Netting Neutrophils Induce Endothelial Damage, Infiltrate Tissues

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Lupus ErythematosusImmunostimulatory Molecules in SystemicDamage, Infiltrate Tissues, and Expose Netting Neutrophils Induce Endothelial

Bruce and Mariana J. KaplanPlumas, Laurence Chaperot, Matthias Kretzler, Allen T.Matthew Klinker, David Shealy, Michael F. Denny, Joel Lin, Cory J. Rubin, Wenpu Zhao, Stephen H. Olsen,Berthier, Jeffrey B. Hodgin, Ritika Khandpur, Andrew M. Eneida Villanueva, Srilakshmi Yalavarthi, Celine C.

http://www.jimmunol.org/content/187/1/538doi: 10.4049/jimmunol.11004502011;

2011; 187:538-552; Prepublished online 25 MayJ Immunol

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The Journal of Immunology

Netting Neutrophils Induce Endothelial Damage, InfiltrateTissues, and Expose Immunostimulatory Molecules inSystemic Lupus Erythematosus

Eneida Villanueva,*,1 Srilakshmi Yalavarthi,*,1 Celine C. Berthier, Jeffrey B. Hodgin,

Ritika Khandpur,x Andrew M. Lin,x Cory J. Rubin,x Wenpu Zhao,* Stephen H. Olsen,

Matthew Klinker,* David Shealy,{ Michael F. Denny,*,2 Joel Plumas,,#,**Laurence Chaperot,,#,** Matthias Kretzler, Allen T. Bruce,x and Mariana J. Kaplan*

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-

density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased

levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array

profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overex-

press mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In con-

trast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to

synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory pro-

teins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial

cells and to stimulate IFN-a synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated

by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These

results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and

its subsequent responses may play a prominent deleterious role. The Journal of Immunology, 2011, 187: 538552.

Recent evidence from our group and others indicates thatneutrophils may play an important role in the induction ofautoimmune responses and organ damage in systemic

lupus erythematosus (SLE) (13). Furthermore, microarray dataindicate that neutrophil-specific genes are highly expressed inPBMCs from lupus patients because of the cosegregation of low-density granulocytes (LDGs) in mononuclear cell fractions (3, 4).These LDGs represent a distinct neutrophil subset that is presentin the peripheral blood of all adult SLE patients analyzed. LupusLDGs are likely to be pathogenic, given their heightened capa-city to induce vascular damage and synthesize type I IFNs uponexposure to specific stimulants, such as G-CSF and polyinosinic:polycytidylic acid, when compared with autologous lupus normal-density neutrophils and healthy control neutrophils (1). Further-

more, lupus patients with higher circulating LDG numbers haveincreased prevalence of skin involvement and/or vasculitis (1).Although currently no specific LDG surface markers have beenidentified that would allow them to be distinguished from normal-density granulocytes, their nuclear morphology suggests that thesecells present a more immature phenotype (1, 3). However, it is stillunknown whether lupus LDGs play prominent roles, when com-pared with normal-density lupus neutrophils, in other aspects ofdisease pathogenesis, including providing a supply of potentialautoantigens or inducing or perpetuating autoimmune responsesand tissue damage.Neutrophils immobilize and kill invading microbes extracellu-

larly through the formation of neutrophil extracellular traps (NETs).As a unique type of neutrophil cell death, recently described as

*Division of Rheumatology, Department of Internal Medicine, University of Mich-igan Medical School, Ann Arbor, MI 48109; Division of Nephrology, Department ofInternal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109;Department of Pathology, University of Michigan Medical School, Ann Arbor, MI48109; xDepartment of Dermatology, University of Michigan Medical School, AnnArbor, MI 48109; {Centocor Research and Development, Radnor, PA 19355; Uni-versite Joseph Fourier, Grenoble 38041, France; #INSERM U823, Immunobiologie etImmunotherapie des Cancers, La Tronche 38706, France; and **Etablissement Fran-cais du Sang Rhone-Alpes, Laboratoire R&D, La Tronche 38701, France

1E.V. and S.Y. contributed equally to the manuscript and share first authorship.

2Current address: Division of Rheumatology and Autoimmunity Center, TempleUniversity, Philadelphia, PA.

Received for publication February 11, 2011. Accepted for publication April 24, 2011.

This work was supported by the National Institutes of Health through Public HealthService Grant HL088419 (to M.J.K.), the Arthritis Foundation (to M.J.K.), the Uni-versity of Michigan/Centocor Postdoctoral Research Program (to M.J.K. and E.V.),the Anthony Gramer Fund in Inflammation Research (to M.J.K.), the Babcock Re-search Endowment (to A.T.B.), and Training Grant in Cell and Molecular Dermatol-ogy 5T32AR007197 (to C.J.R.). This work was supported in part by the NationalInstitutes of Health through the University of Michigans Cancer Center Support

(Grant P30 CA46592), the Rheumatic Disease Core Center (Grant P30 AR48310),and the Applied Systems Biology Core in the OBrien Renal Center (Grant P30DK081943).

The sequences presented in this article have been submitted to the Gene ExpressionOmnibus under accession number GSE26975.

Address correspondence and reprint requests to Dr. Mariana J. Kaplan, Division ofRheumatology, Department of Internal Medicine, University of Michigan MedicalSchool, 5520 MSRBI, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5680.E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: ANCA, anti-neutrophilic cytoplasmic Ab; CTSG,cathepsin-G; DEFA4, defensin a-4; EC, endothelial cell; ELANE, elastase-2; IPA,Ingenuity Pathway Analysis; LCN2, lipocalin-2; LDG, low-density granulocyte; LTF,lactotransferrin; MMP-8, matrix metalloproteinase-8; MNase, micrococcal nuclease;MPO, myeloperoxidase; NET, neutrophil extracellular trap; pDC, plasmacytoid den-dritic cell; RNP, ribonucleoprotein; SLE, systemic lupus erythematosus; SLEDAI,systemic lupus erythematosus disease activity index.

Copyright 2011 by TheAmerican Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100450

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NETosis, this response is distinct from apoptosis and necrosisand characterized by the active release of nuclear chromatin fi-bers (5, 6). NETosis is triggered by a variety of stimuli includingmicroorganisms, proinflammatory cytokines, activated platelets,and endothelial cells (ECs) (6, 7). A variety of putative auto-antigens are present within and attached to NET chromatin fibers,including citrullinated histones (8) and various bactericidal pro-teins and/or enzymes such as the cathelicidin LL-37, neutrophilelastase, and myeloperoxidase (MPO) (9). Upon neutrophil acti-vation, elastase migrates from azurophilic granules to the nucleus,where it partially degrades specific histones and promotes chro-matin decondensation. MPO synergizes with elastase in drivingthis decondensation (9), a phenomenon that is considered key inNET formation.Due to the potential role of netting neutrophils in externalizing

autoantigens and DNA-modifying factors, thereby making thesemolecules more exposed to the adaptive and innate immune sys-tems, a putative link between NETosis and autoimmunity has beenrecently proposed. It has been shown that neutrophils from patientswith anti-neutrophilic cytoplasmic Ab (ANCA)-positive vasculitisrelease NETs enriched in MPO and LL-37 (10). NETs are alsopresent in the kidneys from patients with this disease, where theymay provide a source of antigenic nucleosomes and promoteimmune complex formation (10). Further, impaired NET degra-dation has been identified in a subset of SLE patients, secondary toDNase1 inhibitors and anti-NET Abs that prevent DNase1 accessto NETs (2). LDGs may represent an additional source of NETs,leading to heightened autoantigen exposure and modification oftissue damage. Recent evidence indicates that NETosis may beenhanced in IFN-aprimed lupus neutrophils upon exposure toanti-ribonucleoprotein (RNP) Abs. This is accompanied by therelease of LL-37 and high-mobility group protein B1, which fa-cilitate uptake and recognition of mammalian DNA by plasma-cytoid dendritic cells (pDCs) (11). In addition, recent evidenceindicates that NETs may be harmful to the endothelium andpromote thrombosis (7, 12).To further clarify the origin of LDGs and assess their pathogenic

potential and role in the induction of autoimmune responses, wecompared the gene array expression profiles of purified LDGs withthose of autologous normal-density lupus neutrophils (referred toin the text as lupus neutrophils) or healthy control normal-densityneutrophils (referred to in the text as control neutrophils). Wealso compared the capacity of isolated LDGs to form NETs, ex-ternalize autoantigens and immunostimulatory molecules, stimu-late pDCs, and induce endothelial cytotoxicity through NETosis.Finally, we assessed whether netting neutrophils are present in vivoin involved lupus tissue from various organs as an additional in-dication of their putative pathogenic role.

Materials and MethodsPatient selection

The University of Michigan institutional review board approved this study.Subjects gave informed consent in accordance with the Declaration ofHelsinki. Lupus patients fulfilled the revised American College of Rheu-matology criteria for SLE (13) and were enrolled from the University ofMichigan outpatient rheumatology clinic. Disease activity was assessed bythe SLE disease activity index (SLEDAI) (14). Gender-matched healthycontrols were recruited by advertisement. Demographic and clinical in-formation on the lupus patients enrolled in the study (including medi-cations) is included in Table II.

Reagents

For LDG purification, biotinylated Abs recognizing CD3, CD7, CD19,CD79b, CD56, MHC class II, CD86, and CD235a were obtained fromAncell (Bayport, MN). For immunohistochemical staining, rabbit Abs

recognizing human neutrophil elastase (Abcam, Cambridge, MA), humanRo/SSA and human La/SSB (Santa Cruz Biotechnology, Santa Cruz, CA),mouse Abs recognizing human Smith (Abcam), human Cathelicidin/OSX12 (Abcam), and human dsDNA (Millipore, Temecula, CA), andgoat anti-human IL-17 (R&D Systems, Minneapolis, MN) were used.Secondary detection was performed using goat anti-rabbitFITC (SouthernBiotechnology Associates, Birmingham, AL), donkey anti-goat Alexa 568(Invitrogen, Carlsbad, CA), and/or anti-mouse Cy3 (Sigma-Aldrich, St.Louis, MO). Prolong Gold Antifade and Hoechst 33342 were from In-vitrogen. Micrococcal nuclease (MNase) was from Thermo Scientific(Rockford, IL).

Purification of LDGs

Lupus LDGs were purified as described by our group (1). In brief, PBMCswere isolated by Ficoll/Hypaque gradient, and cell pellets were incubatedwith LDG isolation mixture (equal volumes of biotinylated Abs recog-nizing human CD3, CD7, CD19, CD79b, CD56, MHC class II, CD86, andCD235a), followed by anti-biotin MACS beads (Miltenyi Biotec, Auburn,CA). Cells were applied to an MACS-LS column, and nonimmobilizedcells were recovered by negative selection. The purity of the LDG fractionwas .95%, and cells were identified as CD15+/CD14lo or CD10+/CD14lo.

Isolation of neutrophils

Normal-density neutrophils were isolated by dextran sedimentation of RBCpellets, as described (15). Purity was $95%. In previous studies, we hadcompared the activation status of neutrophils obtained by dextran sedi-mentation versus double gradient and found no differences in their phe-notype or function (1).

NETs immunohistochemical staining and quantification

Neutrophils or LDGs were isolated as above, and 1 to 23 105 cells/ml wereseeded in poly-L-lysine coverslips and incubated at 37C, 5% CO2 for 15min. Cells were washed with ice-cold PBS and either fixed right away with4% paraformaldehyde and then blocked overnight at 4C with 10% FBS/1% BSA/0.05% Tween 20 and 2 mM EDTA/PBS or incubated for 2 h inRPMI 1640/glutamine/2% BSA in the presence or absence of 20 nM PMAto induce NET formation, followed by fixation and overnight blocking at4C. NETs were detected by washing the fixed cells with ice-cold 10%FBS/PBS and incubating with anti-human elastase (1:100) or isotypecontrol for 45 min at 4C, followed by incubation with secondaryfluorochrome-conjugated Abs for 45 min at 4C. Nuclear DNA wasdetected by incubating cells with Hoechst 33342 (1:100) for 10 min atroom temperature. Coverslips were mounted in Prolong Antifade Reagent(Invitrogen) and analyzed using an Olympus microscope (IX70, CenterValley, PA). Statistical background, shading subtraction, and image overlaywere performed with Metamorph v7.7 software (Molecular Devices,Sunnyvale, CA). The recorded images were loaded onto Adobe Photoshop(Adobe Systems) for further analysis, in which NETs were manuallyquantified by two independent observers. The number of cells positive forboth neutrophil elastase and nuclear staining (Hoechst) were considereda NET and digitally recorded to prevent multiple counts. The percentage ofNETs was calculated as the average of five to six fields (340) normalizedto the total number of cells. For LL-37 (1:100), dsDNA (1:10), Ro (1:20),La (1:20), Smith (1:20), and IL-17 (1:10) quantification, the number ofcells positive for either of these markers colocalized with elastase andHoechst were counted as part of the overlay (RGB) image and recordeddigitally to prevent multiple counts.

RNA isolation

Total RNA was isolated with Tripure (Roche, Indianapolis, IN), followingmanufacturers recommendations. For microarray analysis, RNA wasfurther purified and concentrated using an RNeasy micro kit (Qiagen,Valencia, CA). RNA samples were processed on an Agilent 2100 Bio-Analyzer (Agilent Technologies, Santa Clara, CA) to assess integrity.

Microarray data processing, analysis, and pathway mapping

Affymetrix Human U133 Plus 2.0 Genechips (Affymetrix, Santa Clara, CA)were processed at the University of MichiganMicroarray Core Facility. Thesamples analyzed and compared were: normal-density neutrophils fromhealthy individuals (n = 9) and normal- density neutrophils and autologousLDGs from lupus patients (n = 10 for each group). CEL files were nor-malized in GenePattern pipeline (http://www.GenePattern.org) using theRobust MultiChip Average method and the Human Entrez Gene customCDF annotation version 10 (http://brainarray.mbni.med.umich.edu/Brainarray/default.asp). As samples were part of two hybridization rounds, the

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two resulting normalized files were corrected for batch effect (16) withinGenePattern. Of the 17,527 gene identification numbers (corresponding tothe 54,675 Affymetrix probesets), the number of genes expressed abovethe Poly-A Affymetrix control expression baseline (negative controls) andused for further analyses was 15,929. The Gene Expression Omnibus ac-cession number for these arrays is GSE26975 (http://www.ncbi.nlm.nih.gov/geo/).

Statistical paired analyses were performed using Significance Analysis ofMicroarrays method implemented in MultiExperiment Viewer application(17, 18) for comparing lupus neutrophils with lupus LDGs; unpairedanalyses were performed in the comparison of healthy control neutrophilswith lupus neutrophils and healthy control neutrophils with lupus LDGs.The canonical pathways derived from the significantly regulated genesbetween the groups (q , 0.05 depicting the false discovery rate) wereanalyzed using the Ingenuity Pathway Analysis (IPA) Software (http://www.ingenuity.com).

Real-time quantitative PCR

Real-time PCR reactions were run on an ABI Prism 7900HT in duplicateusing 23SYBR Green PCR Master Mix (Applied Biosystems, Foster City,CA). Oligonucleotide primers (IDT, Coralville, IA) used in the reactionswere: lipocalin, 59-CCCAGCCCCACCTCTGA39 (forward), 59-CTTCCC-CTGGAATTGGTTGTC-39 (reverse); matrix metalloproteinase-8 (MMP8),59-GCTGAGGTAGAAAGAGCTATCAAGGA-39 (forward), 59-AGCAAT-GTTGATATCTGCCTCTCC-39 (reverse); MPO, 59-TTTGACAACCTGC-ACGATGAC-39 (forward), 59-CGGTTGTGCTCCCGAAGTAA-39 (reverse);cathepsin-G (CTSG), 59-AGAAGAGTCAGACGGAATCGA-39 (forward),59-CCCTGACGACTTTCCATAGGA-39 (reverse); LL-37, 59-GCAGTCAC-CAGAGGATTGTGAC-39 (forward), 59-CACCGCTTCACCAGCCC-39 (re-verse); IL-17A, 59-ACTCCTGGGAAGACCTCATTGG-39 (forward), 59-GGCCACATGGTGGACAATCG-39 (reverse); and GAPDH, 59-TTGCCA-TCAATGACCCCTTCA-39 (forward), 59-CGCCCCACTTGATTTTGGA-39(reverse).

EC cytotoxicity assay

HUVECs were cultured in MCDB131 basal media (Life Technologies,Carlsbad, CA) supplemented with EGM-2MV (without hydrocortisone)bullet kit (Lonza, Walkersville, MD) on 0.2% gelatin-coated 24-well plates(Fisher, Pittsburgh, PA). Lupus LDGs, their autologous lupus neutrophils,and healthy control neutrophils were incubated with HUVECs at a 2:1 E(neutrophil):T (HUVEC) ratio in MCDB131/EGM-2MV and 0.1 mg/mlPMA in the presence or absence of MNase (10 U/ml) for 1620 h. Al-ternatively, cells were also cocultured with 0.1 mg/ml PMA, with orwithout MNase, for 1 to 2 h, followed by replacement with fresh mediawith or without nuclease. Following the incubation, nuclease reaction wasstopped by adding EDTA to final concentration of 2 mM. Adherent LDGsand neutrophils were collected by gentle pipetting, and HUVECs wereharvested with 0.05% trypsin-EDTA (Life Technologies), followed bycentrifugation at 1600 rpm for 5 min. HUVECs were resuspended in 1%horse serum/1% BSA in PBS, and 104 to 105 cells were incubated withPEanti-human CD146, allophycocyanin-Annexin V, PE/Cy5anti-humanCD45 (BD Pharmingen, San Diego, CA), and/or PE/Cy5-anti-humanCD10 (Biolegend), followed by fixation with 4% paraformaldehyde. Thepercentage of HUVEC cytotoxicity was measured in cells double-positivefor CD146 and Annexin V in the CD45- or CD10-negative gate.

pDC lines and IFN-a quantification

The previously described human pDC cell line GEN 2.2 (19) was cul-tured in RPMI 1640 Glutamax/1 mM sodium pyruvate/4 mM glutamine/nonessential amino acids/10% heat-inactivated FCS. Cells were stimulatedfor 16 h with either CpG (1 mg /ml) or supernatants collected from eithercontrol or lupus neutrophils or autologous lupus LDGs that had beencultured for 1 h, and supernatants were treated in the presence or absenceof MNase (10 U/ml, 10 min). IFN-a mRNA from pDCs was quantified byreal-time PCR as previously described by our group (1).

Human kidney tissue and immunofluorescence

Kidney tissue was obtained from nine renal biopsies from subjects withclinical and histological diagnosis of lupus nephritis according to the newSLE nephritis classification (20) and from one patient with fulminantHenoch-Schonlein purpura with renal involvement. Relevant clinico-histological parameters are in Table II. Immunohistochemical analyseswere carried out at the University of Michigan Histology and Im-munohistochemistry Core. Three-micrometer sections of formalin-fixed,paraffin-embedded tissues were mounted on plus slides, deparaffinized inxylene, and then rehydrated with distilled H2O through graded alcohols.

Ag retrieval was enhanced by microwaving the slides in citrate buffer (pH6; Biogenex) for 10 min. Sections were blocked for 30 min in 10% horseserum prior to 1-h incubation of primary Abs at 4C. Simultaneous stain-ing was performed with mouse monoclonal anti-human MPO (1:1000;Abcam) and rabbit polyclonal anti-human histone H2A (1:500; Abcam)followed by 30 min incubation with matched secondary Abs: anti-mouseIgG-Alexa Fluor 488 (1:300; Invitrogen) or anti-rabbit IgG-Alexa Fluor555 (1:300; Invitrogen). DAPI was added to the slides prior to mountingwith cover slips (Invitrogen). For NETosis detection, images were acquiredby a Leica DMIRB inverted microscope and an RT slider digital camera(model 2.3.1; Diagnostic Instruments) using DP Controller, DP Manager(Olympus, Essex, U.K.) software, and National Institutes of Health ImageJsoftware. RBCs were excluded. NETs were considered as extracellularstructures that costained for MPO, histone H2A, and DAPI.

Skin tissue immunohistochemistry and quantification ofNETosis

Skin biopsies from 11 patients with cutaneous lupus involvement and 10gender-matched healthy controls were analyzed for neutrophil infiltrationand NETosis. In addition, skin biopsies from 10 patients with psoriasis wereincluded as positive controls of IL-17+ cells in the skin. Control donorswere identified from respondents to advertisements, had no personal orfamily history of lupus, and were free of inflammatory skin disease at thetime of biopsy. Five-micrometer sections of skin were deparaffinized ona hot plate at 65C for 1 h. They were rehydrated by incubation and heatretrieval in Cell Conditioning Solution (CC1; Ventana Med, Tucson, AZ).The sections were blocked with 0.2% horse serum (Invitrogen) for 30 min.Simultaneous staining was first performed for 30 min with goat antiIL-17 (100 mg/ml; R&D Systems), mouse anti-human Cathelicidin/OSX12(Abcam), or anti-dsDNA (Millipore) and another primary Ab, either rabbitanti-MPO (1:1500; DakoCytomation) or rabbit anti-human neutrophilelastase (10 mg/ml; Abcam). This was followed by 30-min incubation withmatched secondary Abs: chicken anti-goat IgG-Alexa Fluor 488 (1:300;Invitrogen) or chicken anti-rabbit IgG-Alexa Fluor 594 (1:300; Invitrogen).ProLong Gold antifade reagent with DAPI (Invitrogen) was added to theslides prior to mounting with cover slips. Images were captured witha fluorescent imaging microscope (BX50; Olympus) using DP Controllerand DP Manager (Olympus) software. The recorded images were loadedonto Adobe Photoshop. The number of cells expressing one or bothmarkers of interest in each 3200 field was manually counted by two in-dependent blinded observers.

Statistical analysis

For NET quantification, immunofluorescence was performed in neutrophilsisolated from individual patients (n$ 4 for both SLE and control patients),and counts were averaged, represented as mean 6 SEM, and analyzed bytwo-tailed Student t test, for which p # 0.05 was considered significant.Comparison of NETosis within different areas of the skin was performedby repeated-measures one-way ANOVA with a Tukeys multiple compar-ison posttest. For all other studies, paired or unpaired Student t tests wereperformed. Microarray statistical analysis is detailed above.

ResultsLupus LDGs have a distinct gene expression profile fromautologous lupus neutrophils and control neutrophils

To determine whether LDGs represent a distinct pool of neu-trophils, gene expression profiling of purified neutrophil fractionsof lupus LDGs, autologous lupus neutrophils, and gender-matchedcontrol neutrophils was assessed using Affymetrix genechip mi-croarrays. Samples were matched-paired in comparing lupusLDGs with autologous lupus neutrophils. Although there wereno genes significantly differentially regulated when comparingnormal-density lupus neutrophils with healthy control neutrophils,several genes were differentially expressed in LDGs relative toeither autologous lupus neutrophils or healthy control neutrophils.In all, 302 genes were differentially expressed in lupus LDGs whencompared with control neutrophils, and 281 genes were identifiedas altered by pairwise comparison of each patients LDGs to theirautologous lupus neutrophils (q value ,0.01, fold-change $1.5and #0.7 for the upregulated and downregulated genes, respec-tively) (Fig. 1A, Supplemental Tables I, II). A total of 224 geneswere identified as upregulated in both comparisons (Fig. 1A). Con-

540 ABERRANT NETosis IN LUPUS GRANULOCYTES

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versely, a total of 57 genes were found to be selectively regulated inlupus LDGs when compared with autologous lupus neutrophils, and78 genes were restricted to LDGs alone when compared with controlneutrophils (Fig. 1A, Supplemental Tables I, II).The defined transcripts were associated to canonical pathways

using IPA. The pathways significantly regulated in LDGs comparedwith control neutrophils and autologous lupus neutrophils are listedin Supplemental Table III. Actin cytoskeleton, macropinocytosis,clathrin-mediated endocytosis, and integrin signaling pathwayswere among the most significantly regulated pathways in lupusLDGs compared with control and lupus neutrophils. Inhibition ofangiogenesis by Tsp1 was a pathway significantly regulated only inlupus LDGs compared with control neutrophils, whereas Ephrinreceptor signaling was only significantly regulated in lupus LDGscompared with lupus neutrophils. The top functions identified

using IPA included inflammatory response and hematological andcardiovascular diseases.The top upregulated genes in lupus LDGs in both comparisons

included a number of serine proteases, bactericidal proteins, andother molecules involved in neutrophil regulation of the in-flammatory response (3, 2125) when compared with normal-density lupus and control neutrophils (Table I). Among them,mRNA of the cathelicidin gene CAMP (LL-37) was 3.5- and4.6-fold higher in lupus LDGs when compared with control andlupus neutrophils, respectively (q value ,0.01). In the same group,defensin a-4 (DEFA4) was highly upregulated in LDGs com-pared with control and lupus neutrophils (22.4- and 25.6-fold,respectively). Lactotransferrin (LTF), lipocalin-2 (LCN2), MPO,elastase-2 (ELANE), MMP-8, and CTSG were also significantlyupregulated in the lupus LDGs when compared with control and

FIGURE 1. LDGs overexpress defensins and

proinflammatory molecules. A, Schematic represen-

tation of the sample group and gene list comparisons

made using the gene microarray data (q value ,0.01,fold-change $1.5 and #0.7 for the upregulated and

downregulated genes, respectively). Samples were

matched-paired when comparing lupus neutrophils

with autologous lupus LDGs (n = 10 for each group).

B, Lupus LDGs express elevated levels of azurophilic

granule genes. Log base 2 mRNA mean expression

values of five azurophil genes in control neutrophils

(n = 9), lupus neutrophils (n = 10), and lupus LDGs

(n = 10): MPO, ELANE, DEFA4, CTSG, and azur-

ocidin 1 (AZU1). Data are presented as mean 6 SD.C, Confirmation of enhanced mRNA expression by

real-time PCR of various neutrophils genes in lupus

LDGs when compared with autologous lupus neu-

trophils (n = 712) and control neutrophils (n = 7).

Bar graph represents fold mRNA expression (mean 6SEM) after adjusting for housekeeping gene

(GAPDH). *p , 0.05 LDGs compared with controland/or autologous lupus neutrophils, ***p , 0.0001.


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lupus neutrophils (fold-change range from 10.941.5). Theseresults indicate that lupus LDGs show increased expression ofazurophilic granule genes that is not secondary to a general in-duction of these genes in SLE, as the autologous lupus neutrophilsexpressed levels comparable to control neutrophils (Fig. 1B).These findings were confirmed by real-time PCR for several of themolecules mentioned above (Fig. 1C). Thus, microarray analysisof lupus LDGs confirmed an enhanced bactericidal and activatedsignature compared with control or normal-density lupus neu-trophils.Altogether, the transcriptional analysis results indicate that

LDGs, as a distinct subset of lupus granulocytes, have a specificmolecular pattern that differs from autologous lupus neutrophilsand control neutrophils, whereas lupus neutrophils display nosignificant differences in gene expression compared with controlneutrophils. These results support the hypothesis that LDGs area specific subset of granulocytes with distinct phenotype and func-tional capabilities.

NET formation is increased in lupus LDGs, leading toenhanced externalization of autoantigens andimmunostimulatory molecules

Serine proteases and cathelicidins released from neutrophils areincorporated into NETs, allowing the delivery of high concen-trations of molecules that kill bacteria and degrade their virulencefactors (8). We tested if NET formation differed in lupus LDGsrelative to control neutrophils or autologous neutrophils. Atbaseline, peripheral blood LDGs demonstrated significantly en-hanced NET formation right after isolation when compared withhealthy control neutrophils or autologous lupus neutrophils (Fig.

2). Both control and normal-density lupus neutrophils displayedsignificantly higher NET formation following a 2-h in vitro sti-mulation with PMA when compared with baseline levels (Fig.2). In contrast, the percentage of NETs in lupus LDGs remainedunchanged from baseline (Fig. 2), suggesting that LDGs may beprestimulated in vivo and resistant to further NET induction.When comparing isolation methods, there were no differences inNET formation when neutrophils were isolated through gradientseparation or negative selection with magnetic beads (data notshown). This excluded a potential effect of the isolation techniquein the differences in induction of NETs in vitro.There were no significant correlations between the percentage of

LDGs undergoing NETosis at baseline and lupus disease activity(as determined by SLEDAI) (14) or presence or titers of auto-antibodies. Furthermore, there were no associations between useof various lupus medications (antimalarials, corticosteroids, and/or immunosuppressive drugs) and percentage of LDGs undergoingNETosis (data not shown). These results indicate that enhancedNETosis occurs in lupus LDGs independent of the patients dis-ease activity.To further characterize the NET composition and functional

relevance of extracellular trap formation, the localization andexpression of immunostimulatory and bactericidal proteins wasinvestigated. Control and lupus neutrophil and LDG NETs expressthe bactericidal proteins elastase and LL-37. The latter was de-tected intracellularly in all neutrophils but was predominantlyfound colocalized within the extracellular NETs. Because a higherpercentage of LDGs undergo NETosis, there was significantlyenhanced externalization of LL-37 by these cells when comparedwith control and lupus neutrophils (Fig. 3).

Table I. Affymetrix microarray expression data in lupus LDGs of genes corresponding to enzymes, bactericidal, and other molecules known to beimplicated in reactive oxygen species generation and/or NET formation

Entrez GeneIdentification No. Gene Symbol Gene Name

Lupus LDGsCompared with

ControlNeutrophils

Lupus LDGsCompared with

LupusNeutrophils

FoldChange

qValuea

FoldChange

qValuea

Enzyme and enzyme inhibitor genes6036 RNASE2 RNase, RNaseA family, 2 (liver,

eosinophil-derived neurotoxin)4.24 0.004* 3.06 0.066

4317 MMP8 Matrix metallopeptidase 8(neutrophil collagenase)

38.53 0.000* 41.53 0.000*

4318 MMP9 Matrix metallopeptidase 9 (gelatinase B,92 kDa gelatinase, 92 kDa type IV

collagenase)

1.08 0.999 1.37 0.047*

6037 RNASE3 RNase, RNase A family, 3 (eosinophilcationic protein)

12.34 0.000* 12.07 0.000*

1991 ELANE (Elastase 2) Elastase, neutrophil expressed 11.64 0.000* 10.92 0.000*3934 LCN2 Lipocalin2 24.49 0.000* 19.02 0.000*5476 CTSA Cathepsin-A 1.69 0.004* 1.54 0.043*1511 CTSG Cathepsin-G 13.53 0.000* 17.73 0.000*4353 MPO Myeloperoxidase 12.02 0.000* 14.66 0.000*

Bactericidal molecule genes1669 DEFA4 Defensin, a-4, corticostatin 22.41 0.000* 25.57 0.000*820 CAMP (LL-37) Cathelicidin antimicrobial peptide 3.44 0.003* 4.62 0.000*671 BPI Bactericidal/permeability-increasing protein 13.80 0.000* 16.59 0.000*566 AZU1 Azurocidin 1 11.23 0.000* 12.22 0.000*

Other genes1088 CEACAM8 (CD66b) Carcinoembryonic Ag-related cell adhesion molecule8 16.63 0.000* 18.27 0.000*10321 CRISP3 Cysteine-rich secretory protein3 18.39 0.000* 12.18 0.000*634 CEACAM1 (CD66a) Carcinoembryonic Ag-related cell adhesion

molecule 1 (biliary glycoprotein)1.50 0.099 1.30 0.017*

4057 LTF Lactotransferrin 16.91 0.000* 12.01 0.0001191 CLU Clusterin 21.24 0.000* 17.39 0.000*

aA q value ,0.05 was considered significant (in boldface and with asterisk).


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Because NETs may be sources of autoantigens in other con-ditions (10), we assessed if various targeted autoantigens werepresent in LDG NETs. During quantification, it became evidentthat all NETs expose dsDNA regardless of the neutrophil source.However, as a higher percentage of LDGs undergo NETosis thanhealthy controls or lupus neutrophils, they lead to overall en-hancement in externalization of dsDNA (Fig. 4A, 4C). In contrast,there was no evidence of expression of other common lupusautoantigens (Ro, La, or Smith) within the NETs structure. Indeed,the expression of Ro, La, and Smith was intracellular and equallydetected in LDGs, lupus, and control neutrophils (data not shown).These observations were independent of whether the serum of thepatients was positive or negative for Abs against dsDNA, Smith,Ro, or La (Table II and data not shown). These results indicatethat netting neutrophils externalize dsDNA and that LDGs mayrepresent an enhanced source of extracellular dsDNA throughheightened NET formation.

NET formation leads to enhanced externalization of IL-17 inlupus LDGs

Innate IL-17producing cells are considered an integral part of IL-17mediated immune responses (26). Neutrophils have previouslybeen identified as a source of IL-17, particularly in the contextof autoimmunity (27), although the signaling pathways by whichthese cells synthesize and release this specific cytokine remainuncharacterized. Neutrophils expressing IL-17 have been reportedin diseased tissues such as atherosclerotic plaques (28). Further-more, neutrophils can externalize IL-17 through NETosis in skinand peripheral blood from patients with psoriasis (29).

Recent evidence indicates that IL-17 may be involved in thepathogenesis of SLE through its capacity to amplify local in-flammation by recruiting cells from the innate immune system andits ability to stimulate B cell-adaptive immune responses (30).Indeed, elevated levels of IL-17 and increased numbers of Th17cells have been reported in human and murine lupus, and there isevidence that this cytokine is synthesized in target organs frompatients with this disease, including skin and kidney (3032).However, it is unclear if, in addition to T cells, innate cells in-cluding neutrophils could represent an enhanced source of IL-17production in blood and the periphery in SLE. Indeed, IL-17Awasdetected at the mRNA level in control and lupus neutrophils and inLDGs, and there were no significant differences in expressionamong these three cell subsets (data not shown). To determinewhether NETosis could be a source of enhanced IL-17 external-ization in SLE, the expression of this cytokine in the NETs ofperipheral blood neutrophils and LDGs was compared. Overall,between 8 6 4% to 23 6 5% of all neutrophils isolated fromperipheral blood externalize IL-17 in vitro during NETosis, and allNETS from peripheral blood neutrophils express IL-17. Bothcontrol and lupus neutrophils externalize IL-17 with similar fre-quency. As NETosis was significantly enhanced in lupus LDGs,a higher proportion of these cells externalized IL-17 (Fig. 4B, 4C).These results suggest that NET formation by LDGs in situ maypotentiate IL-17dependent responses.Overall, these results indicate that, through enhanced NETosis,

LDGs externalize various immunostimulatory molecules and au-toantigens that may be crucial in stimulation of adaptive andinnate immunity.

FIGURE 2. Circulating lupus LDGs undergo in-

creased NETosis. A, Representative images of control

neutrophils, lupus neutrophils, and lupus LDGs iso-

lated from peripheral blood and analyzed at baseline

(T0) or after stimulation for 2 h with DMSO or PMA.

Top panels show immunofluorescent merged images

of NETs, which were detected by neutrophil elastase

(green), and DNA was labeled with Hoechst 33342

(blue). Original magnification340. Scale bar, 20 mm.B, Quantification of the percentage of NETs (elastase-

labeled cells over total number of cells) are plotted as

mean 6 SEM (n = 6 patients/group). *p # 0.05.


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Netting neutrophils infiltrate lupus kidneys affected byglomerulonephritis

Previous studies have indicated that impaired NET degradation inSLE is associated with the development of lupus nephritis (2).Furthermore, patients with ANCA+ vasculitis have evidence ofnetting neutrophils in their kidneys (10). Although a potential rolefor neutrophils in lupus nephritis was proposed decades ago (33),it is unclear if NETosis is observed in lupus nephritis and if NETsplay a pathogenic role in associated renal damage. We analyzedkidney biopsies from nine lupus patients with World Health Or-ganization class III or IV glomerulonephritis (Table II). NETsvisualized as weblike or granular structures costaining with MPO,histone H2A, and DAPI were observed in the majority of lupusnephritis cases (67%) (Fig. 5). Overall, 17% of glomeruli exam-ined displayed netting neutrophils (range 050%). Patients withclass IV lupus nephritis had a higher percentage of glomeruliinfiltrated by netting neutrophils than patients with class III ne-phritis (mean 6 SEM, 27.5 6 10% versus 9.84 6 3%, re-spectively; p = 0.05). Renal biopsies with higher activity index, asper World Health Organization classification, had a higher per-centage of glomeruli infiltrated by netting neutrophils (19.5 6 5%for biopsies with activity index $10 versus 6.6 6 1.5% for bi-opsies with activity index ,10). In addition, those patients withnetting neutrophils in glomeruli had on average higher levels ofanti-dsDNA Abs in serum than those without evidence of thesecells (669 6 380 versus 189 6 150 IU/ml). In contrast, noNETosis was observed in the kidney biopsy from a patient withfulminant Henoch-Schonlein purpura and no lupus (not shown).

Netting neutrophils infiltrate lupus skin and expose dsDNA andLL-37

In previous studies, we identified that SLE patients with high levelsof LDGs in the circulation had increased prevalence of skin in-volvement (1). After identifying increased NETosis in peripheralblood lupus LDGs, we proceeded to assess if lupus skin lesionsshow evidence of infiltration by netting neutrophils. We analyzedskin biopsies from 11 patients with several forms of cutaneouslupus including discoid lupus, acute cutaneous lupus, subacutecutaneous lupus, and lupus panniculitis (Table II). High-powerexamination of dual-color immunofluorescence slides of SLEpatient and control skin revealed numerous NETs visualizedas weblike structures costaining with MPO and DAPI (Fig. 6).Similar distribution was seen when skin biopsies were stainedwith a neutrophil elastase Ab (not shown). Frequently observedin all cutaneous lupus lesions, these NETS were particularlyenriched near the dermal-epidermal junction in the papillary der-mis and around blood vessels and adnexae, including hair fol-licles and eccrine glands (Fig. 6AC). In the two patients withlupus panniculitis, aggregates of netting neutrophils were fre-quently observed in the lobular adipose tissue with extension intoadjacent reticular dermis (Fig. 6DG). Overall, in lupus biopsies,7% of all neutrophils in the epidermis, 24.3% of neutrophils in thepapillary dermis, and 25.4% of neutrophils in the reticular dermisand subcutis had formed NETs. NETosis was particularly enrichedin areas with large aggregates of neutrophils (Fig. 6). In contrast,neutrophil infiltration and NETosis were not observed in skin bi-opsies from 10 healthy control individuals (data not shown).

FIGURE 3. LL-37 externalization in NETs

is increased in lupus LDGs. A, Representative

images of control neutrophils, lupus neutrophils,

and lupus LDGs after isolation from peripheral

blood. Cells were stained for detection of LL-37

(red) and neutrophil elastase (green), and DNA

was labeled with Hoechst 33342 (blue). Top pan-

els show images of LL-37 and Hoechst (left

panels), elastase and Hoechst (middle panels), and

merged LL-37, elastase, and Hoechst (right pan-

els). Original magnification 340. Scale bar, 20mm. Arrows represent areas of LL-37 localization

within the NETs. B, Quantification of the per-

centage of cells containing LL-37 colocalized with

elastase over total number of cells are plotted as

mean 6 SEM (n $ 3 patients). *p # 0.05.


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Interestingly, large aggregates of NETs were seen in the skinfrom all five lupus patients who had increased levels of anti-dsDNAAbs in sera (Table II). Because SLE is frequently associated withthe presence of circulating anti-dsDNA Abs, we tested if largeaggregates of NETs in skin could be a potential antigenic sourcefor formation of these autoantibodies, especially because LL-37DNA complexes present in the NETs may activate pDCs (34).Confirming the findings from peripheral blood neutrophils, analy-sis of these cells in affected lupus skin revealed that NETs ex-pressed dsDNA (data not shown). Furthermore, LL-37 was clearlypresent in the NETs in lupus skin biopsies (Fig. 6H) and wasmost prominent in netting neutrophils from the deep dermis areas.These results indicate that NETs extrude dsDNA and LL-37, pos-sibly providing antigenic stimuli for anti-dsDNA Ab formation.

Skin biopsies from lupus patients display higher numbers ofIL-17+ neutrophils

Because IL-17 was detected in the NETs from peripheral bloodneutrophils, we also assessed if IL-17positive (IL-17+) neu-trophils were detected in the affected lupus skin. Using dual-colorimmunofluorescence for IL-17 and MPO, we observed that 51.566.8% of all IL-17expressing cells in lupus skin were neutrophils.Further, 19.7 6 7.5% of intact neutrophils stained brightly withIL-17. These intact neutrophils were predominantly observed indermal blood vessels and in the dermal interstitium of lupuslesions (Fig. 7). Only a small proportion (1 6 0.8%) of NETslocated in lupus skin were IL-17+. Although the temporal eventssurrounding neutrophil extravasation, migration in tissue, andNETosis are unclear, our observations are consistent with a modelin which intact neutrophils containing IL-17 circulate in bloodvessels and then release IL-17 upon extravasation and migrationinto tissue, with all IL-17 released by the completion of extra-cellular trap formation. The percentage of neutrophils expressing

IL-17 in lupus skin was significantly higher than in healthy controlskin (1.9%, n = 8; p , 0.001) and similar to that seen in skin frompatients with psoriasis (32%, n = 12) (Fig. 7 and data not shown).Overall, these results demonstrate that enhanced NETosis and

exposure of autoantigens and immunostimulatory proteins occursin vivo in affected organs of SLE patients.

Netting neutrophils stimulate IFN-a synthesis by pDCs

Recent work indicates that antimicrobial products, includingLL-37, are immunostimulatory when bound to DNA and induceIFN-a synthesis by pDCs (23). Additionally, others have shownthat NETosis stimulates pDCs to synthesize increased levels ofIFN-a (11). There is also evidence that type I IFNs can induceNET formation (35). Given the important pathogenic role ofIFN-a in SLE and the increased NET formation and LL-37 exter-nalization by LDGs, we tested if these cells also induce pDCsto synthesize more IFN-a. Supernatants from control neutrophilsdid not induce the pDC cell line Gen2.2 (19) to synthesize IFN-amRNAwhen compared with pDCs alone. In contrast, supernatantsfrom both lupus neutrophils and LDGs induced significantly en-hanced IFN-a mRNA synthesis in pDCs when compared withhealthy controls (Fig. 8A). IFN-a induction by neutrophil super-natants was significantly decreased in lupus neutrophils and LDGsafter MNAse treatment, indicating that NETosis is involved in thepDC activation. The capacity to stimulate IFN-a in pDCs didnot significantly differ between LDGs and autologous lupusneutrophils. These results confirm that both lupus neutrophils andLDGs have heightened capacity to induce IFN-a synthesis inpDCs, at least in part, through a NET-mediated effect.

Lupus netting neutrophils induce endothelial cytotoxicity

Patients with SLE display evidence of accelerated EC apoptosis(36), which strongly correlates with the development of aberrant

FIGURE 4. Lupus LDGs externalize dsDNA and

IL-17 through NETosis. Representative images of

control neutrophils, lupus neutrophils, and lupus

LDGs after isolation from peripheral blood. Cells

were stained for detection of neutrophil elastase

(green), DNA (Hoechst 33342, blue), and either

dsDNA (red) or IL-17 (red). Merged images of

dsDNA, elastase, and Hoechst (A) and merged

images of IL-17, elastase, and Hoechst (B). C,

Quantification of the percentage of cells containing

dsDNA or IL-17 colocalized with elastase over total

number of cells is plotted as mean 6 SEM (n $ 5patients). Scale bars, 20 mm. *p # 0.05.


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Table

II.

Clinicalcharacteristicsoflupuspatientsincluded

inthestudy

Peripheral

Blood

Patient

No.

SLEDAI

Score

dsD

NA-A

bC3,C4

ENA

ANA

Clinical

Manifestations

Medications

1B

2+

Norm

alNegative

1:160

Skin,joints,CNS

MMF,

antimalarials

2B

10

+Norm

alNegative

1:320

Nephritis,arthritis

None

3B

8+

Low

Sm,RNP

1:2560

Cytopenias,nephritis

PDN,antimalarials

4B

02

Norm

alRo

1:2560

Neuropathy,Raynauds

Antimalarials

5B

02

Norm

alNegative

1:320

Arthritis,skin,mucositis

Antimalarials

6B

2+

Norm

alSm,RNP

1:320

Skin,cytopenias,fever

PDN,antimalarials

7B

20

+Low

Sm,RNP

1:2560

Arthritis,CNS,cytopenias,skin

PDN,antimalarials,MMF

8B

22

Low

Sm,RNP

1:320

Nephritis,synovitis,serositis


9B

4+

Norm

alNegative

1:160

Serositis,synovitis,mucositis,skin

Antimalarials,MMF

10B

22

Norm

alNegative

1:160

Skin,nephritis,arthritis


11B

4+

Norm

alRo

1:640

Arthritis,cytopenias

Antimalarials

12B

02

Norm

alNegative

1:320

Arthritis,eye

Antimalarials

13B

10

+Low

Ro,

La

1:2560

Arthritis,nephritis,Raynauds

MMF,

PDN

14B

12

+Low

Ro,Sm,RNP

1:2560

Arthritis,skin,serositis,kidney,CNS,

MMF,

MTX,PDN

15B

4+

Low

Sm,RNP

1:320

Skin,arthritis,serositis,leukopenia

Antimalarials

16B

62

Norm

alSm,RNP,Ro

1:2560

Nephritis,cytopenias,arthritis

PDN,antimalarials,AZA

17B

42

Norm

alNegative

1:2560

Serositis,arthritis,skin

Antimalarials

18B

02

Norm

alSm,RNP

Negative

Arthritis

PDN,antimalarials

19B

10

+Low

Ro

1:320

Nephritis,arthritis

PDN,MMF,

antimalarials

20B

62

Norm

alNegative

1:320

Arthritis,skin

PDN,antimalarials

Skin

Patient

No.

Diagnosis

dsD

NA-A

bC3,C4

ENA

ANA

Other

Clinical

Manifestations

Medications

1S

DL

+Low

Negative

1:160

CNS,nephritis

MMF,

PDN

2S

DL

+Low

RNP,Ro

1:2560

CNS,Raynauds

PDN,antimalarials

3S

Interfacedermatitis

+Low

SmRNP

1:2560

Nephritis

PDN,antimalarials

4S

Lupuspanniculitis

+Low

Negative

1:640

None

5S

Lupuspanniculitis

+Low

SmRNP,RNP

1:2560

Arthritis

PDN,antimalarials

6S

SCLE

2Norm

alRo

Negative

Sicca

Antimalarials

7S

DL

NA

NA

Negative

Negative

None

8S

DL

NA

NA

NA

Negative

None

9S

DL

NA

Norm

alNA

Negative

None

10S

DL

NA

Norm

alNegative

1:640

Arthritis

None

11S

Interfacedermatitis

2NA

Ro,

La

Negative

None

Kidney

Patient

No.

Kidney

Diagnosis;

Activity/Chronicity

dsD

NA-A

bC3,C4

ENA

ANA

Other

Clinical

Manifestations

Medicationsat

Tim

eofBiopsy

1N

Class

IV/S;12/8

+Low

Sm,RNP

1:160

Serositis,arthritis,

PDN,AZA

2N

Class

IV/S;11/3

+Low

Sm

1:320

Skin,serositis,arthritis,cytopenia

PDN,antimalarials

3N

Class

IV/S;13/2

NA

Low

NA

1:640

Cytopenia

None

4N

Class

III/V;13/2

+Low

Ro,

La,Sm,RNP

1:1280

Skin,joints,serositis

PDN,MMF

5N

Class

IV;17/11

+Norm

alSm

1:2560

CNS

PDN

6N

Class

III/V;6/8

+Low

Ro,Sm,RNP

1:1280

PDN,AZA

(Table

continues)


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vascular function and may predispose to the development ofatherosclerosis. Our group had previously reported that lupusLDGs, and less so lupus neutrophils, induce enhanced EC cyto-toxicity using a coculture system with HUVECs (1). However, themechanisms implicated in this enhanced EC cytotoxicity have notbeen identified. Because EC activation may induce NET for-mation, which in turn promotes endothelial cytotoxicity (7), wetested if NET formation by lupus LDGs could enhance EC death.Incubation of HUVEC monolayer with lupus LDGs induces sig-nificantly elevated levels of EC cytotoxicity, relative to control andlupus neutrophils (Fig. 8B). As previously reported, lupus neu-trophils also showed enhanced EC cytotoxicity when comparedwith healthy control neutrophils, but significantly less when com-pared with lupus LDGs (1). A component of this enhanced cy-totoxicity was mediated by NET formation because disruptingthese structures by treating lupus LDGs and neutrophils withMNase (Fig. 8C) significantly downregulated EC apoptosis (Fig.8B). These results indicate that lupus LDGs mediated more ex-tensive EC killing, at least in part, through their enhanced capacityto form NETs.

DiscussionRecent work from various groups indicates that NET formationmay be an important phenomenon in autoantigen modification andexposure to the immune system, as well as in the induction oftissue damage (9, 10). As such, aberrant NET formation mayplay an important role in the development and perpetuation ofautoimmune diseases and organ damage observed in chronic in-flammatory disorders. Our work now expands and reinforces thisconcept by reporting that a distinct subset of neutrophils found inSLE patients (LDGs) have enhanced capacity to form NETs andupregulate expression of various neutrophil proteins and enzymesimplicated in NET formation and autoimmunity induction. TheseNETs also expose dsDNA, an autoantigen considered key in lupuspathogenesis. Furthermore, lupus neutrophils and, in particular,LDGs elicit enhanced EC cytotoxicity through NET formation.This phenomenon also appears to play a role in the induction ofIFN-a synthesis by pDCs. We have also identified that enhancedNETosis occurs in vivo in SLE in affected skin and kidney. Fur-thermore, neutrophils from blood and skin from SLE patientsfrequently externalize IL-17 as part of the NETosis process, whichTa

ble

II.(Continued

)

7N

Class

III;6/2

+Low

Negative

1:160

Cytopenias,arthritis

PDN,MMF,

antimalarials

8N

Class

III;8/1

+Low

Negative

1:160

Arthritis,cytopenias

PDN,AZA,antimalarials

9N

Class

IV;17/4

+Low

Ro,Sm,RNP

1:2560

Arthritis,lung

PDN

10N

Henoch-Schonlein;

crescenticglomerulonephritis

NA

Low

NA

NA

Skin,arthritis

Steroids

MicroarrayAnalysis

Patient

No.

SLEDAI

Score

dsD

NA-A

bC3,C4

ENA

ANA

Clinical

Manifestations

Medications

1M

42

Norm

alRNP,Sm

Negative

Skin

Antimalarials

2M

22

Low

Negative

1:160

Skin

Antimalarials

3M

4+

Low

Sm,RNP

1:2560

Skin,arthritis

PDN,antimalarials

4M

2+

Norm

alNegative

1:160

Skin,arthritis,pleuritis

Antimalarials

5M

10

2Low

Ro

1:160

Arthritis,skin,fever

PDN,antimalarials

6M

42

Norm

alNegative

1:2560

Cytopenias,arthritis

None

7M

14

+Low

La,RNP

1:2560

Skin,arthritis,serositis

PDN,antimalarials

8M

42

Norm

alNegative

1:2560

Arthritis,CNS

Antimalarials

9M

8+

Low

Negative

1:640

Arthritis

None

10M

10

+Low

Sm,RNP

1:2560

Nephritis,skin,arthritis

Antimalarials

Thedashes

inthedsD

NA

Abcolumnmeannegative.Thedashes

intheclinical

manifestationscolumnmeannone.

+,positive;ANA,anti-nuclearAb;AZA,azathioprine;DL,discoid

lupus;ENA,extractable

nuclearAgs;MMF,mycophenolatemofetil;MTX,methotrexate;NA,notavailable;PDN,prednisone;SCLE,subacutecutaneouslupus;SM,Smith;

SmRNP,Smith/RNP.

FIGURE 5. Netting neutrophils are present in glomeruli from patients

with lupus nephritis. Colocalization of histone H2A (green), MPO (red),

and DNA (white) by direct immunofluorescence reveals in vivo evidence

of NET formation in a glomerulus from a representative kidney micro-

photograph from a patient with class IV lupus nephritis. Yellow arrows and

inset boxes highlight intraglomerular NET formation. Original magnifi-

cation 320.


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may contribute to tissue damage and immune dysregulation. Over-all, these observations further support a pathogenic role for neu-trophils in organ damage in SLE.One of the findings from our study is that no differences in gene

expression were found when normal density lupus neutrophils werecompared with gender-matched healthy control neutrophils. Thus,previous reports examining alterations in lupus neutrophils mayin part reflect responses specifically elicited in the LDG pool. Incontrast, lupus LDGs obtained from the same patients from whomthe normal-density neutrophils were obtained showed significantdifferences in gene expression when compared with healthy controland lupus neutrophils. The pairwise comparison of gene expressionin LDGs and autologous neutrophils in SLE patients providesa control for many potential sources of variability such as medi-cations, disease activity, clinical manifestations, and exposure to

environmental factors. As it is expected that LDGs and autologouslupus neutrophils were exposed to a very similar cytokine milieu,these results indicate that LDGs may indeed represent a distinctsubset of proinflammatory and pathogenic cells within the gran-ulocyte spectrum.It is unclear why LDGs upregulate mRNA of various serine

proteases and bactericidal proteins present in azurophilic granules.One possibility is that these findings are indicative of a moreimmature phenotype of the LDGs, further supported by theirimmature nuclear morphology (1). Indeed, proteins synthesized atthe same time during neutrophil differentiation are colocalized inthe same granules. The levels of expression of the mRNA thatencode the neutrophil serine proteases are greatest at the pro-myelocytic stage of neutrophil differentiation in the marrow andare downregulated as neutrophils mature (37). This observation

FIGURE 6. Netting neutrophils are present in

affected lupus dermis and subcutis. Direct im-

munofluorescence staining of DNA (blue) and

MPO (red) reveal NETs throughout affected lupus

skin. A, Low-power view of a punch biopsy from

lesional lupus skin. Scale bar, 200 mm. B, Arrows

highlight perifollicular infiltration of NETs. Scale

bar, 50 mm. C, NETs within the papillary dermis.

Scale bar, 20 mm. Low-power view of lupus re-

ticular dermis (D; scale bar, 200 mm) with arrows

highlighting infiltration by NETs (E; scale bar, 50

mm). Low-power view of a large blood vessel in

affected lupus subcutis (F; scale bar, 200 mm),

with arrows highlighting NETs in an area of

panniculitis (G; scale bar, 20 mm). Dotted line

delineates the dermal-epidermal junction (in A, C,

D) and circumscribes the follicle (f) in B.

Epidermis is designated e and dermis by d. H,

Cathelicidin (LL-37) is present in NETs within

inflamed lupus skin lesions. Expression of LL-37

in neutrophils in lupus tissue was examined by

dual-color immunofluorescence staining for LL-

37 (green) and MPO (red) with DAPI counterstain

(blue). Representative images from one of three

sections stained with LL-37 and MPO (original

magnification 3600). Scale bar, 100 mm. I, Fre-quency of NETosis in cutaneous lupus lesions.

NETs and neutrophils were counted after immu-

nofluorescence staining with MPO and DAPI.

Percentage of neutrophils undergoing NETosis of

all neutrophils was calculated for the epidermis,

papillary dermis, reticular dermis, and subcutis.

*p , 0.05.


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could indicate that LDGs are indeed a more immature neutrophilsubset, despite their apparent expression of markers of fully ma-tured neutrophils, including CD16 and CD10. Indeed, a previousstudy has shown that, among the lupus bone marrow upregulatedgenes (when compared with controls), the highest overexpressionoccurs in granulopoiesis-related genes. These genes include sev-eral of the early granulopoiesis genes upregulated in the LDGmicroarray in our study, including MPO, ELA2, CTSG, DEFA4,and LTF (24). This is also confirmed by a previous study thatshowed that the PBMC granulocyte signatures observed in pedi-atric SLE patients were for genes preferentially transcribed withinthe earliest granulocytes (myeloblast and promyelocytes) and withthe presence of immature neutrophils in their peripheral blood (3).These observations further support that LDGs could represent anaberrant immature subset originating from lupus bone marrow thatmay persist or expand in the blood and/or other tissues from SLEpatients.The functional consequences of high serine protease expression

in LDGs may be varied. It has been proposed that all serineproteases of azurophilic granules (CTSG, proteinase 3, and neu-

trophil elastase), released after encountering immune complexes,may potentiate a positive autocrine feedback on neutrophil acti-vation (38, 39). Further, these molecules have been implicated inthe activation of the proforms of proinflammatory cytokines in-cluding TNF and IL-1b (40). Neutrophil elastase can activateTLR4, eventually resulting in IL-8 production (41). IL-8 levels areelevated in SLE, but the exact mechanisms leading to this increasehave been unclear (42). One could propose that enhanced expo-sure to extracellular elastase through NET formation in SLELDGs could promote enhanced synthesis of IL-8. This cytokinecould in turn activate neutrophil recruitment and promote damagein various organs. In addition, we previously showed that LDGssynthesize enhanced levels of IL-8 and TNF upon activation whencompared with control and normal-density lupus neutrophils (1).Themechanisms by which LDGs are more primed tomake NETs

are unclear. To date, the exact molecular mechanisms and sub-cellular events leading to NETosis remain elusive. Although acrucial role for elastase, reactive oxygen species, and the cyto-skeleton has been proposed, recent reports suggest that NETosis isquite complex. The observed higher expression of elastase andMPO in LDGs could play an important role in enhancing extra-cellular trap synthesis, based on what other groups have recentlyreported (9). There is also evidence that type I and II IFNs can actas priming factors on mature neutrophils, allowing the formationof NETs upon subsequent stimulation with complement factor 5a(35). One possibility may be that LDGs are more sensitive to theeffects of type I IFNs and/or to IFN signaling than normal-densitylupus neutrophils. However, this would go against the hypothesisthat LDGs represent a more immature subset of neutrophils, be-cause previous evidence indicates that granulocyte precursors andless mature cells are fairly insensitive to type I IFN effects whencompared with fully differentiated neutrophils (35). Further, al-though one possibility is that LDGs represent cells that have beenexposed to elevated levels of type I IFNs in the bone marrow, wedo not consider this is likely the case because these cells do notdisplay evidence of increased type I IFN gene signature. Futurestudies need to explore whether the LDGs are derived from normalneutrophils or result from alterations in granulocyte development.Previous studies have shown that the antimicrobial peptide

LL-37 is a key factor that mediates pDC activation in psoriasis (23).LL-37 converts inert self-DNA into a potent trigger of type I IFNproduction. This occurs by binding the DNA to form condensedstructures that are delivered to and retained within early endocyticcompartments in pDCs to trigger TLR9 (23). LL-37 also convertsself-RNA into a trigger of TLR7 and TLR8 in human DCs (22). Ingeneral, LL-37 is involved in a myriad of important immunefunctions, including chemoattraction of immune cells and releaseof inflammatory mediators from epithelial cells (43). Our findingssupport that the enhanced release of LL-37 through NETosis couldpromote enhanced inflammatory responses in SLE organs. Othermolecules overexpressed in lupus LDGs, including various de-fensins, may also be immunostimulatory (44). Our findings arein agreement with a recent study that reported that SLE patientshave elevated serum levels of various neutrophil peptides includ-ing MPO and defensins (45, 46). It is possible that these are de-rived from LDGs. Further, increased anti-defensin and CTSGANCA Abs are found in lupus patients (47). Given their immu-nomodulatory role, overproduction of alarmins might activate theadaptive immune and promote autoimmune responses, as is mani-fested in SLE (48, 49). Another overexpressed molecule in LDGs,neutrophil-gelatinase associated lipocalin/LCN2, has recentlybeen proposed as a biomarker of and to have a pathogenic rolein lupus nephritis (50). Finally, the role of MMP-8 overex-pression in LDGs remains to be determined, but this metallo-

FIGURE 7. IL-17positive neutrophils infiltrate SLE skin. Direct im-

munofluorescence staining of IL-17 (green) and MPO (red) in affected

lupus dermis. A, Arrows highlight intact IL-17+ neutrophils in a blood

vessel. Scale bar, 200 mm. B, IL-17 present in a NET (arrow). Scale bar, 10

mm. C, Frequency of IL-17 expression in intact neutrophils and NETs in

SLE skin. Percentages of: 1) IL-17+ neutrophils of all IL-17+ cells (d); 2)

IL-17+ neutrophils of all intact neutrophils (n); and 3) IL-17+ NETs of all

NETs (:) are reported in the graph.


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proteinase may play a key role in vascular damage in other con-ditions (51).Lupus patients develop accelerated atherosclerosis, leading to

premature cardiovascular disease. We had previously found thatECs from lupus patients undergo accelerated apoptosis in vivo andthat this phenomenon may be pathogenic, as it correlates withendothelial dysfunction development (36). LDGs are capable ofkilling ECs, but the mechanism involved was unclear (1). Thecurrent study shows that, through enhanced NET formation, LDGsacquire a heightened capability to damage the endothelium, ascytotoxicity was abrogated with MNAse treatment. As such, ac-celerated NETosis may represent an important mechanism ofpremature vascular damage in SLE.Death by NETosis may also represent an important immunos-

timulatory event with regards to activation of innate immunity inSLE, given the enhanced capacity of netting lupus neutrophils toactivate pDCs. This could promote chronic activation of the im-mune system and perpetuation of type I IFN synthesis characteristicof this disease (52). Enhanced NETosis in SLE may be one of themechanisms explaining why infections may trigger flares in thisdisease (53), with NET induction by microorganisms that in turnleads to autoantigen externalization and immune system activa-tion. The observation that both lupus neutrophils and LDGs in-duced comparable induction of IFN-a synthesis by pDCs mayindicate that this process cannot be fully explained by pure en-hancement of NETosis in LDGs and that other variables are in-volved in this phenomenon. Future studies are needed to betterunderstand how this process occurs.The skin is an important target organ of the immune system in

SLE, and a significant proportion of lupus patients have cutane-

ous involvement. Although mechanisms of skin damage in SLEare likely multifactorial (54), recent evidence has identified anenhanced type I interferogenic signature and IFN-aproducingpDCs in lupus skin (55). The role of pDCs in the inflammationobserved in autoimmune skin disease in animal models has re-cently supported the notion that IFN-a may be crucial in cuta-neous involvement in SLE (56). In a lupus murine model, tapestripping led to an influx into skin of neutrophils forming NETs,which contain DNA and RNA associated with LL-37 (56). Ourdata confirm a similar pattern in human lupus skin biopsies, inwhich abundant dsDNA+, LL-37+ NETs are seen in multiplelayers of affected skin. Interestingly, these findings are associatedwith the presence of elevated anti-dsDNA Abs in the circulation,supporting the notion that autoantigen exposure in the NETs couldpromote autoantibody formation in vivo.Similar findings were seen in the kidneys of lupus patients af-

fected by class III and IV glomerulonephritis. This supports recentfindings that impaired NET degradation in SLE is associated withrenal involvement in this disease (2). Indeed, IgG deposition onNETs in tubuli and glomeruli in the kidney of an SLE patient whodegraded NETs poorly was reported in that study (2). We havenow expanded this observation by studying a larger number ofpatients with lupus nephritis, and the presence of netting neu-trophils in glomeruli was observed in a majority of them. In-terestingly, patients with a higher proportion of glomeruli in-filtrated by netting neutrophils had higher levels of circulatinganti-dsDNA Abs and higher activity index in renal biopsies. It hasbeen previously proposed that anti-NET Abs and persistent NETscould form NET immune complexes that could be relevant inlupus disease severity (2). It is possible that the presence of in-

FIGURE 8. Netting lupus LDGs stimulate IFN-a

synthesis by pDCs and kill ECs. A, Gen2.2 pDCs

were stimulated for 16 h with supernatants from

control neutrophils (n = 4), lupus neutrophils (n = 6),

and autologous lupus LDGs (n = 6) that had been

previously cultured in the presence or absence of

MNase for 1 h. Results represent mean6 SEM IFN-amRNA expression in Gen2.2 cells. *p , 0.05 forcontrol neutrophils compared with lupus neutrophils

and with LDGs and for lupus and LDGs compared

with lupus or LDGs+MNAse. B, Bar graph represents

mean 6 SEM percentage of apoptotic HUVECs afterexposure to activated LDGs, autologous neutrophils,

and control neutrophils (n = 4 to 5/group) in the

presence or absence of MNase. *p , 0.05. C, Rep-resentative images of LDGs isolated from peripheral

blood cultured for 2 h followed by either no MNase

treatment (T2) or with MNase (T2+MNase) for 10

min at room temperature and processed for immu-

nofluorescence staining of elastase (green) and DNA

(Hoechst 33342, blue). Original magnification 340.Scale bar, 20 mm.


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filtrating netting neutrophils in lupus tissue samples representeda combination of enhanced NETosis and impaired NET degrada-tion. We could then propose a model of profound imbalance inNET production and degradation. On the one hand, NETosiswould be enhanced in lupus LDGs. On the other hand, the activityof DNase1 (an enzyme important in NET degradation) is decreasedin a subset of SLE patients (2). This may provide the conditionsby which NETs may persist and constitute a prolonged sourceof autoantigen exposure in an immunostimulatory context, leadingto enhanced formation of immune complexes and induction ofautoantibodies that could further contribute to tissue damage.Whether the infiltrating neutrophils in the skin and kidney cor-

respond primarily to LDGs is unclear at this point, as no specificcell marker has to this date been identified to distinguish these cellsfrom normal-density neutrophils. A better understanding of thehoming characteristics of LDGs versus other neutrophil subsetswill be important to assess if, through enhanced NETosis or otheryet-unidentified mechanism, LDGs could have an increased ca-pacity to migrate to various tissues and induce damage. It is rel-evant that patients with high levels of LDGs in their circulationhave higher prevalence of skin involvement, and this associationcould also support that this cell subset is pathogenic to cutaneoustissue (1). Future studies in a larger number of patients are re-quired to assess the specific role that these cells play in tissuedamage and progression of disease in SLE.IL-17 has recently been linked to the pathogenesis of SLE (31)

by participation in the amplification of autoimmune responses bystimulating autoantibody production by B cells (30). Further, itaugments tissue injury and target organ damage in this disease(32). Most studies have focused on Th17+ cells, which are ele-vated in SLE (57) and infiltrate renal tissue (58, 59). A recentreport showed IL-17+ cells infiltrating lupus-affected skin, butneutrophils were not specifically studied (60). Similar to what hasbeen shown in other conditions (28) (Lin et al., submitted forpublication), neutrophils expressing IL-17 are seen at significantlyenhanced levels in blood and affected skin from lupus patients.This phenomenon could initiate a cycle in which IL-17secretingcells in lupus skin lesions (including innate and adaptive im-mune cells) would recruit additional neutrophils. IL-17 can stimu-late ECs to produce chemoattractants (IL-8) that selectively driveneutrophil but not lymphocyte chemotaxis. IL-17 increases neu-trophil adhesion to the endothelium, which may also enhanceneutrophil recruitment to organs (61). We had previously beenunable to detect IL-17A in LDG supernatants (1). Given that wehave now found evidence of IL-17A expression both at the mRNAand protein levels, it is possible that the technique previously usedwas not sensitive enough or that the NET-bound IL-17 had notbeen fully released to the supernatants to allow for quantificationby ELISA. Overall, these results further support a pathogenic rolefor IL-17 in organ damage in SLE and that neutrophils representan important subset of IL-17producing cells.Whether enhanced NETosis is a phenomenon present in other

autoimmune diseases associated to autoantibody production, inter-ferogenic signatures, and/or vascular damage remains to be de-termined and should be the focus of future investigations. Theseconditions may include inflammatory myopathies, Sjogren syn-drome, and rheumatoid arthritis (6264).In conclusion, we have identified that LDGs isolated from lupus

patients have a higher capacity to synthesize NETs with immu-nostimulatory and cytotoxic properties. These results further ex-pand the potential pathogenic role of aberrant lupus neutrophilsthrough a NET-mediated effect and indicate that strategies at-tempting to modulate NET formation with the objective of abro-gating autoimmune responses should be investigated.

Note added in proof.While this manuscript was in the process ofbeing accepted, two articles that also demonstrate the putativeimportance of NETs in lupus have been published (65, 66).

AcknowledgmentsWe thank Dr. Michelle Kahlenberg for critical review of the manuscript.

DisclosuresD.S. is an employee of Centocor Research & Development and owns stock

in Johnson & Johnson. The other authors have no financial conflicts of

interest.

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