte glucocorticoid receptors and the apparent affinity of dexamethasone for receptors are not related to the presence or severity of asthma; however, a significant sex effect exists which should be corrected for in future studies of lymphocyte glucocorticoid receptors.     

Inhaled salbutamol decreases blood ammonia levels during exercise in normal subjects

The present study examines the effect of salbutamol, a β2-adrenoreceptor agonist, on blood ammonia levels during an incremental cycle exercise test in healthy non-asthmatic subjects. Blood ammonia levels were lower after inhalation of 400 mcg of salbutamol than after placebo during submaximal exercise: 33?±?2 μmol?·?l^?1 vs 48?±?9 μmol?·?l^?1 at 220?W and 39?±?2 μmol?·?l^?1 vs 50?±?4?μmol?·?l^?1 at 260?W. At peak exercise there were no significant differences. The results suggest that β2-adrenoreceptors are involved in the regulation of blood ammonia during exercise.     

Interrelationships among asthma, atopy, rhinitis and exhaled nitric oxide in a population-based sample of children

BACKGROUND: Exhaled nitric oxide (eNO) has attracted increasing interest as a non-invasive marker of airway inflammation in asthma. However, little evidence exists on the influences exerted on eNO by the interrelations among atopic status, asthma and rhinitis. METHODS: Among the 1156 children who participated in a large-scale epidemiological survey on asthma and allergies (ISAAC II: International Study of Asthma and Allergies in Childhood Phase II) in the city of Clermont-Ferrand, 53 asthmatics without corticosteroid treatment and 96 non-asthmatics were invited to perform eNO and skin prick tests (SPTs) to 12 common allergens. RESULTS: Atopic asthmatic children had higher eNO than non-atopic asthmatic children (28.9+/-9.1 vs. 17.1+/-13.1 p.p.b.; P=0.0004) with a significant increase when one SPT or more are positive (26.5+/-7.8 vs. 17.1+/-13.1 p.p.b.; P=0.03). Similarly, non-asthmatic, atopic subjects had higher eNO than non-atopic subjects with a significant increase when two SPTs or more are positive (19.4+/-9.8 vs. 11.7 +/-6.7 p.p.b.; P=0.003). In the case of equal levels of positive SPTs (0, 1, >/=2), asthmatic children always had higher eNO than non-asthmatic ones. Furthermore, among non-asthmatic children, the eNO level increased only in atopics who had rhinitis (20.7+/-13 vs. 12.5+/-6.4 p.p.b. in atopic controls (subjects without rhinitis and asthma) and 12.3+/-6.6 p.p.b. in non-atopic controls; P=0.001), whereas among asthmatic children, eNO level increased in atopics independently of rhinitis (28.2+/-9.5 p.p.b. in those with rhinitis and 30.9+/-8.1 p.p.b. in those without) as well as in non-atopics with rhinitis (22.5+/-17.2 p.p.b.). CONCLUSIONS: Our data suggest that besides atopy and asthma, allergic rhinitis should also be taken into account in the assessment of eNO.     

Enhanced cytokine generation by peripheral blood mononuclear cells in allergic and asthma subjects.

BACKGROUND: Although preferential expression of the Th2 cytokines, IL-4 and IL-5, has been described in atopic asthma, the role of IFN-gamma and IL-10 are less clear. OBJECTIVE: To determine the cytokine pattern of T cell mitogen-induced peripheral blood mononuclear cells obtained from atopic asthmatic (AA) subjects. METHODS: Peripheral blood mononuclear cells obtained from AA (n = 24), allergic rhinitis (AR) (n = 9), and normals (NL) (n = 9) were stimulated with phytohemagglutinin (PHA) and the generation of IL-4, IL-5, IFN-gamma, IL-10, and GM-CSF was quantified using ELISA. RESULTS: Compared with NL subjects, peripheral blood mononuclear cells from the atopic groups had increased generation of both IL-5 (AA, P = .001 and AR, P = .024) and IFN-gamma (AA, P = .037 and AR, P = .048) and decreased generation of IL-10 (AA, P = .038 and AR, P = .036). The absolute levels of cytokines did not differ between the two atopic groups; however, the ratio of IL-5/IL-10 was significantly higher in AA (P < .05), but not in AR when compared with NL subjects. CONCLUSION: The concomitant increase in the generation of IL-5 and IFN-gamma, with a decrease in IL-10 in the atopic groups suggests that in, at least a subset of these patients, there is potential expression of both Th2- and Th1-type cytokines. Furthermore, the increased IL-5 to IL-10 ratio could represent a key feature that distinguishes atopic asthmatic from non-asthmatic atopic subjects.     

TCR usage and cytokine expression in peripheral blood and BAL T cells

T cells are thought to play an important regulatory role in atopic asthma. We hypothesized that human blood and BAL T cell subsets bearing various TCR-Vbeta genes might show selective differences in their cytokine profile. Peripheral blood (PB) and bronchoalveolar lavage (BAL) T cells from seven atopic asthmatic and six non-atopic non-asthmatic subjects were stimulated with PMA and ionomycin in the presence of monensin and analysed for TCR-Vbeta expression and production of cytokines at the single cell level. The percentage of IFN-gamma- and IL-2-producing BAL T cells was elevated compared with PB T cells from both the asthmatic subjects and the non-atopic, non-asthmatic controls. A small percentage of PB and BAL T cells produced IL-4 and IL-5, in asthmatic and normal subjects. In peripheral blood, the percentage of T cells expressing each cytokine was similar in the various TCR-Vbeta subsets and in total CD3+ T cells in all normal and six of seven asthmatic subjects. However, there was a substantial degree of heterogeneity in the cytokine profile of BAL TCR-Vbeta subsets compared with the total CD3+ T cells. This was more obvious in the asthmatic subjects with a reduction in the percentage of IFN-gamma- and IL-2-expressing T cells (five of seven asthmatic subjects) and an increase in the percentage of IL-4- and IL-5-expressing T cells (two of seven asthmatic subjects). These data confirm previous findings of an elevated proportion of IFN-gamma- and IL-2-producing BAL T cells while only a small proportion of PB and BAL T cells produce IL-4 and IL-5. Moreover, subsets of BAL T cells, defined by their TCR-Vbeta usage, may differ in their cytokine profile compared with the total CD3+ T cells, implying that T cells expressing different Vbeta elements may play different roles in regulating the airway inflammation in asthma.     

Activities of superoxide dismutases and NADPH oxidase in neutrophils obtained from asthmatic and normal donors

PMN obtained from asthmatic subjects demonstrate a heightened respiratory burst with increased superoxide generation compared to normals. This enhanced superoxide anion generation could be secondary to increased activity of the respiratory burst NADPH oxidase or diminished metabolism of superoxide via superoxide dismutase (SOD). The two forms of SOD expressed in PMN, CuZnSOD expressed constitutively in the cytosol and inducible mitochondrial MnSOD, were investigated in asthmatics. Resting ?MN from asthmatics (N = 9) contained significantly less MnSOD activity compared to controls (0.46 ± 0.16 vs. 0.79 ± 0.17 units/10⁷ PMN, respectively;P=0.0002). As several cytokines including interleukins (IL) -1, -4, and -6 as well as granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF) enhance the PMN respiratory burst and are synthesized in the asthmatic lung, their effects on PMN MnSOD activity were assayed. In contrast to its effects on lymphocytes, both IL-1 and IL-6 significantly inhibited in a dosedependent fashion the induction of MnSOD in PMN from normals (0.42 ± 0.12 and 0.45 ± 0.05 units/10⁷ PMN, respectively, at 10 units/ml of each cytokine;P=0.02 compared to resting cells) but failed to further modulate MnSOD production in asthmatic PMN. IL-4 and GM-CSF had no effect on MnSOD production, and TNF effects could not be studied because of its effects on cell viability. There were no differences in the activity of CuZnSOD (N=9) or NADPH oxidase (N = 4) in the two groups. Inhibition of MnSOD activity in PMN secondary to cytokine exposure in the asthmatic lung could explain, at least in part, the increased generation of superoxide from PMN obtained from asthmatics. This would promote the presence and severity of inflammation in the asthmatic lung. These data further support a role for IL-1 and IL-6 in allergic inflammation.     

Inhaled allergen-driven CD1c up-regulation and enhanced antigen uptake by activated human respiratory-tract dendritic cells in atopic asthma

Background Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory‐tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis. Objective To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation. Methods Sputum cells were induced from steroid‐naïve, allergen‐challenged and allergen‐naïve subjects (atopic asthmatics, atopic non‐asthmatics and non‐atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry. Results hRTDC stained HLA‐DR⁺ (negative for markers of other cell lineages) were predominantly myeloid and comprised ∼0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4⁺ naïve T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC‐dextran was enhanced in cells from asthmatics (P<0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P<0.05–P<0.001), CD1c, CD1d and langerin (P<0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24 h; P<0.05). CD11c⁻CD123^high hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge. Conclusion Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.     

Lack of role for mononuclear cell-derived histamine releasing factors in occupational asthma due to western red cedar

Occupational asthma due to Western Red Cedar (WRCA) is attributed to sensitization to plicatic acid (PA), but does not appear to be dependent on PA-specific IgE antibodies. Exposure to PA induces histamine release in vivo and in vitro, so if IgE is not important, other mechanisms of histamine release must presumably operate in WRCA. To explore the possible role of histamine-releasing factors in WRCA, peripheral blood mononuclear cells were obtained and cultured with PA, PA-albumin conjugate plicatic acid-human serum albumin (PA-HSA), grass pollen or Concanavalin A using a standard histamine releasing factor (HRF) generation protocol. Supernatants were dialysed to remove endogenous histamine and then assayed for histamine releasing activity using human basophils as targets and a Con A-induced bulk supernatant as an internal HRF standard. In contrast to some previous reports, spontaneous HRF release from the peripheral blood mononuclear cells (PBMC) of WRCA patients (n=9) and atopic asthmatic subjects (n=5) was not elevated compared with the non-asthmatic controls (n=11; five atopic and six non-atopic). Both PA and PA-HSA induced the production of small amounts of HRF by PBMC of WRCA patients, but a similar degree of HRF generation was also observed in PBMC from the atopic asthmatic, atopic nonasthmatic, and non-atopic subjects. In contrast, grass pollen induced the production of HRF by PBMC from the subjects with positive skin tests to grass pollen but not by PBMC of non-atopic subjects, confirming that our methods and assay were capable of detecting antigen-specific HRF production. Since neither PA nor PA-HSA induced significantly elevated HRF production from PBMC of WRCA patients, it seems unlikely that PA-induced HRFs play a substantial role in the pathogenesis of WRCA.