Sputum Antineutrophil Cytoplasmic Antibodies in Serum Antineutrophil Cytoplasmic Antibody-Negative Eosinophilic Granulomatosis with Polyangiitis.

RATIONALE: Eosinophilic granulomatosis with polyangiitis (eGPA) is a small-vessel vasculitis where 40% of patients present with serum antineutrophil cytoplasmic antibodies (ANCAs). We examined the presence and clinical relevance of sputum ANCAs in the serum ANCA- patients with eGPA. METHODS: ANCA was investigated in matched sputum and blood samples collected from 23 patients with eGPA (n = 10, serum ANCA+), 19 patients with eosinophilic asthma (prednisone dependent), and 13 healthy volunteers. IgG reactivity to common target antigens and cytokine profiles in sputum samples were examined. Pathogenicity of detected sputum ANCA was assessed using in vitro degranulation assays. MEASUREMENTS AND MAIN RESULTS: Most patients with eGPA (17 of 23, 74%) showed significantly increased sputum ANCAs compared with patients with eosinophilic asthma (P = 0.002) and healthy controls (P < 0.0001), irrespective of their serum ANCA status. In addition, 16 of 17 (94%) of sputum ANCA+ patients had clinical manifestations of severe asthma compared with 3 of 6 (50%) in the sputum ANCA- subset (P = 0.04). Microarray analysis of 123 common antigens failed to reveal a specific target for the ANCA IgG. However, immunoprecipitated immunoglobulins from ANCA+ sputum allowed extensive extracellular trap formations from both neutrophils and eosinophils in vitro, indicating pathogenicity of detected IgG autoantibodies. Cytokine analysis showed lung-localized increases in CXCL8 (neutrophil/eosinophil chemotaxis), CCL24 (eosinophil recruitment), and CXCL12 (lymphocyte recruitment) in the sputa from ANCA+ patients (P < 0.01). CONCLUSIONS: We report a novel finding of ANCA reactivity in the sputa of patients with eGPA in whom disease severity is driven by respiratory complications. Investigating localized autoimmunity may lead to the discovery of novel pathomechanisms, therapeutic targets, and optimal biomarkers for diagnosing and managing eGPA.

Sputum autoantibodies in patients with severe eosinophilic asthma.

BACKGROUND: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. OBJECTIVES: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. METHODS: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. RESULTS: We report a "polyclonal" autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a TH2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. CONCLUSION: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.

Chlorophyll revisited: anti-inflammatory activities of chlorophyll a and inhibition of expression of TNF-α gene by the same.

In view of the folklore use of green leaves to treat inflammation, the anti-inflammatory property of chlorophylls and their degradation products were studied. Chlorophyll a and pheophytin a (magnesium-free chlorophyll a) from fresh leaves showed potent anti-inflammatory activity against carrageenan-induced paw edema in mice and formalin-induced paw edema in rats. Chlorophyll a inhibited bacterial lipopolysaccharide-induced TNF-α (a pro-inflammatory cytokine) gene expression in HEK293 cells, but it did not influence the expression of inducible nitric acid synthase and cyclooxygenase-2 genes. Chlorophyll b only marginally inhibited both inflammation and TNF-α gene expression. But both chlorophyll a and chlorophyll b showed the same level of marginal inhibition on 12-O-tetradecanoyl-phorbol-13-acetate-induced NF-κB activation. Chlorophylls and pheophytins showed in vitro anti-oxidant activity. The study shows that chlorophyll a and its degradation products are valuable and abundantly available anti-inflammatory agents and promising for the development of phytomedicine or conventional medicine to treat inflammation and related diseases.

Effect of obesity on airway inflammation: a cross-sectional analysis of body mass index and sputum cell counts.

BACKGROUND: Several observational studies have demonstrated an association between obesity and asthma. Studies evaluating exhaled nitric oxide levels and obesity have revealed that a higher body mass index (BMI) is associated with elevated exhaled nitric oxide levels. Airway inflammation using sputum cell counts has not been assessed in obese patients with airway diseases. OBJECTIVE: The primary aim of this study was to determine whether obesity (based on BMI) is associated with eosinophilic or neutrophilic bronchitis. METHODS: The results from a database of induced sputum cell counts were compared with BMI and analysed using correlation statistics, regression and parametric and non-parametric analysis. RESULTS: Seven-hundred and twenty-seven adult participants with an equal number of sputum samples were included in the analysis. BMI varied from 14.5 to 55 kg/m(2). Sputum total cell count (mean+/-SD: 12.9 x 10(6) cell/g+/-21.5), eosinophil percent (median; min to max: 0.3%; 0-89.0), and neutrophil percent (mean+/-SD: 63.5+/-26.6%) were within normal limits. Participants with asthma had a higher percentage of sputum eosinophils than those without asthma (P=0.01). However, there was no difference in the total or differential cell counts among the obese and non-obese participants, when the data were analysed according to BMI category, gender, dose of inhaled corticosteroid, and presence or absence of asthma. CONCLUSION: In this large sample of adult asthmatic and non-asthmatic participants, there was no association between BMI and airway inflammation measured by sputum cell counts. Other mechanisms to explain the relationship between obesity and asthma will need to be explored if this association is to be better understood.

Effects of intranasal fluticasone and salmeterol on allergen-induced nasal responses.

BACKGROUND: Combination of inhaled steroid and long-acting beta-agonists has synergistic effects in asthma. OBJECTIVE: To investigate whether nasal corticosteroid and long-acting beta-agonists have synergistic effects on allergen-induced nasal responses. METHODS: The effects of intranasal treatment with fluticasone p-MDI (50 microg bid), salmeterol p-MDI (25 microg bid), their combination, and placebo, on nasal symptoms, eosinophil differential cell count and albumin in nasal lavage fluid (measures of inflammation and leakage respectively) and nasal electrical potential difference (measure of epithelial integrity) were studied in 11 atopic subjects with rhinitis, in a randomized, partially-blinded, 4-period, cross-over study. The measurements were made at baseline, at the end of 1 week of treatment, and immediately after a nasal allergen provocation. RESULTS: Allergen-induced sneeze, postnasal drip and nasal obstruction were significantly reduced by fluticasone, but not by salmeterol. Eosinophil count in postallergen nasal lavage fluid was significantly less after fluticasone (median 1.9%, IQR 4.6) and salmeterol treatment (median 2.5%, IQR 8.5) compared with placebo (median 12.5%, IQR 27.9). Compared with placebo, both fluticasone and salmeterol attenuated allergen-induced change in nasal potential (mean change from baseline -18.5, +0.4 and -7.2% respectively) and the increase in nasal albumin (median 154, 119 and 130 ng/ml respectively). Combination treatment did not have any additional benefits over the individual therapies. CONCLUSIONS: Although salmeterol has anti-inflammatory properties, intranasal salmeterol or its combination with fluticasone do not offer any added benefit over intranasal fluticasone alone for allergen-induced nasal responses.