Diabetes Volume 63, December 2014 4239 Yudong Wang,1,2 Yang Xiao,3 Ling Zhong,1,2 Dewei Ye,1,2,4 Jialiang Zhang,1,2 Yiting Tu,3 Stefan R. Bornstein,5 Zhiguang Zhou,3 Karen S.L. Lam,1,2 and Aimin Xu1,2,4 Increased Neutrophil Elastase and Proteinase 3 and Augmented NETosis Are Closely Associated With b-Cell Autoimmunity in Patients With Type 1 Diabetes Diabetes 2014;63:4239–4248 | DOI: 10.2337/db14-0480 IMMUNOLOGY AND TRANSPLANTATION Type 1 diabetes (T1D) is an autoimmune disease resulting serine proteases activities in the pathogenesis of b-cell au- from the self-destruction of insulin-producing b-cells. Re- toimmunity and also suggest that circulating NE and PR3 duced neutrophil counts have been observed in patients may serve as sensitive biomarkers for the diagnosis of T1D. with T1D. However, the pathological roles of neutrophils in the development of T1D remain unknown. Here we show that circulating protein levels and enzymatic activities of The global incidence of type 1 diabetes (T1D), an autoim- neutrophil elastase (NE) and proteinase 3 (PR3), both of mune disease caused by an interactive combination of which are neutrophil serine proteases stored in neutrophil genetic and environmental factors, has more than doubled primary granules, were markedly elevated in patients with in the past two decades (1,2). Although the triggering fac- T1D, especially those with disease duration of less than tors that are involved in the initiation of T1D remain un- 1 year. Furthermore, circulating NE and PR3 levels in- clear, it is widely accepted that organ-specific autoimmune creased progressively with the increase of the positive destruction of the insulin-producing b-cells in the pancre- numbers and titers of the autoantibodies against b-cell atic islets of Langerhans is mediated primarily by autoreac- antigens. An obvious elevation of NE and PR3 was detected tive T cells, which is accompanied by the production of even in those autoantibody-negative patients. Increased different autoantibodies to b-cell antigens, including glutamic NE and PR3 in T1D patients are closely associated with acid decarboxylase autoantibody (GADA), insulinoma- elevated formation of neutrophil extracellular traps. associated protein 2 autoantibody (IA2A), and zinc transporter-8 By contrast, the circulating levels of a1-antitrypsin, an autoantibody (ZnT8A) (3–5). These autoantibodies have endogenous inhibitor of neutrophil serine proteases, are been proven to be instrumental for the prediction and di- decreased in T1D patients. These findings support an early agnosis of T1D but are deemed not to be pathogenic (6,7). role of neutrophil activation and augmented neutrophil A number of other immune cells, including dendritic cells, 1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Received 24 March 2014 and accepted 28 July 2014. Kong, Hong Kong, China This article contains Supplementary Data online at http://diabetes 2 Department of Medicine, The University of Hong Kong, Hong Kong, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0480/-/DC1. 3Diabetes Center, Institute of Metabolism and Endocrinology, Second Xiangya Y.W., Y.X., and L.Z. contributed equally to this work. Hospital, Key Laboratory of Diabetes Immunology, Ministry of Education, Central South University, National Clinical Research Center for Metabolic Diseases, © 2014 by the American Diabetes Association. Readers may use this article as Changsha, Hunan, China long as the work is properly cited, the use is educational and not for profit, and 4Department of Pharmacology & Pharmacy, The University of Hong Kong, Hong the work is not altered. Kong, China See accompanying article, p. 4018. 5Department of Medicine, University of Dresden, Dresden, Germany Corresponding author: Aimin Xu, [email protected]; Karen S.L. Lam, [email protected]; or Zhiguang Zhou, [email protected]. 4240 Neutrophil Serine Proteases, NETosis, and T1D Diabetes Volume 63, December 2014 macrophages, B cells, and neutrophils, are also implicated October 2000 to October 2013. Patients with T1D were in the development of insulitis in T1D (8,9). diagnosed according to the criteria of the American Neutrophils, which are the most abundant (40–75%) Diabetes Association (21). All patients were treated with type of white blood cells, have recently been implicated in insulin. The median disease duration of T1D was 4.2 the onset and progression of T1D (10,11). The primary (interquartile range 1.7–7.1) years. functions of neutrophils are to eliminate extracellular patho- A total of 77 age- and sex-matched healthy control gens by multiple strategies, including phagocytosis, degran- subjects were recruited from children in the community ulation to release lytic enzymes, and neutrophil extracellular participating in a health screening at the Children Health traps (NETs) that are formed through a unique cell death Center of the Second Xiangya Hospital, Central South process clearly differentiated from apoptosis and necrosis, University, using the following inclusion criteria: fasting termed “NETosis” (12–14). However, improper activation of plasma glucose of less than 5.6 mmol/L and 2-h plasma neutrophils may lead to tissue damage during autoimmune glucose of less than 7.8 mmol/L, and no family history of or exaggerated inflammatory responses (15). Notably, circu- diabetes or other autoimmune or chronic diseases. lating neutrophil counts are reduced in patients with T1D A total of 25 adults with type 2 diabetes diagnosed within and in their nondiabetic first-degree relatives but not in 1 year and 25 age- and sex-matched healthy control subjects patients with type 2 diabetes (11). In nonobese diabetic were recruited at the Diabetes Center, Second Xiangya (NOD) mice (a spontaneous model of T1D), neutrophil in- Hospital of Central South University, and the inclusion filtration and NET formation in the islets were observed as criteria were described in our previous study (22). early as 2 weeks after birth, and blockage of neutrophil The Second Xiangya Hospital of Central South Univer- activities with an anti-Ly6G antibody reduced the subse- sity Institutional Review Board approved the study, and quent development of insulitis and diabetes (9). written informed consents were obtained from the patients Neutrophil serine proteases, including neutrophil elas- and healthy control subjects. tase (NE), proteinase 3 (PR3), and cathepsin G (CG), are the major components of neutrophil azurophilic granules Clinical and Biochemical Assessments that participate in the elimination of engulfed micro- After overnight fasting, a venous blood specimen was organisms (16). Neutrophil activation and degranulation collected in the morning (;0800) for analysis of various can result in the release of neutrophil serine proteases biochemical parameters. Plasma glucose was measured into the extracellular medium and circulation, where they enzymatically on a Hitachi 7170 analyzer (Boehringer not only help to eliminate the invaded pathogens but also Mannheim GmbH, Mannheim, Germany). HbA1c was mea- serve as the humoral regulators of the immune responses sured by automated liquid chromatography (VARIANT II during acute and chronic inflammation, modulating cellular Hemoglobin Testing System; Bio-Rad, Hercules, CA). Serum signaling network by processing chemokines, and activat- levels of C-peptide and C-reactive protein were quantified ing specific cell-surface receptors (17–19). Abnormal activ- using a chemiluminescence immunoassay on a Bayer 180SE ities of neutrophil serine proteases have been implicated in Automated Chemiluminescence System (Bayer AG, Leverkusen, the pathogenesis of several inflammatory and autoimmune Germany) and an immunoturbidimetric assay (Orion diseases, including chronic obstructive pulmonary disease, Diagnostica, Espoo, Finland), respectively. The titers of cystic fibrosis, Wegener granulomatosis, Papillon-Lefèvre GADA, IA2A, and ZnT8A were determined by in-house syndrome, and small-vessel vasculitis (20). However, their radioligand assays, as previously described (22,23). association with T1D has not been explored so far. Circulating protein levels of NE, PR3, and A1AT were In this study, we measured circulating levels of two main measured using ELISA kits established in our laboratory types of neutrophil serine proteases (NE and PR3) and their (Antibody and Immunoassay Services, The University of enzymatic activities in T1D patients with different disease Hong Kong). The limits of detection for the NE, PR3, and duration together with age- and sex-matched healthy control A1AT ELISA kits were 0.156 ng/mL. No cross-reactivity subjects. Furthermore, we explored whether altered NET among these proteins or with other proteins was detected. formation and a1-antitrypsin (A1AT), a major endogenous The intra- and interassay variations were, respectively, inhibitor of neutrophil serine proteases, are associated with 4.5% and 5.1% for the NE ELISA kit, 3.9% and 4.3% for the reduced neutrophil counts and markedly increased activities PR3 ELISA kit, and 4.9% and 5.3% for the A1AT ELISA kit. of neutrophil serine proteases in patients with T1D. We also The combined enzymatic activities of PR3 and NE in measured the dynamic changes of circulating NE/PR3 activ- serum were determined with a chromogen-based assay ities during the development of autoimmune diabetes in using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide NOD mice. (Sigma-Aldrich, St. Louis, MO) as the substrate, which has 21 21 a catalytic constant Kcat/Km of 33,915 M s for NE 2 2 RESEARCH