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.

Endogenous peroxidases in sputum interfere with horse-radish peroxidase-based ELISAs.

Peroxidase-based immunoassays are commonly used for detecting inflammatory mediators in biological samples. We suggest caution while interpreting assays particularly in sputum samples that have endogenous peroxidases like eosinophil peroxidase (EPX), which may interact with a horseradish peroxidase (HRP)-based ELISA. Using IL-8 as an example, we demonstrate that values generated with an HRP-ELISA (n=47) show significant positive correlation with the sputum EPX content (r=0.6, P=0.0004), which can be misconstrued to be affiliated with an eosinophilic event. The data-set generated with the same samples (n=47) using alkaline phosphatase (AP)-based ELISA and a non-enzymatic Milliplex system do not show any correlation with sputum EPX (Milliplex r=-0.24, P=0.13; AP r=0.26, P=0.09). Moreover, sub-group analysis shows significantly increased IL-8 levels detected by HRP-ELISA in eosinophilic patient sputa (n=28) compared to AP-ELISA (P=0.0001). We, therefore, recommend the use of AP-based ELISA or Multiplex system rather than peroxidase-based ELISA for detecting soluble mediators, and more importantly for non-Th2 related mediators in sputum samples with increased eosinophil activity.