Histone Deimination As a Response to Inflammatory Stimuli in Neutrophils 1 Indira Neeli, Salar N. Khan, and Marko Radic 2 Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stim- ulate autoimmunity. To examine conditions leading to histone deimination in neutrophils, we used Abs that detect citrullines in the N terminus of histone H3. Deimination was investigated in human neutrophils and HL-60 cells differentiated into granulocytes. We observed rapid and robust H3 deimination in HL-60 cells exposed to LPS, TNF, lipoteichoic acid, f-MLP, or hydrogen peroxide, which are stimuli that activate neutrophils. Importantly, we also observed H3 deimination in human neutrophils exposed to these stimuli. Citrullinated histones were identified as components of extracellular chromatin traps (NETs) produced by degranulating neutrophils. In contrast, apoptosis proceeded without detectable H3 deimination in HL-60 cells exposed to stau- rosporine or camptothecin. We conclude that histone deimination in neutrophils is induced in response to inflammatory stimuli and not by treatments that induce apoptosis. Our results further suggest that deiminated histone H3, a covalently modified form of a prominent nuclear autoantigen, is released to the extracellular space as part of the neutrophil response to infections. The possible association of a modified autoantigen with microbial components could, in predisposed individuals, increase the risk of autoimmunity. The Journal of Immunology, 2008, 180: 1895–1902. H istones acquire a number of posttranslational modifica- tions (1). One intriguing histone posttranslational mod- ification is the deimination of specific arginines in his- tones H2A, H3, and H4 (2–4). The conversion of arginine to citrulline, an atypical amino acid that lacks arginine’s positive charge, is accomplished by a member of the peptidylarginine deiminase (PAD) 3 family of enzymes, PAD4 (5). Although PAD4-mediated modifications of histones are involved in regulating chromatin struc- ture and gene expression in a variety of cell types (6–8), PAD4 may assume an additional role in eosinophil and neutrophil re- sponses to infections. In these cells, PAD4 is an abundant com- ponent of cytoplasmic granules (9) and its activation leads to wide- spread histone deimination. However, a granulocyte-specific role of PAD4 has not yet been defined. Interest in PAD4 intensified following the identification of ge- netic polymorphisms that enhance the expression of PAD4 and constitute a risk factor for rheumatoid arthritis (10). PAD4 poly- morphisms are associated with rheumatoid arthritis susceptibility in Asian and North American populations (11), and thus may pro- vide insights into the pathogenesis of this important autoimmune disease (12). Inflammation in the joints of rheumatoid arthritis pa- tients was demonstrated to involve PAD4 activation in the infil- trating leukocytes and the in situ generation of deiminated proteins (13). In animal models of rheumatoid arthritis, PAD4 expression was correlated with the induction or progression of disease (14). However, the conditions that lead to PAD4 activation in the in- flamed synovium have not been clearly identified. The activity of PAD4 has been examined in HL-60 cells that, following treatment with all-trans retinoic acid (ATRA), acquire properties of mature neutrophils. Addition of calcium ionophore to ATRA-treated HL-60 cells induces rapid histone deimination (2, 15). Because elevated intracellular calcium levels promote apopto- sis (16), it was concluded that histone deimination is an early step in apoptosis. In this study, we examine conditions that favor pro- duction of citrullinated histones in HL-60 cells and human blood neutrophils. By establishing that PAD4 activation is an inflamma- tory response that does not require caspase activity, we define the relationship between granulocyte apoptosis and histone deimina- tion. Our results indicate that deimination of histones, rather than constituting an essential component of neutrophil apoptosis, is a convergence point for diverse signals that trigger the neutrophil response to infections. Materials and Methods Abs and supplies Anti-citrullinated histone H3 rabbit Abs (ab 5103, lot no. 122699) were obtained from Abcam, anti-phospho-H2B (Ser 14 ) rabbit Abs (07-191) from Upstate Biotechnology, and anti-poly(ADP-ribose) polymerase (PARP) IgG1 mouse mAb (clone 42) from BD Biosciences. ATRA, calcium iono- phore (A23187), staurosporine, camptothecin, f-MLP, LPS, cyclohexi- mide, and HRP-conjugated secondary Abs to rabbit or mouse Ig were purchased from Sigma-Aldrich. The pan-caspase inhibitor, z-VAD-fmk, was obtained from Calbiochem. Goat anti-rabbit AF648, goat anti-rabbit AF488, annexin V AF488, and Sytox orange were obtained from Invitro- gen Life Technologies. H 2 O 2 was obtained from Fisher Scientific. Recom- binant human TNF was a gift of Dr. L. Pfeffer (University of Tennessee Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163 Received for publication June 12, 2007. Accepted for publication November 28, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Lupus Research Institute, the Univer- sity of Tennessee Center of Excellence for Microbial Pathogenesis, the Center of Excellence for Diseases of Connective Tissue, and the University of Tennessee Rheu- matic Disease Research Core Center, National Institutes of Health. 2 Address correspondence and reprint requests to Dr. Marko Radic, Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163. E-mail address: mradic@utmem.edu 3 Abbreviations used in this paper: PAD, peptidylarginine deiminase; ATRA, all- trans retinoic acid; LTA, lipoteichoic acid; NET, neutrophil extracellular trap; PARP, poly(ADP-ribose) polymerase. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org