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Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps JCB: Article Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps Venizelos Papayannopoulos, Kathleen D. Metzler, Abdul Hakkim, and Arturo Zychlinsky Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Berlin 10117, Germany eutrophils release decondensed chromatin decondensation independent of its enzymatic activity. termed neutrophil extracellular traps (NETs) to Accordingly, NE knockout mice do not form NETs in a N trap and kill pathogens extracellularly. Reactive pulmonary model of Klebsiella pneumoniae infection, oxygen species are required to initiate NET formation which suggests that this defect may contribute to the but the downstream molecular mechanism is unknown. immune deficiency of these mice. This mechanism pro ­ We show that upon activation, neutrophil elastase (NE) vides for a novel function for serine proteases and highly escapes from azurophilic granules and translocates to charged granular proteins in the regulation of chromatin the nucleus, where it partially degrades specific histones, density, and reveals that the oxidative burst induces a promoting chromatin decondensation. Subsequently, myelo­ selective release of granular proteins into the cytoplasm peroxidase synergizes with NE in driving chromatin through an unknown mechanism. Introduction Neutrophils are the first line of immune defense (Lekstrom- virulence factors and kills bacteria (Lehrer and Ganz, 1990; Himes and Gallin, 2000; Nathan, 2006), and they combat patho- Belaaouaj et al., 2000; Weinrauch et al., 2002). MPO catalyzes the gens by phagocytosis, degranulation, and the release of neutrophil oxidation of halides by hydrogen peroxide (Hazen et al., 1996; extracellular traps (NETs; Brinkmann et al., 2004; Nauseef, Eiserich et al., 1998; Nauseef, 2007). NE and MPO knockout mice 2007; Papayannopoulos and Zychlinsky, 2009). NETs are com- are susceptible to bacterial and fungal infections (Belaaouaj posed of decondensed chromatin and antimicrobial factors, in- et al., 1998; Aratani et al., 1999; Tkalcevic et al., 2000; Gaut cluding neutrophil elastase (NE) and myeloperoxidase (MPO; et al., 2001; Belaaouaj, 2002). Interestingly, histones are the most Brinkmann et al., 2004; Urban et al., 2009), and capture and kill abundant NET component and are potent antimicrobials (Hirsch, bacteria, fungi, and parasites (Urban et al., 2006; Guimarães- 1958; Kawasaki and Iwamuro, 2008; Urban et al., 2009). Costa et al., 2009; Ramos-Kichik et al. 2009). NETs are impli- Isolated human neutrophils release NETs 2–4 h after stim- cated in immune defense, sepsis, and autoimmunity (Clark ulation with microbes or activators of PKC such as PMA (Fuchs et al., 2007; Kessenbrock et al., 2009; Papayannopoulos and et al., 2007), but respond much faster when activated by platelet Zychlinsky, 2009; Hakkim et al., 2010). Mast cells, eosinophils, cells stimulated with LPS, a process thought to be relevant during and plant cells also release DNA, which suggests that this may sepsis (Clark et al., 2007). be a common strategy in immunity (von Köckritz-Blickwede NETs form via a novel form of cell death (Fuchs et al., et al., 2008; Yousefi et al., 2008; Wen et al., 2009). 2007) that requires the production of reactive oxygen spe- NE and MPO are stored in azurophilic granules of naive cies (ROS). Neutrophils from chronic granulomatous disease neutrophils (Borregaard and Cowland, 1997; Lominadze et al., patients with mutations in the NADPH oxidase that disrupt 2005). NE is a neutrophil-specic fi serine protease that degrades ROS production (Clark and Klebanoff, 1978) fail to form NETs (Fuchs et al., 2007; Bianchi et al., 2009). In neutrophils from healthy donors, ROS production is followed by the disassembly Correspondence to Arturo Zychlinsky: [email protected] of the nuclear envelope. Chromatin decondenses in the cyto- Abbreviations used in this paper: ABAH, 4-aminobenzoic acid hydrazide; CG, cathepsin G; CGi, CG inhibitor I; DPI, diphenyleneiodonium; G-CSF, plasm and binds to granular and cytoplasmic antimicrobial granulocyte colony-stimulating factor; HSP, high-speed pellet; HSS, high-speed supernatant; LSS, low-speed supernatant; MPO, myeloperoxidase; NE, neutro- © 2010 Papayannopoulos et al. This article is distributed under the terms of an Attribution– phil elastase; NET, neutrophil extracellular trap; PAD4, peptidylarginine deimi- Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub - nase 4; PBMC, peripheral blood mononuclear cell; PR3, proteinase 3; PVDF, lication date (see http://www.rupress.org/terms). After six months it is available under a polyvinylidene fluoride; ROS, reactive oxygen species; SLPI, serum leukocyte Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, protease inhibitor. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 191 No. 3 677–691 www.jcb.org/cgi/doi/10.1083/jcb.201006052 JCB 677 THE JOURNAL OF CELL BIOLOGY Figure 1. NE cleaves histones and promotes nuclear decondensation in vitro. (A) Nuclei isolated from neutrophils were incubated in buffer or in neutrophil- derived LSS lysates for 120 min at 37°C and labeled with Sytox green. Bar, 10 µm. (B) Neutrophil extracts are sufficient to decondense nuclei from other cell types. Nuclear decondensation of LSS extracts from HL-60 cells differentiated with RA, HeLa cells, PBMCs, and neutrophils were tested with nuclei 678 JCB • VOLUME 191 • NUMBER 3 • 2010 proteins before NET release. Chromatin decondensation and two NE inhibitors, GW311616A (NEi) and serum leukocyte the association with antimicrobial proteins are two essential protease inhibitor (SLPI; Macdonald et al., 2001), but not by the steps during NET formation. The molecular mechanism linking MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH; Fig. 1 C; ROS production to chromatin decondensation and binding to Kettle et al., 1997). antimicrobial proteins is unknown. Azurophilic granules store two additional NE-related pro- Here we show that NE is essential to initiate NET for- teases, proteinase 3 (PR3) and cathepsin G (CG). Using protease- mation and that it synergizes with MPO to drive chromatin specific chromogenic peptides and histones as substrates, we decondensation. Our findings reveal a novel mechanism to show that NEi inhibits NE and PR3, but not CG (Fig. S1). SLPI drive massive chromatin decondensation, and provide evidence is known to inhibit NE and CG but not PR3 (Rao et al., 1991). for a novel pathway that allows granular proteins to leak into Considering the specificity of these inhibitors, these results in - the cytoplasm. dicate that PR3 is not sufficient to drive decondensation in vitro. In addition, CG inhibitor I (CGi), a CG-specific inhibitor, did not inhibit nuclear decondensation. Collectively, these data sug- Results gest that among these serine proteases, only NE is required for Neutrophil extracts promote the decondensation of the nucleus. chromatin decondensation To identify factors involved in NET formation, we developed a NE degrades histones to promote cell-free nuclear decondensation assay using intact nuclei and nuclear decondensation cytoplasmic extracts from neutrophils and other control cells. Because histones pack DNA, we examined whether histones Only the neutrophil-derived low-speed supernatant (LSS), con- H3 and H4 are degraded in nuclei treated with LSS. To dis- taining cytoplasm and granules, decondensed nuclei from neu- tinguish between histone degradation and histone release, we trophils, peripheral blood mononuclear cells (PBMCs), human separated the soluble unbound proteins (SF) from the nuclear leukemia-60 (HL-60), and HeLa cells (Fig. 1, A and B), which fraction (NF) by filtration. Interestingly, H4, but not H3, was indicates that neutrophil LSS contains specific factors that de - degraded in an NE-dependent manner (Fig. 1 E). We also mon- condense nuclei. Further separation of the LSS into cytoplasmic itored the kinetics of nuclear localization of NE and MPO in (high-speed supernatant [HSS]) and membrane/granule (high- relation to histone degradation and nuclear decondensation. Both speed pellet [HSP]) fractions showed that the decondensation NE and MPO were detected in the nuclear fraction within the activity partitioned with the HSP (Fig. 1 C). Neutrophils con- r fi st 30 min. Notably, H1 was degraded early but decondensation tain azurophilic, specific, and gelatinase granules (Borregaard coincided with H4 degradation at 150 min (Fig. 1 F), which and Cowland, 1997). The decondensation activity fractionated suggests that nuclear decondensation is driven primarily by the with the azurophilic granules (Fig. 1 C, fraction 3; Kjeldsen et al., degradation of core histones (Fig. 1 G). However, H1 may have 1994), which suggests that granular factors are not only com- to be degraded first to allow for the subsequent degradation of ponents of NETs but are also involved in their formation. core histones. Moreover, purified NE and PR3, but not CG, promoted NE is necessary and sufficient for nuclear decondensation in vitro (Fig. 1 H, Fig. S1 F, and not de- chromatin decondensation picted). NE degraded H4 processively, whereas the other his- NE and MPO are two of the primary enzymes stored in azuro- tones were only partially degraded (Fig. 1 I). Histone degradation philic granules and are found in abundance in NETs. Un- was detectable by 30 min and coincided with nuclear deconden- like MPO, only NE sedimented exclusively to the azurophilic sation (Fig. 1, H and I). As a control, we showed that NE com- fraction (Fig. 1 D, lane 3; Lominadze et al., 2005). We tested pletely degraded soluble histones purified from neutrophils whether the activity of NE and MPO is required for nuclear (Fig. 1 I), which indicates that the pattern of histone degra- decondensation. Chromatin decondensation was blocked by dation depends on chromatin structure and not on the intrinsic isolated from neutrophils (Neut). The nuclear area was quantified using ImageJ image processing software. Circles denote the median area and the bars indicate the range of the nuclear area values, calculated from the standard deviation of each dataset. (C) Decondensation activity is stored in azurophilic granules. Decondensation of nuclei treated with buffer, neutrophil LSS, neutrophil HSS, HSP, and the granular subfractions of gelatinase (1), specific (2), and azurophilic (3) granules were purified over a discontinuous Percoll gradient. Nuclei were also incubated with LSS or HSP in the presence or absence of NEi, SLPI, CGi, or ABAH. Inhibitors were used at the indicated concentrations. (D) The purity of gelatinase (1), specific (2), and azurophilic (3) granule fractions was tested by SDS-PAGE immunoblot analysis using antibodies against gelatinase, lactoferrin, MPO, and NE. NE fractionates exclusively with azurophilic granules (3). (E) NEi inhibits the degradation of nuclear H4 in vitro. After 120 min at 37°C, decondensation reactions were passed over a 0.65-µm filter to separate the nuclei from the extract. The total reaction (T), flowthrough (SF), or nuclear (NF) fractions were analyzed by immunoblotting. Top, nuclei in buffer; middle, nuclei in LSS; bottom, nuclei incubated with LSS and 5 µM NEi. (F) NE and MPO translocate to nuclei in vitro. LSS (L), nuclei alone (N), or LSS + nuclei (L+N), were incubated for the indicated time and separated over filters as in E. The levels of H1, H4, NE, and MPO in the nuclear fraction at different time points were analyzed by immunoblotting. (G) A plot of the corresponding nuclear decondensation, measured as in A, for the experiment in F. (H) Purified NE is sufficient to decondense nuclei in vitro. Nuclei were incubated with buffer (B) or 1 µM purified NE (NE) in the absence of MPO (no MPO, open circles) for the indicated duration (0–240 min). In parallel, 1 µM MPO was added to the same samples (+MPO, closed circles). Decondensation was measured by quantifying the nuclear area and was significantly enhanced in the presence of both NE and MPO (closed circles). (I) Purified NE cleaves nuclear histones in vitro. The samples from H along with NE incubated with purified neutrophil histones were resolved by SDS-PAGE and analyzed by immunoblotting. H4 was processively degraded but H2A, H2B, and H3 were only partially degraded by NE. Soluble purified histones were completely degraded. Notably, the addition of 1 µM MPO had no effect on histone degradation by NE (third column). ***, P < 0.0001. Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 679 susceptibility of histones to this protease. Similarly, purified To investigate whether high concentrations of NE de- PR3 cleaved nuclear histones in vitro (Fig. S1 E). In con- creased histone degradation, we incubated nuclei with increasing trast, and consistent with our previous observations, nuclear NE concentrations and separated the nuclear (NF) and soluble and purified histones were poor substrates for CG (unpub - (SF) fractions over filters (Fig. 2 E). H4 was degraded under lished data). optimal, but not autoinhibitory, concentrations of NE. Notably, To examine whether the degradation of histones is caus- NE activity is not required for NE binding to nuclei, as it parti- ing chromatin decondensation, we preincubated permeabilized tioned with nuclei in the presence of NEi. The lower levels of nuclei with antihistone or a control antibody against CD63, a NE in the NF fraction in the absence of NEi suggest that NE transmembrane granular protein. Nuclear decondensation was degrades itself in the nucleus (Fig. 2 E, left, asterisks). reduced by 50% in nuclei incubated with antibodies against H4, We asked whether NE and MPO interact inside the nu- and to a lesser extent with antibodies against other core histones cleus by incubating nuclei with NE and low concentrations of (Fig. S2 A), but not by the control antibody. Accordingly, H4 MPO. MPO nuclear localization was independent of NE activ- degradation was reduced in nuclei treated with antihistone anti- ity, as significant amounts of MPO were found in the nuclear bodies (Fig. S2 B). fraction in the presence of NEi (Fig. 2 E, right). Moreover, we Interestingly, in a fractionation experiment, we purified observed NE-dependent MPO degradation in the nuclear, but H1 as an inhibitor of NET formation (unpublished data). H1 not in the soluble fraction, which indicates that these proteins promotes the condensed, closed state of chromatin, which re- come into close proximity in the nucleus. stricts its accessibility (Roche et al., 1985; Bustin et al., 2005; Woodcock et al., 2006). Nuclei pretreated or coincubated with NE activity is required for NET formation H1 were resistant to decondensation and histone degradation in neutrophils by LSS and HSP (Fig. S2, C–E). Moreover, MPO failed to par- To investigate whether NE activity is required for NET forma- tition with the NF in nuclei pretreated with H1, which indicates tion, we pretreated neutrophils isolated from healthy donors that chromatin is the primary binding site of MPO in vitro with NEi or CGi, and induced NET formation with PMA in the (Fig. S2 D). Therefore, NE and MPO require access to the core presence of Sytox, a cell-impermeable DNA dye. Untreated histones to degrade them and induce decondensation. neutrophils and cells treated with CGi formed NETs efficiently 2–4 h after activation. Notably, NEi-treated neutrophils failed to NE and MPO synergize to promote make NETs, and appeared necrotic as determined by the Sytox- nuclear decondensation positive condensed nuclei (Fig. 3 A). To quantitate NET formation, Because MPO localizes to NETs and binds to nuclei in vitro we measured the DNA area of each Sytox-positive neutrophil (Fig. 1 F), we tested whether MPO promotes nuclear deconden- and plotted the percentage of Sytox-positive cells against their sation (Brinkmann et al., 2004). At 1 µM, MPO alone had little corresponding nuclear area (Fig. 3 B). Our quantitation con- effect, but it dramatically enhanced chromatin decondensation firmed that neutrophil death is dramatically reduced in the in the presence of NE (Fig. 1 H). Interestingly, MPO did not presence of NEi (11.2% vs. 63.7%), whereas the low nuclear affect histone degradation, which suggests that it does not up- areas of the dead cells indicate that cells did not make NETs regulate NE activity (Fig. 1 I). MPO promoted nuclear de- but were necrotic. condensation in a dose-dependent manner that did not require Candida albicans is a potent physiological activator of its substrate H O and was not inhibited by ABAH (Fig. 2, NET formation in isolated primary neutrophils. Neutrophils 2 2 A and B). Furthermore, horseradish peroxidase, used as a control treated with NEi failed to make NETs efficiently when ex - for enzymatic activity, did not promote chromatin deconden- posed to C. albicans (Fig. 3 C). The number of neutrophils sation (unpublished data). We concluded that MPO enhances that died without making NETs increased dramatically and NET formation independent of its enzymatic activity. was comparable to neutrophils treated with NEi and stimu- To better understand the synergy between NE and MPO, lated with PMA (Fig. 3 D). Interestingly, C. albicans induced we measured its effect on nuclear decondensation driven by NE. NET formation with lower efficiency than PMA (Fig. 3 D), Increasing amounts of MPO enhanced NE-mediated nuclear which indicates that when presented with microbes, neutro- decondensation (Fig. 2 C). Surprisingly, high concentrations phils may respond in different ways, with some neutrophils of NE inhibited nuclear decondensation (Fig. 2 D). This auto- consuming NE and MPO through phagocytosis or degranula- inhibition could be caused by NE autodegradation. Alternatively, tion while others form NETs. at high concentrations, NE could inhibit histone degradation. As controls, we show that NEi was not cytotoxic (Fig. 3 E) Accordingly, DNA has been shown to inhibit NE (Belorgey and and did not inhibit ROS production, which was potently blocked Bieth, 1995). We confirmed the inhibitory effect of DNA on NE by the NADPH oxidase inhibitor diphenyleneiodonium (DPI; and CG, and found that it was reversed by the addition of NaCl Fig. 3 F; Cross and Jones, 1986). (Fig. S3, A and B). Intact nuclei also inhibited NE activity, but this effect was not reversed by the addition of NaCl. Therefore, NE cleaves histones during NET formation after histone cleavage, NE activity may be down-regulated by Because PMA is a more potent inducer of NETs than C. albi- binding to DNA. Autoinhibition was not observed for PR3, cans and other microbes, we used it to examine whether his- which completely dissolved the nuclei at high concentrations tones are degraded in an NE-dependent manner during NET (Fig. S1, E and F). formation. Consistent with our in vitro data, H2B and H4 were 680 JCB • VOLUME 191 • NUMBER 3 • 2010 Figure 2. NE and MPO synergize. (A) Effect of MPO titration on nuclear decondensation in vitro. Nuclei were incubated with the indicated concentra- tions of MPO for 30 (open circles), 60 (gray circles), or 120 min (closed circles). Decondensation was concentration- but not time-dependent. (B) MPO promotes decondensation independent of its enzymatic activity. Nuclei were treated with 5 µM MPO alone or MPO in the presence of 100 µM ABAH (an MPO inhibitor), 100 µM H O (MPO substrate), or both. (C) Nuclear decondensation was driven by titration of NE at different concentrations of MPO. 2 2 MPO increases NE-mediated decondensation. (D) The effect of NE titration on nuclear decondensation. Nuclei were incubated with the indicated concen- trations of NE in the absence (black line) or presence (red line) of 10 µM NEi for 120 min before measuring nuclear decondensation. (E) NE-mediated degradation of histones is dose-dependent during nuclear decondensation. Western immunoblot analysis of MPO, H4, and NE was performed. Nuclei mixed with increasing concentrations of NE and 0.3 µM MPO for 120 min were separated into nuclear (NF, left) and soluble (SF, right) fractions. Reactions were performed in the absence or presence of NEi. The samples where nuclei decondensed are indicated by positive signs below the NF blot. Asterisks mark the levels of NE bound to nuclei, in the presence or absence of NEi, at the highest concentration of NE. ***, P < 0.0001. partially degraded during NET formation, whereas H3 shifted the degradation of H2B. NEi also prevented the formation of to a higher molecular weight species (Fig. 4 A). The presence of an H2A degradation product, which was observed in both naive NEi inhibited the degradation of H4 and significantly reduced and activated cells in the absence of the inhibitor. The higher Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 681 Figure 3. NE is required for NET formation. (A) NEi but not CGi, a CG inhibitor, blocks NET formation. Purified human neutrophils, untreated or pretreated with NEi or CGi, were either activated with PMA for 4 h or left unactivated (naive) in the presence of 10% FCS. Shown are fluorescence images of cells in the presence of the cell-impermeable DNA dye Sytox green (left), and phase contrast images (right). Bar, 50 µm. (B) Quantitation of chromatin decondensation 682 JCB • VOLUME 191 • NUMBER 3 • 2010 Figure 4. NE partially degrades core histones during NET formation. (A) Histone cleavage during NET formation is inhibited by NEi. Western immuno- blotting against histones in lysates of naive (N) and PMA-activated neutrophils (PMA) in the presence (+NEi) or in the absence of NEi (untreated). (B and C) Quantitation of chromatin decondensation for the samples shown in A. (B) Untreated neutrophils. (C) Neutrophils treated with NEi. Shown are naive neutrophils at 0 h (gray) or 4 h (black), activated with PMA for 1 (yellow), 2 (orange), 3 (red), or 4 h (blue). (D) NE is not significantly externalized before NET formation. The time course of the release of NE into the medium by neutrophils activated with PMA measured by ELISA is shown. MNase was added to solubilize NE bound to DNA. Samples were normalized to NE levels from plated naive neutrophils lysed with 0.1% Triton X-100 (Total). molecular weight H3 species in both activated and naive neu- Histone degradation was detected before cell death and trophils were independent of NE activity and may represent peaked during NET formation (Fig. 4 B). NET release and other posttranslational modifications. Histones were degraded neutrophil death occurred simultaneously between 2 h and 3 h 2–4 h after stimulation, coinciding with the peak of NET re- after stimulation, as indicated by the large DNA area of all lease (Fig. 4 B). Sytox-positive neutrophils, which was blocked in the presence in samples from A. A plot of the distribution of Sytox-positive neutrophils with respect to the chromatin area is shown. Naive cells at 4 h (orange), cells stimulated with PMA alone (black), or stimulated with PMA in the presence of NEi (yellow) or CGi (purple) were quantified. The overall percentage of Sytox-positive cells for each sample is shown in parentheses. Representative data out of six independent experiments are shown. (C) NEi blocks NET forma- tion stimulated by C. albicans. Neutrophils were untreated or pretreated with 10 µM NEi in 10% human serum, and incubated with C. albicans for 3 h at MOI = 10 in the presence of Sytox. Extracellular DNA was visualized by Sytox fluorescence. PMA-stimulated and PMA + NEi control neutrophils from the same donor are shown as well. NET formation by C. albicans is less efficient than with PMA, and is blocked by NEi. (D) Quantitation of C. The distribution of Sytox-positive cells against their DNA area is shown. The right inset depicts the percentage of NETs as the number of cells whose DNA area exceeds 400 µm . Representative data out of three independent experiments are shown. (E) NEi is not cytotoxic. Release of LDH, a cytoplasmic protein, is used to monitor cell lysis. LDH levels in the medium of naive neutrophils after a 5-h incubation in the presence of increasing levels of NEi (0, 1, 10, and 30 µM). LDH levels are normalized to the total LDH content of an equivalent number of neutrophils lysed with detergent (total). (F) 5 µM NEi and 5 µM CGi have no effect on the production of ROS in response to PMA. Neutrophils were either untreated or pretreated with NEi, CGi, or the NADPH oxidase inhibitor DPI as a control. Subsequently, cells were stimulated with PMA, and ROS were detected by monitoring luminol chemiluminescence in the presence of horseradish peroxidase. Mean values and the standard deviation from triplicate samples for each condition are presented (error bars). ***, P < 0.0001. Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 683 Figure 5. NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules 684 JCB • VOLUME 191 • NUMBER 3 • 2010 of NEi (Fig. 4 C). By monitoring the release of NE during stim- dose of Klebsiella pneumoniae, a Gram-negative bacterium that ulation, we conr fi med that signic fi ant levels of the NE remained causes pneumonia. Neutrophils were massively recruited to the intracellular during the first 2 h (Fig. 4 D). lungs 48 h after infection in both groups of animals, confirming that the loss of NE does not interfere with neutrophil recruit- NE translocates to the nucleus during ment (Hirche et al., 2004). The lungs of WT mice contained de- NET formation condensed web-like structures that stain with antibodies against During NET formation, NE localized to the nucleus 60 min after MPO and an H2A-H2B-DNA complex (Fig. 6 A, i; Brinkmann stimulation, accompanied by reduced granular staining (Fig. 5 A, ii). et al., 2004). NETs have also been observed in a mouse pneumo- In contrast, only low levels of PR3 were detected in the hetero- coccal pneumonia model (Beiter et al., 2006). In contrast, in the chromatin areas of the nucleus, arguing for a selective trans- lungs of infected NE knockout animals, neutrophils exhibited location of NE. After 120 min of stimulation, NE, but not PR3, condensed nuclei, and MPO did not colocalize with the DNA was found predominantly in the decondensing nucleus in a diffuse marker but remained granular (Fig. 6 A, ii). gradient-like pattern (Fig. 5 A, iii). Interestingly, when neutro- We also analyzed these sections by scanning electron phils were treated with NEi, NE remained granular (Fig. 5 A, v), microscopy. NETs entrapping bacteria were present in abun- which suggests that NE activity is required for NE translocation. dance in the lungs of WT animals (Fig. 6 B, i and ii). In contrast, Because NE bound efc fi iently to isolated nuclei in the presence of the lungs of NE knockout mice were devoid of structures re- NEi, NE activity may be required for granular release (Fig. 2 E). sembling NETs on lung tissues and bacterial surfaces (Fig. 6 B, In contrast to NE, MPO remained granular until the iii and iv). The absence of NETs in NE single knockout animals later stages of NET formation (Fig. 5 B, i), where it colocal- indicates that the extracellular DNA detected in the lungs of WT ized with NE and DNA in neutrophils undergoing NET release mice does not originate from bacterial biofilm, as such struc - (Fig. 5 B, ii). Consistently, NE translocated faster than MPO tures would be present in both groups of mice. Furthermore, in vitro (Fig. 1 F). Furthermore, the translocation of MPO was these results suggest that PR3 cannot complement the loss of also blocked by NEi (Fig. 5 C). NE in NET formation. We detected high levels of NE in the nuclei of neutrophils In addition, we examined nuclear decondensation by undergoing NET formation by immunoblot analysis, whereas extracts derived from mouse peritoneal neutrophils. LSS ex- NE activity was selectively decreased in the granules compared tracts from WT, but not from NE knockout mouse peritoneal with MPO (Fig. 5 D). We did not detect NE activity in the nu- neutrophils, decondensed nuclei in vitro (Fig. 6 C). Despite clear fraction of either sample, as expected from the inhibitory our efforts, peritoneal mouse neutrophils responded poorly to effect of DNA (Fig. S3 A; Belorgey and Bieth, 1995). More- stimulation ex vivo, reflecting our current inability to potently over, CD63, a transmembrane protein found in azurophilic stimulate NET formation in these isolated cells (Fig. S5). granule membranes, was not perinuclear at any point during Collectively, these results indicate that NE is the major pro- NET formation. This indicates that the granules do not fuse tease driving nuclear decondensation during NET formation with the nuclear membrane. Instead, NE is released from the in vivo. granules and then translocates to the nucleus, a hypothesis that remains to be confirmed ( Fig. S4). Discussion Although it is conceivable that in isolated neutrophils, NE is r fi st externalized and then binds to the nucleus of necrotic neu - According to our model, ROS production leads to the release trophils, our observations argue against this possibility. The trans- of NE and subsequently MPO from azurophilic granules. NE location of NE to the nucleus is selective and occurs within 1 h after translocates first to the nucleus, where it digests nucleosomal stimulation (Fig. 5 A, iii), whereas neutrophil death occurs only histones and promotes extensive chromatin decondensation after 3 h of stimulation with PMA (Fig. 4 B). Moreover, NE is not (Fig. 7). In contrast to this crude approach, posttranslational signic fi antly externalized during that time (Fig. 4 D). Finally, cell histone modifications are well-suited for fine and reversible death is primarily caused by NET formation, as neutrophil death spatiotemporal control. Our data also imply that the late bind- is strikingly reduced when NE activity is inhibited (Fig. 3 B). ing of MPO to chromatin enhances decondensation indepen- However, such a mechanism may represent an alternative route to dent of its enzymatic activity. This synergy may constitute an NET formation during an infection where externalized NE may important trigger for timely cell rupture and NET release. MPO process nuclei derived from necrotic bystander cells. may drive chromatin decondensation by promoting a relaxed chromatin state, but the details of the molecular mechanism re- NE is required for NET formation in vivo main to be addressed. We examined NET formation in the lungs of wild type (WT) In addition to histone degradation, posttranslational modifi - and NE knockout mice infected intranasally with a sublethal cations may also play a role during NET formation. We observed during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by immunoblotting against MPO, NE, and histone H2B. Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 685 Figure 6. NE knockout mice fail to form NETs. (A) Representative fluorescence images of the lungs of WT (i) and NE knockout (ii) mice infected with K. pneumoniae, and stained with antibodies against MPO (green) and against a DNA/histone complex (red). The lungs of WT mice (i) contain decon- densed web-like chromatin structures that stain for MPO (arrow). In contrast, in the lungs of NE knockout mice, all neutrophils appear naive, with condensed nuclei and granular MPO staining (asterisks). Bar, 20 µm. (B) NETs (arrows) trapping bacteria are detected in scanning electron micrographs of WT mouse lungs (i and ii) infected with K. pneumoniae. The lungs of NE knockout animals are devoid of NETs (iii and iv). Bars: (i and iii) 100 nm; (ii and iv) 1 µm. (C) Lysates from NE knockout mouse peritoneal neutrophils lack nuclear decondensation activity in vitro. Cell-free nuclear decondensation assays of mouse peritoneal nuclei treated for 2 h with LSS extracts from peritoneal neutrophils derived from WT and NE knockout mice are shown. ***, P < 0.0001. 686 JCB • VOLUME 191 • NUMBER 3 • 2010 Figure 7. Model of NET formation. In resting neutrophils, NE and MPO are stored in the azurophilic granules. Upon activation and ROS production, NE escapes the granules and translocates to the nucleus. In the nucleus, NE cleaves histones and promotes chromatin decondensation. MPO binds to chromatin in the late stages of the process. MPO binding promotes further decondensation. NE and MPO cooperatively enhance chromatin decondensation, leading to cell rupture and NET release. several shifts in the mobility of histone H3 that may represent resulting increase in membrane permeability is thought to apply such modifications (Fig. 4 A). In particular, histone citrullina - to ions and not larger molecules (Lemasters et al., 2002). Alter- tion has been observed in chromatin released by neutrophil- natively, the membrane of some granules may be more exten- like human leukemia 60 (HL-60) cells and primary neutrophils sively disrupted through an unidentified mechanism. In support (Neeli et al., 2008; Wang et al., 2009). The peptidylarginine of a membrane breakdown mechanism, we have previously deiminase 4 (PAD4) inhibitor Cl -amidine was tested against reported that the nuclear envelope and granular membranes dis- HL-60 cells, where it was shown to block hypercitrullination appear during NET formation (Fuchs et al., 2007). Understanding and limit chromatin release in response to IL-8 and Shigella how NE and MPO translocate from the granules to the nucleus e fl xneri (Wang et al., 2009). However, Wang et al. (2009) did not is a complex question that awaits further investigation. test Cl -amidine in primary neutrophils but showed that over- This release mechanism may represent a new concept in expression of active PAD4 is sufc fi ient to induce NET formation cell biology associated with the oxidative burst that may not be in HL-60 cells, although the overall NET yield is low in these restricted to neutrophils. However, the release of NE is reminis- cells. Citrullination may account for some of the NE-independent cent of the lysosomal membrane permeabilization observed shifts observed in histone H3 during NET formation only in con- during cell death (Boya and Kroemer, 2008). As in the case junction with additional modifications because deimination has of NET formation, lysosomal lysis implicates ROS and requires been reported to increase the mobility of these proteins (Wang protease activation. In such a case, neutrophils may have adapted et al., 2004). Alternatively, in the absence of NEi, NE may the same concept for NET formation. first cleave histone H3 to a lower molecular weight moiety. An important role of NETs may be the sequestration of Over time, the cleaved histone H3 may accumulate additional the neutrophil’s toxic antimicrobials to minimize tissue dam- modifications, which may account for the apparent higher age (Henson and Johnston, 1987; Weiss, 1989; Xu et al., 2009). molecular weight shifts. Whether PAD4 is required for NET Using antimicrobials as key factors in NET formation ensures formation remains to be tested in primary neutrophils and in vivo. that these toxic proteins are sequestered by chromatin. A two-step Nevertheless, it is interesting to speculate that posttranslational mechanism provides the necessary time for these proteins to bind histone modifications may regulate NET formation by modulat - to chromatin before neutrophil rupture. Allowing NE to operate ing the ability of NE to cleave histones. early protects other NET proteins from being degraded by NE The intracellular release of NE underlies the existence of while it is still active inside the nucleus. Moreover, the inhibitory an as yet unknown mechanism that allows granular proteins to effect of DNA on NE activity may slow down the degradation be selectively released into the cytoplasm. ROS could promote of DNA-bound proteins, preserving their antimicrobial capacity. the release of NE directly by disrupting the association of NE Interestingly, although histone degradation fragments are more with the proteoglycan matrix that is thought to down-regulate potent antimicrobials than the intact proteins (Kawasaki and protease activity in resting cells (Serafin et al., 1986; Kolset and Iwamuro, 2008), the degradation of histones reduces cytotoxicity Gallagher, 1990; Reeves et al., 2002). Consistently, we find that against the host during sepsis (Xu et al., 2009). NE activity is required for NE translocation. NE and MPO may The requirement of neutrophil-specific factors explains be differentially released from the matrix because of differences why thus far extracellular trap formation is restricted to granu- in net surface charge and size. Alternatively, the release of these locytes, which also produce the highest levels of ROS (Segal, proteins may not involve such interactions because Niemann 2005). Mast cells express mast cell protease II, and eosino- et al. (2004) did not detect serglycin in mature circulating neu- phil elastase is found in eosinophils (Woodbury et al., 1978; trophils. In addition, ROS may drive lipid peroxidation, but the Lungarella et al., 1992). However, eosinophils may not require Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 687 proteases, as they release their mitochondrial DNA, which lacks humidified chamber at 37°C for 120 min (unless otherwise specified). Sytox-labeled nuclei were analyzed by fluorescence microscopy. histones (Yousefi et al., 2008). Moreover, the contribution of other cell-specific peroxidases (Henderson and Kaliner, 1979; Cell-free nuclear decondensation assay over filters 100-µl reactions containing 10 nuclei and 90 µl LSS extract, buffer, or Ten et al., 1989) in ET formation may be closely associated with granules were incubated for 2 h at 37°C. The reactions were then centri- their ability to interact with DNA. fuged through Ultrafree-MC Amicon polyvinylidene fluoride (PVDF) filters The immunodeficiency of NE and MPO knockout mice with a 0.65-µm pore size (Millipore) to separate the soluble flowthrough, containing the cytoplasm and the granules, from the nuclear material and may be due in part to a defect in NET formation. Our findings bound proteins, which are retained by the filter. The filters were washed may help explain the nonredundancy of NE with other related three times in cell lysis buffer, and the nuclear material was eluted with serine proteases. Mutations in NE are associated with neutro- 120 µl of 1× SDS sample buffer. 20 µl of 6× SDS sample buffer was added to the 100 µl FT. Before the initiation of the reactions, a 3-µl aliquot was penias that are treatable with granulocyte colony-stimulating removed and placed on diagnostic slides, and 0.3 µl of 10× Sytox was factor (G-CSF; Ancliff et al., 2001; Aprikyan and Dale, 2001). added to measure nuclear decondensation by microscopy at the indicated Patients treated with G-CSF display neutrophils devoid of time points. granular proteins (Horwitz et al., 2007). In spite of having near Quantitation of chromatin decondensation and NET formation normal neutrophil counts, death from sepsis still poses a high Sytox images of unfixed neutrophils or nuclei were analyzed using ImageJ risk in patients treated with G-CSF. With G-CSF treatment, image processing software. The area of Sytox signal for 300–500 cells per sample was individually measured. For quantitation of nuclear deconden- mortality caused by sepsis has decreased from 6% to 0.9% per sation, we plotted the mean DNA area derived from each nucleus (circles) year (Rosenberg et al., 2006). The remaining risk in neutropenic and the standard deviation of the values (bars) denoting the range of areas patients with mutations in NE may be due in part to a defect in for each condition. For NET formation, the distribution of the number of cells across the range of nuclear area was obtained using the frequency NET formation. Because the molecules involved in NET for- function in Excel (Microsoft). The data were converted to a percentage of mation play important roles in other neutrophil antimicrobial Sytox-positive cells by dividing the Sytox-positive counts by the total num- mechanisms, it is difficult to dissect the contribution of NETs ber of cells as determined from corresponding phase-contrast images, and plotted as the percentage of all cells that were positive for Sytox for each in immune defense. Notably, microbes have evolved virulence DNA area range. factors to degrade NETs (Beiter et al., 2006; Buchanan et al., 2006; Walker et al., 2007; Lauth et al., 2009). Therefore, it NET formation We seeded 5 × 10 neutrophils per well in 24-well plates, in HBSS(+) could be informative to document the types of microbes that (including calcium and magnesium), supplemented with 10% FCS. Cells infect patients with different immunodeficiencies, and associate were allowed to settle onto uncoated plates for 1 h before stimulation with them to their susceptibility to NETs. 100 nM PMA. Sytox green (1:15,000) was added and NETs were visu- alized by fluorescence microscopy. Wherever indicated, cells were pre - The advances presented here uncover a novel mechanism treated with 5 µM NEi (GW311616A; Sigma-Aldrich) or CGi (219372; for NET formation that may help to better understand and treat EMD) for 1 h before stimulation. Stimulation with C. albicans was per- human immunodeficiency, sepsis, and autoimmune disease. formed in HBSS(+) medium supplemented with 10% human serum at an MOI of 10. C. albicans were grown overnight at 30°C in YPD media and subcultured to reach an exponential phase. Sytox images were taken 3 h Materials and methods after infection. Cell isolation and NET formation Histone degradation during NET formation Blood was drawn from healthy volunteers, and neutrophils were isolated At each time point, the medium was removed and 5 × 10 neutrophils were over a Histopaque 1119 bed and a discontinuous Percoll gradient as de- resuspended in 300 µl of 1× SDS loading buffer. Samples were resolved scribed previously (Fuchs et al., 2007). Cells were stored in HBSS() (with- by SDS-PAGE electrophoresis, transferred to PVDF, blocked in 5% BSA, out calcium or magnesium) before experiments. PBMCs were isolated from and labeled with primary antibodies and secondary horseradish peroxidase– the same preparation. conjugated antibodies (see Antibodies for Western immunoblotting). Extracellular NE was measured by removing half the volume of Preparation of extracts, nuclei, and granular fractionation media. NE levels were quantitated by ELISA. MNase (2 U/ml) was pres- Extracts were prepared from 5 × 10 neutrophils/ml, which were lysed ent throughout the time course to solubilize any extracellular NE bound by douncing in 20 mM Hepes, pH 7.4, 100 mM KCl, 100 mM sucrose, to DNA. 100 mM NaCl, 3 mM MgCl , 1 mM EGTA, protease inhibitor cocktail pellets (Roche), and 0.1 mM PMSF. LSS was prepared by removal of Antibodies for Western immunoblotting nuclei by centrifugation at 300 g for 10 min. HSS was prepared by 1:1,000 rabbit anti-H2A (2578, recognizes the C terminus; Cell Signaling centrifugation of LSS at 100,000 g for 1 h. For granule preparations, Technology), 1:5,000 anti-H2B (07-371, recognizes aa 118–126; Milli- cells were lysed by nitrogen cavitation followed by light douncing. pore), 1:10,000 anti-H3 (07-690, recognizes the C terminus; Millipore), The granule fractionation was performed by centrifugation (37,000 g, 1:5,000 anti-H4 (04-858, epitope mapped to aa 25–28; Millipore), and 20 min) of LSS prepared in 20 mM Hepes, pH 7.4, 100 mM KCl, 100 mM the pan-histone antibody MAB052 (1:500; Millipore) for analysis of H1. sucrose, 3 mM NaCl, 3 mM MgCl , and 1 mM EGTA, over a discon- 2 Rabbit anti-NE (ab21595; Abcam) was used at 1:200, or rabbit anti-NE tinuous (1.050, 1.090, and 1.120 g/ml) Percoll gradient as described (EMD) was used at 1:500. 1:10,000 anti-MPO (A0398; Dako), 1:500 previously (Kjeldsen et al., 1994; Lominadze et al., 2005). Gradient anti-gelatinase (Dako), and 1:500 anti-lactoferrin (Sigma-Aldrich) were also fractions were isolated and centrifuged at 100,000 g for 1 h to remove used. Secondary antibodies conjugated to horseradish peroxidase (Jackson the residual Percoll. Granule fractions were resuspended into the original ImmunoResearch Laboratories, Inc.) were used at a 1:20,000 dilution. volume of LSS. HL-60 lysates and nuclei were prepared from HL-60 cells differentiated with 5 µM retinoic acid for 96 h. Intact nuclei were isolated Immunostaining and microscopy as described by Celis (1998). Cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 3% BSA, and stained with the following primary anti- Cell-free nuclear decondensation assay bodies: 1:500 mouse anti-NE (in house), 1:200 rabbit anti-NE (EMD), Reactions of 10 µl of LSS extract at 10 mg/ml total protein (derived from 1:200 rabbit anti-MPO (Dako), 1:200 EPC rabbit anti-PR3, 1:200 mouse 3–5 × 10 neutrophils/ml), the equivalent amount of HSP, or isolated gran- anti-CD63 (CBL 553; Millipore),and DRAQ5 (Biostatus Limited). Second- ules, were mixed with 10 nuclei and Sytox. Before incubation, 3-µl ali- ary antibodies conjugated with Cy2 and Cy3 (Invitrogen) were used. Con- quots were transferred onto 12-well, 5-mm diagnostic slides (Menzel-Glaser) focal images were obtained using a confocal fluorescence microscope and covered with 20 × 50 mm coverslips. Reactions were performed in a (TCS-SP; Leica) and a Fluotar 100× objective lens; images were captured 688 JCB • VOLUME 191 • NUMBER 3 • 2010 using Leica software. Epifluorescence images were obtained using an epi - of 1 mM H O . Where indicated (Fig. S3), 0.3 mg/ml of plasmid DNA or 2 2 fluorescence microscope (Axioplan; Carl Zeiss, Inc.) and 10×, 20×, or 2 × 10 /ml nuclei derived from RA-differentiated HL-60 cells were added 40× objective lenses. Images were captured using a ProgRes camera and to protease activity reactions. NE ELISA was performed using the Human software (Jenoptik). Elastase ELISA test kit (Hycult biotechnology). Statistical analysis Luminol assay Raw measurements were analyzed in Graph Pad Prism 5 software using For ROS production, 10 neutrophils, either untreated or treated with 5 µM Kruskal-Wallis analysis of variance and Dunn’s multiple comparisons test NEi, 5 µM CGi, or 10 µM of the NADPH inhibitor DPI, were activated with for pairwise comparisons (***, P < 0.0001). All experiments have been 100 nM PMA. ROS was measured by monitoring luminol fluorescence repeated at least three times unless indicated. (50 µM) in the presence of 1.2 U/ml horseradish peroxidase. Online supplemental material Cytotoxicity assay Fig. S1 shows the specificity of the NE inhibitor. Fig. S2 shows that histone Cells were either untreated or treated with NEi at the indicated concentra- degradation mediates NE-mediated nuclear decondensation. Fig. S3 tions. After 5 h of incubation, the medium was collected, and LDH release shows that DNA and nuclear binding inhibits NE activity. Fig. S4 shows was measured using the LDH cytotoxicity detection kit (Takara Bio, Inc.) and that azurophilic granules do not fuse with the nuclear membrane during normalized to the total LDH of an equivalent number of lysed neutrophils. NET formation. Fig. S5 shows that mouse peritoneal neutrophils do not form NETs in response to PMA. Online supplemental material is available Mouse lung infections at http://www.jcb.org/cgi/content/full/jcb.201006052/DC1. Five WT 129S2 mice and five NE (129S2 NE KO) knockout mice were infected with 2 × 10 Klebsiella pneumonia (KP52145; Benghezal et al., We thank Volker Brinkmann for obtaining scanning electron micrograph 2007). The bacteria were picked from an overnight plate and diluted into images of mouse lungs, Kaaweh Molawi and Juana de Diego for useful com- PBS. Mice were anesthetized and infected intranasally with 50 µl of PBS ments on the manuscript, and Marc Monestier for his generous gift of the solution containing bacteria. 48 h later, mice were sacrificed, and lungs antibody against the histone-DNA complex. V. Papayannopoulos designed were removed and fixed with 2% PFA overnight. Lungs were embedded the project and all the experiments. V. Papayannopoulos performed all experi- into paraffin and sectioned into 5-µm sections. Paraffin was removed with ments with the exception of experiments performed by K.D. Metzler and where Neo Clear (Merck) and samples were rehydrated in ethanol. Samples assisted by A. Hakkim. V. Papayannopoulos wrote the manuscript. K.D. Metzler were incubated in PBS containing 0.075% Tween 20 for 30 min at 50°C performed all human neutrophil immunofluorescence stains and imaging to expose the antigens, blocked with 5% FCS/5% donkey serum, and (Fig. 4), the nuclear digestion with purified NE (Fig. 1, H and I), the blots of stained with rabbit anti-MPO (1:50; Dako) and a mouse Fabs against granule fractions (Fig. 1 D), and the NE ELISA (Fig. 4 B). A. Hakkim developed a H2A–H2B–DNA complex (a gift from Marc Monestier, Department of the mouse infection model and performed the mouse lung infections and lung Microbiology and Immunology, School of Medicine, Temple University, isolations. A. Hakkim and V. Papayannopoulos adjusted and optimized the Philadelphia, Pennsylvania; Losman et al., 1992) fused to ATT0 550. Lung sublethal dose of K. pneumoniae for knockout animals. V. Papayannopoulos inflammation and neutrophil recruitment were evaluated by hematoxylin performed lung processing, staining, and image analysis of lung sections. and eosin stains. The experiment was repeated twice with five and seven A. Zychlinsky advised and coordinated the project. A. Zychlinksy supervised mice per group, respectively. the writing of the manuscript. For scanning electron microscopy, paraffin-embedded samples This work was funded by the Max Planck Society. V. Papayannopoulos were rehydrated, postx fi ed with glutaraldehyde, contrasted using repeated was supported by an EMBO Long term fellowship. None of the authors have changes of 0.5% OsO and 0.05% tannic acid, dehydrated in a graded any commercial or other conflict of interest. ethanol series, critical-point dried, and coated with 5 nm platinum/carbon. 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Nat. Med. 15:1318–1321. doi:10.1038/nm.2053 Yousefi, S., J.A. Gold, N. Andina, J.J. Lee, A.M. Kelly, E. Kozlowski, I. Schmid, A. Straumann, J. Reichenbach, G.J. Gleich, and H.U. Simon. 2008. Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Nat. Med. 14:949–953. doi:10.1038/nm.1855 Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 691 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

Papayannopoulos, Venizelos; Metzler, Kathleen D.; Hakkim, Abdul; Zychlinsky, Arturo
The Journal of Cell Biology , Volume 191 (3) – Nov 1, 2010
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Abstract

JCB: Article Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps Venizelos Papayannopoulos, Kathleen D. Metzler, Abdul Hakkim, and Arturo Zychlinsky Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Berlin 10117, Germany eutrophils release decondensed chromatin decondensation independent of its enzymatic activity. termed neutrophil extracellular traps (NETs) to Accordingly, NE knockout mice do not form NETs in a N trap and kill pathogens extracellularly. Reactive pulmonary model of Klebsiella pneumoniae infection, oxygen species are required to initiate NET formation which suggests that this defect may contribute to the but the downstream molecular mechanism is unknown. immune deficiency of these mice. This mechanism pro ­ We show that upon activation, neutrophil elastase (NE) vides for a novel function for serine proteases and highly escapes from azurophilic granules and translocates to charged granular proteins in the regulation of chromatin the nucleus, where it partially degrades specific histones, density, and reveals that the oxidative burst induces a promoting chromatin decondensation. Subsequently, myelo­ selective release of granular proteins into the cytoplasm peroxidase synergizes with NE in driving chromatin through an unknown mechanism. Introduction Neutrophils are the first line of immune defense (Lekstrom- virulence factors and kills bacteria (Lehrer and Ganz, 1990; Himes and Gallin, 2000; Nathan, 2006), and they combat patho- Belaaouaj et al., 2000; Weinrauch et al., 2002). MPO catalyzes the gens by phagocytosis, degranulation, and the release of neutrophil oxidation of halides by hydrogen peroxide (Hazen et al., 1996; extracellular traps (NETs; Brinkmann et al., 2004; Nauseef, Eiserich et al., 1998; Nauseef, 2007). NE and MPO knockout mice 2007; Papayannopoulos and Zychlinsky, 2009). NETs are com- are susceptible to bacterial and fungal infections (Belaaouaj posed of decondensed chromatin and antimicrobial factors, in- et al., 1998; Aratani et al., 1999; Tkalcevic et al., 2000; Gaut cluding neutrophil elastase (NE) and myeloperoxidase (MPO; et al., 2001; Belaaouaj, 2002). Interestingly, histones are the most Brinkmann et al., 2004; Urban et al., 2009), and capture and kill abundant NET component and are potent antimicrobials (Hirsch, bacteria, fungi, and parasites (Urban et al., 2006; Guimarães- 1958; Kawasaki and Iwamuro, 2008; Urban et al., 2009). Costa et al., 2009; Ramos-Kichik et al. 2009). NETs are impli- Isolated human neutrophils release NETs 2–4 h after stim- cated in immune defense, sepsis, and autoimmunity (Clark ulation with microbes or activators of PKC such as PMA (Fuchs et al., 2007; Kessenbrock et al., 2009; Papayannopoulos and et al., 2007), but respond much faster when activated by platelet Zychlinsky, 2009; Hakkim et al., 2010). Mast cells, eosinophils, cells stimulated with LPS, a process thought to be relevant during and plant cells also release DNA, which suggests that this may sepsis (Clark et al., 2007). be a common strategy in immunity (von Köckritz-Blickwede NETs form via a novel form of cell death (Fuchs et al., et al., 2008; Yousefi et al., 2008; Wen et al., 2009). 2007) that requires the production of reactive oxygen spe- NE and MPO are stored in azurophilic granules of naive cies (ROS). Neutrophils from chronic granulomatous disease neutrophils (Borregaard and Cowland, 1997; Lominadze et al., patients with mutations in the NADPH oxidase that disrupt 2005). NE is a neutrophil-specic fi serine protease that degrades ROS production (Clark and Klebanoff, 1978) fail to form NETs (Fuchs et al., 2007; Bianchi et al., 2009). In neutrophils from healthy donors, ROS production is followed by the disassembly Correspondence to Arturo Zychlinsky: [email protected] of the nuclear envelope. Chromatin decondenses in the cyto- Abbreviations used in this paper: ABAH, 4-aminobenzoic acid hydrazide; CG, cathepsin G; CGi, CG inhibitor I; DPI, diphenyleneiodonium; G-CSF, plasm and binds to granular and cytoplasmic antimicrobial granulocyte colony-stimulating factor; HSP, high-speed pellet; HSS, high-speed supernatant; LSS, low-speed supernatant; MPO, myeloperoxidase; NE, neutro- © 2010 Papayannopoulos et al. This article is distributed under the terms of an Attribution– phil elastase; NET, neutrophil extracellular trap; PAD4, peptidylarginine deimi- Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub - nase 4; PBMC, peripheral blood mononuclear cell; PR3, proteinase 3; PVDF, lication date (see http://www.rupress.org/terms). After six months it is available under a polyvinylidene fluoride; ROS, reactive oxygen species; SLPI, serum leukocyte Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, protease inhibitor. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 191 No. 3 677–691 www.jcb.org/cgi/doi/10.1083/jcb.201006052 JCB 677 THE JOURNAL OF CELL BIOLOGY Figure 1. NE cleaves histones and promotes nuclear decondensation in vitro. (A) Nuclei isolated from neutrophils were incubated in buffer or in neutrophil- derived LSS lysates for 120 min at 37°C and labeled with Sytox green. Bar, 10 µm. (B) Neutrophil extracts are sufficient to decondense nuclei from other cell types. Nuclear decondensation of LSS extracts from HL-60 cells differentiated with RA, HeLa cells, PBMCs, and neutrophils were tested with nuclei 678 JCB • VOLUME 191 • NUMBER 3 • 2010 proteins before NET release. Chromatin decondensation and two NE inhibitors, GW311616A (NEi) and serum leukocyte the association with antimicrobial proteins are two essential protease inhibitor (SLPI; Macdonald et al., 2001), but not by the steps during NET formation. The molecular mechanism linking MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH; Fig. 1 C; ROS production to chromatin decondensation and binding to Kettle et al., 1997). antimicrobial proteins is unknown. Azurophilic granules store two additional NE-related pro- Here we show that NE is essential to initiate NET for- teases, proteinase 3 (PR3) and cathepsin G (CG). Using protease- mation and that it synergizes with MPO to drive chromatin specific chromogenic peptides and histones as substrates, we decondensation. Our findings reveal a novel mechanism to show that NEi inhibits NE and PR3, but not CG (Fig. S1). SLPI drive massive chromatin decondensation, and provide evidence is known to inhibit NE and CG but not PR3 (Rao et al., 1991). for a novel pathway that allows granular proteins to leak into Considering the specificity of these inhibitors, these results in - the cytoplasm. dicate that PR3 is not sufficient to drive decondensation in vitro. In addition, CG inhibitor I (CGi), a CG-specific inhibitor, did not inhibit nuclear decondensation. Collectively, these data sug- Results gest that among these serine proteases, only NE is required for Neutrophil extracts promote the decondensation of the nucleus. chromatin decondensation To identify factors involved in NET formation, we developed a NE degrades histones to promote cell-free nuclear decondensation assay using intact nuclei and nuclear decondensation cytoplasmic extracts from neutrophils and other control cells. Because histones pack DNA, we examined whether histones Only the neutrophil-derived low-speed supernatant (LSS), con- H3 and H4 are degraded in nuclei treated with LSS. To dis- taining cytoplasm and granules, decondensed nuclei from neu- tinguish between histone degradation and histone release, we trophils, peripheral blood mononuclear cells (PBMCs), human separated the soluble unbound proteins (SF) from the nuclear leukemia-60 (HL-60), and HeLa cells (Fig. 1, A and B), which fraction (NF) by filtration. Interestingly, H4, but not H3, was indicates that neutrophil LSS contains specific factors that de - degraded in an NE-dependent manner (Fig. 1 E). We also mon- condense nuclei. Further separation of the LSS into cytoplasmic itored the kinetics of nuclear localization of NE and MPO in (high-speed supernatant [HSS]) and membrane/granule (high- relation to histone degradation and nuclear decondensation. Both speed pellet [HSP]) fractions showed that the decondensation NE and MPO were detected in the nuclear fraction within the activity partitioned with the HSP (Fig. 1 C). Neutrophils con- r fi st 30 min. Notably, H1 was degraded early but decondensation tain azurophilic, specific, and gelatinase granules (Borregaard coincided with H4 degradation at 150 min (Fig. 1 F), which and Cowland, 1997). The decondensation activity fractionated suggests that nuclear decondensation is driven primarily by the with the azurophilic granules (Fig. 1 C, fraction 3; Kjeldsen et al., degradation of core histones (Fig. 1 G). However, H1 may have 1994), which suggests that granular factors are not only com- to be degraded first to allow for the subsequent degradation of ponents of NETs but are also involved in their formation. core histones. Moreover, purified NE and PR3, but not CG, promoted NE is necessary and sufficient for nuclear decondensation in vitro (Fig. 1 H, Fig. S1 F, and not de- chromatin decondensation picted). NE degraded H4 processively, whereas the other his- NE and MPO are two of the primary enzymes stored in azuro- tones were only partially degraded (Fig. 1 I). Histone degradation philic granules and are found in abundance in NETs. Un- was detectable by 30 min and coincided with nuclear deconden- like MPO, only NE sedimented exclusively to the azurophilic sation (Fig. 1, H and I). As a control, we showed that NE com- fraction (Fig. 1 D, lane 3; Lominadze et al., 2005). We tested pletely degraded soluble histones purified from neutrophils whether the activity of NE and MPO is required for nuclear (Fig. 1 I), which indicates that the pattern of histone degra- decondensation. Chromatin decondensation was blocked by dation depends on chromatin structure and not on the intrinsic isolated from neutrophils (Neut). The nuclear area was quantified using ImageJ image processing software. Circles denote the median area and the bars indicate the range of the nuclear area values, calculated from the standard deviation of each dataset. (C) Decondensation activity is stored in azurophilic granules. Decondensation of nuclei treated with buffer, neutrophil LSS, neutrophil HSS, HSP, and the granular subfractions of gelatinase (1), specific (2), and azurophilic (3) granules were purified over a discontinuous Percoll gradient. Nuclei were also incubated with LSS or HSP in the presence or absence of NEi, SLPI, CGi, or ABAH. Inhibitors were used at the indicated concentrations. (D) The purity of gelatinase (1), specific (2), and azurophilic (3) granule fractions was tested by SDS-PAGE immunoblot analysis using antibodies against gelatinase, lactoferrin, MPO, and NE. NE fractionates exclusively with azurophilic granules (3). (E) NEi inhibits the degradation of nuclear H4 in vitro. After 120 min at 37°C, decondensation reactions were passed over a 0.65-µm filter to separate the nuclei from the extract. The total reaction (T), flowthrough (SF), or nuclear (NF) fractions were analyzed by immunoblotting. Top, nuclei in buffer; middle, nuclei in LSS; bottom, nuclei incubated with LSS and 5 µM NEi. (F) NE and MPO translocate to nuclei in vitro. LSS (L), nuclei alone (N), or LSS + nuclei (L+N), were incubated for the indicated time and separated over filters as in E. The levels of H1, H4, NE, and MPO in the nuclear fraction at different time points were analyzed by immunoblotting. (G) A plot of the corresponding nuclear decondensation, measured as in A, for the experiment in F. (H) Purified NE is sufficient to decondense nuclei in vitro. Nuclei were incubated with buffer (B) or 1 µM purified NE (NE) in the absence of MPO (no MPO, open circles) for the indicated duration (0–240 min). In parallel, 1 µM MPO was added to the same samples (+MPO, closed circles). Decondensation was measured by quantifying the nuclear area and was significantly enhanced in the presence of both NE and MPO (closed circles). (I) Purified NE cleaves nuclear histones in vitro. The samples from H along with NE incubated with purified neutrophil histones were resolved by SDS-PAGE and analyzed by immunoblotting. H4 was processively degraded but H2A, H2B, and H3 were only partially degraded by NE. Soluble purified histones were completely degraded. Notably, the addition of 1 µM MPO had no effect on histone degradation by NE (third column). ***, P < 0.0001. Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 679 susceptibility of histones to this protease. Similarly, purified To investigate whether high concentrations of NE de- PR3 cleaved nuclear histones in vitro (Fig. S1 E). In con- creased histone degradation, we incubated nuclei with increasing trast, and consistent with our previous observations, nuclear NE concentrations and separated the nuclear (NF) and soluble and purified histones were poor substrates for CG (unpub - (SF) fractions over filters (Fig. 2 E). H4 was degraded under lished data). optimal, but not autoinhibitory, concentrations of NE. Notably, To examine whether the degradation of histones is caus- NE activity is not required for NE binding to nuclei, as it parti- ing chromatin decondensation, we preincubated permeabilized tioned with nuclei in the presence of NEi. The lower levels of nuclei with antihistone or a control antibody against CD63, a NE in the NF fraction in the absence of NEi suggest that NE transmembrane granular protein. Nuclear decondensation was degrades itself in the nucleus (Fig. 2 E, left, asterisks). reduced by 50% in nuclei incubated with antibodies against H4, We asked whether NE and MPO interact inside the nu- and to a lesser extent with antibodies against other core histones cleus by incubating nuclei with NE and low concentrations of (Fig. S2 A), but not by the control antibody. Accordingly, H4 MPO. MPO nuclear localization was independent of NE activ- degradation was reduced in nuclei treated with antihistone anti- ity, as significant amounts of MPO were found in the nuclear bodies (Fig. S2 B). fraction in the presence of NEi (Fig. 2 E, right). Moreover, we Interestingly, in a fractionation experiment, we purified observed NE-dependent MPO degradation in the nuclear, but H1 as an inhibitor of NET formation (unpublished data). H1 not in the soluble fraction, which indicates that these proteins promotes the condensed, closed state of chromatin, which re- come into close proximity in the nucleus. stricts its accessibility (Roche et al., 1985; Bustin et al., 2005; Woodcock et al., 2006). Nuclei pretreated or coincubated with NE activity is required for NET formation H1 were resistant to decondensation and histone degradation in neutrophils by LSS and HSP (Fig. S2, C–E). Moreover, MPO failed to par- To investigate whether NE activity is required for NET forma- tition with the NF in nuclei pretreated with H1, which indicates tion, we pretreated neutrophils isolated from healthy donors that chromatin is the primary binding site of MPO in vitro with NEi or CGi, and induced NET formation with PMA in the (Fig. S2 D). Therefore, NE and MPO require access to the core presence of Sytox, a cell-impermeable DNA dye. Untreated histones to degrade them and induce decondensation. neutrophils and cells treated with CGi formed NETs efficiently 2–4 h after activation. Notably, NEi-treated neutrophils failed to NE and MPO synergize to promote make NETs, and appeared necrotic as determined by the Sytox- nuclear decondensation positive condensed nuclei (Fig. 3 A). To quantitate NET formation, Because MPO localizes to NETs and binds to nuclei in vitro we measured the DNA area of each Sytox-positive neutrophil (Fig. 1 F), we tested whether MPO promotes nuclear deconden- and plotted the percentage of Sytox-positive cells against their sation (Brinkmann et al., 2004). At 1 µM, MPO alone had little corresponding nuclear area (Fig. 3 B). Our quantitation con- effect, but it dramatically enhanced chromatin decondensation firmed that neutrophil death is dramatically reduced in the in the presence of NE (Fig. 1 H). Interestingly, MPO did not presence of NEi (11.2% vs. 63.7%), whereas the low nuclear affect histone degradation, which suggests that it does not up- areas of the dead cells indicate that cells did not make NETs regulate NE activity (Fig. 1 I). MPO promoted nuclear de- but were necrotic. condensation in a dose-dependent manner that did not require Candida albicans is a potent physiological activator of its substrate H O and was not inhibited by ABAH (Fig. 2, NET formation in isolated primary neutrophils. Neutrophils 2 2 A and B). Furthermore, horseradish peroxidase, used as a control treated with NEi failed to make NETs efficiently when ex - for enzymatic activity, did not promote chromatin deconden- posed to C. albicans (Fig. 3 C). The number of neutrophils sation (unpublished data). We concluded that MPO enhances that died without making NETs increased dramatically and NET formation independent of its enzymatic activity. was comparable to neutrophils treated with NEi and stimu- To better understand the synergy between NE and MPO, lated with PMA (Fig. 3 D). Interestingly, C. albicans induced we measured its effect on nuclear decondensation driven by NE. NET formation with lower efficiency than PMA (Fig. 3 D), Increasing amounts of MPO enhanced NE-mediated nuclear which indicates that when presented with microbes, neutro- decondensation (Fig. 2 C). Surprisingly, high concentrations phils may respond in different ways, with some neutrophils of NE inhibited nuclear decondensation (Fig. 2 D). This auto- consuming NE and MPO through phagocytosis or degranula- inhibition could be caused by NE autodegradation. Alternatively, tion while others form NETs. at high concentrations, NE could inhibit histone degradation. As controls, we show that NEi was not cytotoxic (Fig. 3 E) Accordingly, DNA has been shown to inhibit NE (Belorgey and and did not inhibit ROS production, which was potently blocked Bieth, 1995). We confirmed the inhibitory effect of DNA on NE by the NADPH oxidase inhibitor diphenyleneiodonium (DPI; and CG, and found that it was reversed by the addition of NaCl Fig. 3 F; Cross and Jones, 1986). (Fig. S3, A and B). Intact nuclei also inhibited NE activity, but this effect was not reversed by the addition of NaCl. Therefore, NE cleaves histones during NET formation after histone cleavage, NE activity may be down-regulated by Because PMA is a more potent inducer of NETs than C. albi- binding to DNA. Autoinhibition was not observed for PR3, cans and other microbes, we used it to examine whether his- which completely dissolved the nuclei at high concentrations tones are degraded in an NE-dependent manner during NET (Fig. S1, E and F). formation. Consistent with our in vitro data, H2B and H4 were 680 JCB • VOLUME 191 • NUMBER 3 • 2010 Figure 2. NE and MPO synergize. (A) Effect of MPO titration on nuclear decondensation in vitro. Nuclei were incubated with the indicated concentra- tions of MPO for 30 (open circles), 60 (gray circles), or 120 min (closed circles). Decondensation was concentration- but not time-dependent. (B) MPO promotes decondensation independent of its enzymatic activity. Nuclei were treated with 5 µM MPO alone or MPO in the presence of 100 µM ABAH (an MPO inhibitor), 100 µM H O (MPO substrate), or both. (C) Nuclear decondensation was driven by titration of NE at different concentrations of MPO. 2 2 MPO increases NE-mediated decondensation. (D) The effect of NE titration on nuclear decondensation. Nuclei were incubated with the indicated concen- trations of NE in the absence (black line) or presence (red line) of 10 µM NEi for 120 min before measuring nuclear decondensation. (E) NE-mediated degradation of histones is dose-dependent during nuclear decondensation. Western immunoblot analysis of MPO, H4, and NE was performed. Nuclei mixed with increasing concentrations of NE and 0.3 µM MPO for 120 min were separated into nuclear (NF, left) and soluble (SF, right) fractions. Reactions were performed in the absence or presence of NEi. The samples where nuclei decondensed are indicated by positive signs below the NF blot. Asterisks mark the levels of NE bound to nuclei, in the presence or absence of NEi, at the highest concentration of NE. ***, P < 0.0001. partially degraded during NET formation, whereas H3 shifted the degradation of H2B. NEi also prevented the formation of to a higher molecular weight species (Fig. 4 A). The presence of an H2A degradation product, which was observed in both naive NEi inhibited the degradation of H4 and significantly reduced and activated cells in the absence of the inhibitor. The higher Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 681 Figure 3. NE is required for NET formation. (A) NEi but not CGi, a CG inhibitor, blocks NET formation. Purified human neutrophils, untreated or pretreated with NEi or CGi, were either activated with PMA for 4 h or left unactivated (naive) in the presence of 10% FCS. Shown are fluorescence images of cells in the presence of the cell-impermeable DNA dye Sytox green (left), and phase contrast images (right). Bar, 50 µm. (B) Quantitation of chromatin decondensation 682 JCB • VOLUME 191 • NUMBER 3 • 2010 Figure 4. NE partially degrades core histones during NET formation. (A) Histone cleavage during NET formation is inhibited by NEi. Western immuno- blotting against histones in lysates of naive (N) and PMA-activated neutrophils (PMA) in the presence (+NEi) or in the absence of NEi (untreated). (B and C) Quantitation of chromatin decondensation for the samples shown in A. (B) Untreated neutrophils. (C) Neutrophils treated with NEi. Shown are naive neutrophils at 0 h (gray) or 4 h (black), activated with PMA for 1 (yellow), 2 (orange), 3 (red), or 4 h (blue). (D) NE is not significantly externalized before NET formation. The time course of the release of NE into the medium by neutrophils activated with PMA measured by ELISA is shown. MNase was added to solubilize NE bound to DNA. Samples were normalized to NE levels from plated naive neutrophils lysed with 0.1% Triton X-100 (Total). molecular weight H3 species in both activated and naive neu- Histone degradation was detected before cell death and trophils were independent of NE activity and may represent peaked during NET formation (Fig. 4 B). NET release and other posttranslational modifications. Histones were degraded neutrophil death occurred simultaneously between 2 h and 3 h 2–4 h after stimulation, coinciding with the peak of NET re- after stimulation, as indicated by the large DNA area of all lease (Fig. 4 B). Sytox-positive neutrophils, which was blocked in the presence in samples from A. A plot of the distribution of Sytox-positive neutrophils with respect to the chromatin area is shown. Naive cells at 4 h (orange), cells stimulated with PMA alone (black), or stimulated with PMA in the presence of NEi (yellow) or CGi (purple) were quantified. The overall percentage of Sytox-positive cells for each sample is shown in parentheses. Representative data out of six independent experiments are shown. (C) NEi blocks NET forma- tion stimulated by C. albicans. Neutrophils were untreated or pretreated with 10 µM NEi in 10% human serum, and incubated with C. albicans for 3 h at MOI = 10 in the presence of Sytox. Extracellular DNA was visualized by Sytox fluorescence. PMA-stimulated and PMA + NEi control neutrophils from the same donor are shown as well. NET formation by C. albicans is less efficient than with PMA, and is blocked by NEi. (D) Quantitation of C. The distribution of Sytox-positive cells against their DNA area is shown. The right inset depicts the percentage of NETs as the number of cells whose DNA area exceeds 400 µm . Representative data out of three independent experiments are shown. (E) NEi is not cytotoxic. Release of LDH, a cytoplasmic protein, is used to monitor cell lysis. LDH levels in the medium of naive neutrophils after a 5-h incubation in the presence of increasing levels of NEi (0, 1, 10, and 30 µM). LDH levels are normalized to the total LDH content of an equivalent number of neutrophils lysed with detergent (total). (F) 5 µM NEi and 5 µM CGi have no effect on the production of ROS in response to PMA. Neutrophils were either untreated or pretreated with NEi, CGi, or the NADPH oxidase inhibitor DPI as a control. Subsequently, cells were stimulated with PMA, and ROS were detected by monitoring luminol chemiluminescence in the presence of horseradish peroxidase. Mean values and the standard deviation from triplicate samples for each condition are presented (error bars). ***, P < 0.0001. Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 683 Figure 5. NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules 684 JCB • VOLUME 191 • NUMBER 3 • 2010 of NEi (Fig. 4 C). By monitoring the release of NE during stim- dose of Klebsiella pneumoniae, a Gram-negative bacterium that ulation, we conr fi med that signic fi ant levels of the NE remained causes pneumonia. Neutrophils were massively recruited to the intracellular during the first 2 h (Fig. 4 D). lungs 48 h after infection in both groups of animals, confirming that the loss of NE does not interfere with neutrophil recruit- NE translocates to the nucleus during ment (Hirche et al., 2004). The lungs of WT mice contained de- NET formation condensed web-like structures that stain with antibodies against During NET formation, NE localized to the nucleus 60 min after MPO and an H2A-H2B-DNA complex (Fig. 6 A, i; Brinkmann stimulation, accompanied by reduced granular staining (Fig. 5 A, ii). et al., 2004). NETs have also been observed in a mouse pneumo- In contrast, only low levels of PR3 were detected in the hetero- coccal pneumonia model (Beiter et al., 2006). In contrast, in the chromatin areas of the nucleus, arguing for a selective trans- lungs of infected NE knockout animals, neutrophils exhibited location of NE. After 120 min of stimulation, NE, but not PR3, condensed nuclei, and MPO did not colocalize with the DNA was found predominantly in the decondensing nucleus in a diffuse marker but remained granular (Fig. 6 A, ii). gradient-like pattern (Fig. 5 A, iii). Interestingly, when neutro- We also analyzed these sections by scanning electron phils were treated with NEi, NE remained granular (Fig. 5 A, v), microscopy. NETs entrapping bacteria were present in abun- which suggests that NE activity is required for NE translocation. dance in the lungs of WT animals (Fig. 6 B, i and ii). In contrast, Because NE bound efc fi iently to isolated nuclei in the presence of the lungs of NE knockout mice were devoid of structures re- NEi, NE activity may be required for granular release (Fig. 2 E). sembling NETs on lung tissues and bacterial surfaces (Fig. 6 B, In contrast to NE, MPO remained granular until the iii and iv). The absence of NETs in NE single knockout animals later stages of NET formation (Fig. 5 B, i), where it colocal- indicates that the extracellular DNA detected in the lungs of WT ized with NE and DNA in neutrophils undergoing NET release mice does not originate from bacterial biofilm, as such struc - (Fig. 5 B, ii). Consistently, NE translocated faster than MPO tures would be present in both groups of mice. Furthermore, in vitro (Fig. 1 F). Furthermore, the translocation of MPO was these results suggest that PR3 cannot complement the loss of also blocked by NEi (Fig. 5 C). NE in NET formation. We detected high levels of NE in the nuclei of neutrophils In addition, we examined nuclear decondensation by undergoing NET formation by immunoblot analysis, whereas extracts derived from mouse peritoneal neutrophils. LSS ex- NE activity was selectively decreased in the granules compared tracts from WT, but not from NE knockout mouse peritoneal with MPO (Fig. 5 D). We did not detect NE activity in the nu- neutrophils, decondensed nuclei in vitro (Fig. 6 C). Despite clear fraction of either sample, as expected from the inhibitory our efforts, peritoneal mouse neutrophils responded poorly to effect of DNA (Fig. S3 A; Belorgey and Bieth, 1995). More- stimulation ex vivo, reflecting our current inability to potently over, CD63, a transmembrane protein found in azurophilic stimulate NET formation in these isolated cells (Fig. S5). granule membranes, was not perinuclear at any point during Collectively, these results indicate that NE is the major pro- NET formation. This indicates that the granules do not fuse tease driving nuclear decondensation during NET formation with the nuclear membrane. Instead, NE is released from the in vivo. granules and then translocates to the nucleus, a hypothesis that remains to be confirmed ( Fig. S4). Discussion Although it is conceivable that in isolated neutrophils, NE is r fi st externalized and then binds to the nucleus of necrotic neu - According to our model, ROS production leads to the release trophils, our observations argue against this possibility. The trans- of NE and subsequently MPO from azurophilic granules. NE location of NE to the nucleus is selective and occurs within 1 h after translocates first to the nucleus, where it digests nucleosomal stimulation (Fig. 5 A, iii), whereas neutrophil death occurs only histones and promotes extensive chromatin decondensation after 3 h of stimulation with PMA (Fig. 4 B). Moreover, NE is not (Fig. 7). In contrast to this crude approach, posttranslational signic fi antly externalized during that time (Fig. 4 D). Finally, cell histone modifications are well-suited for fine and reversible death is primarily caused by NET formation, as neutrophil death spatiotemporal control. Our data also imply that the late bind- is strikingly reduced when NE activity is inhibited (Fig. 3 B). ing of MPO to chromatin enhances decondensation indepen- However, such a mechanism may represent an alternative route to dent of its enzymatic activity. This synergy may constitute an NET formation during an infection where externalized NE may important trigger for timely cell rupture and NET release. MPO process nuclei derived from necrotic bystander cells. may drive chromatin decondensation by promoting a relaxed chromatin state, but the details of the molecular mechanism re- NE is required for NET formation in vivo main to be addressed. We examined NET formation in the lungs of wild type (WT) In addition to histone degradation, posttranslational modifi - and NE knockout mice infected intranasally with a sublethal cations may also play a role during NET formation. We observed during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by immunoblotting against MPO, NE, and histone H2B. Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 685 Figure 6. NE knockout mice fail to form NETs. (A) Representative fluorescence images of the lungs of WT (i) and NE knockout (ii) mice infected with K. pneumoniae, and stained with antibodies against MPO (green) and against a DNA/histone complex (red). The lungs of WT mice (i) contain decon- densed web-like chromatin structures that stain for MPO (arrow). In contrast, in the lungs of NE knockout mice, all neutrophils appear naive, with condensed nuclei and granular MPO staining (asterisks). Bar, 20 µm. (B) NETs (arrows) trapping bacteria are detected in scanning electron micrographs of WT mouse lungs (i and ii) infected with K. pneumoniae. The lungs of NE knockout animals are devoid of NETs (iii and iv). Bars: (i and iii) 100 nm; (ii and iv) 1 µm. (C) Lysates from NE knockout mouse peritoneal neutrophils lack nuclear decondensation activity in vitro. Cell-free nuclear decondensation assays of mouse peritoneal nuclei treated for 2 h with LSS extracts from peritoneal neutrophils derived from WT and NE knockout mice are shown. ***, P < 0.0001. 686 JCB • VOLUME 191 • NUMBER 3 • 2010 Figure 7. Model of NET formation. In resting neutrophils, NE and MPO are stored in the azurophilic granules. Upon activation and ROS production, NE escapes the granules and translocates to the nucleus. In the nucleus, NE cleaves histones and promotes chromatin decondensation. MPO binds to chromatin in the late stages of the process. MPO binding promotes further decondensation. NE and MPO cooperatively enhance chromatin decondensation, leading to cell rupture and NET release. several shifts in the mobility of histone H3 that may represent resulting increase in membrane permeability is thought to apply such modifications (Fig. 4 A). In particular, histone citrullina - to ions and not larger molecules (Lemasters et al., 2002). Alter- tion has been observed in chromatin released by neutrophil- natively, the membrane of some granules may be more exten- like human leukemia 60 (HL-60) cells and primary neutrophils sively disrupted through an unidentified mechanism. In support (Neeli et al., 2008; Wang et al., 2009). The peptidylarginine of a membrane breakdown mechanism, we have previously deiminase 4 (PAD4) inhibitor Cl -amidine was tested against reported that the nuclear envelope and granular membranes dis- HL-60 cells, where it was shown to block hypercitrullination appear during NET formation (Fuchs et al., 2007). Understanding and limit chromatin release in response to IL-8 and Shigella how NE and MPO translocate from the granules to the nucleus e fl xneri (Wang et al., 2009). However, Wang et al. (2009) did not is a complex question that awaits further investigation. test Cl -amidine in primary neutrophils but showed that over- This release mechanism may represent a new concept in expression of active PAD4 is sufc fi ient to induce NET formation cell biology associated with the oxidative burst that may not be in HL-60 cells, although the overall NET yield is low in these restricted to neutrophils. However, the release of NE is reminis- cells. Citrullination may account for some of the NE-independent cent of the lysosomal membrane permeabilization observed shifts observed in histone H3 during NET formation only in con- during cell death (Boya and Kroemer, 2008). As in the case junction with additional modifications because deimination has of NET formation, lysosomal lysis implicates ROS and requires been reported to increase the mobility of these proteins (Wang protease activation. In such a case, neutrophils may have adapted et al., 2004). Alternatively, in the absence of NEi, NE may the same concept for NET formation. first cleave histone H3 to a lower molecular weight moiety. An important role of NETs may be the sequestration of Over time, the cleaved histone H3 may accumulate additional the neutrophil’s toxic antimicrobials to minimize tissue dam- modifications, which may account for the apparent higher age (Henson and Johnston, 1987; Weiss, 1989; Xu et al., 2009). molecular weight shifts. Whether PAD4 is required for NET Using antimicrobials as key factors in NET formation ensures formation remains to be tested in primary neutrophils and in vivo. that these toxic proteins are sequestered by chromatin. A two-step Nevertheless, it is interesting to speculate that posttranslational mechanism provides the necessary time for these proteins to bind histone modifications may regulate NET formation by modulat - to chromatin before neutrophil rupture. Allowing NE to operate ing the ability of NE to cleave histones. early protects other NET proteins from being degraded by NE The intracellular release of NE underlies the existence of while it is still active inside the nucleus. Moreover, the inhibitory an as yet unknown mechanism that allows granular proteins to effect of DNA on NE activity may slow down the degradation be selectively released into the cytoplasm. ROS could promote of DNA-bound proteins, preserving their antimicrobial capacity. the release of NE directly by disrupting the association of NE Interestingly, although histone degradation fragments are more with the proteoglycan matrix that is thought to down-regulate potent antimicrobials than the intact proteins (Kawasaki and protease activity in resting cells (Serafin et al., 1986; Kolset and Iwamuro, 2008), the degradation of histones reduces cytotoxicity Gallagher, 1990; Reeves et al., 2002). Consistently, we find that against the host during sepsis (Xu et al., 2009). NE activity is required for NE translocation. NE and MPO may The requirement of neutrophil-specific factors explains be differentially released from the matrix because of differences why thus far extracellular trap formation is restricted to granu- in net surface charge and size. Alternatively, the release of these locytes, which also produce the highest levels of ROS (Segal, proteins may not involve such interactions because Niemann 2005). Mast cells express mast cell protease II, and eosino- et al. (2004) did not detect serglycin in mature circulating neu- phil elastase is found in eosinophils (Woodbury et al., 1978; trophils. In addition, ROS may drive lipid peroxidation, but the Lungarella et al., 1992). However, eosinophils may not require Neutrophil elastase in extracellular trap formation • Papayannopoulos et al. 687 proteases, as they release their mitochondrial DNA, which lacks humidified chamber at 37°C for 120 min (unless otherwise specified). Sytox-labeled nuclei were analyzed by fluorescence microscopy. histones (Yousefi et al., 2008). Moreover, the contribution of other cell-specific peroxidases (Henderson and Kaliner, 1979; Cell-free nuclear decondensation assay over filters 100-µl reactions containing 10 nuclei and 90 µl LSS extract, buffer, or Ten et al., 1989) in ET formation may be closely associated with granules were incubated for 2 h at 37°C. The reactions were then centri- their ability to interact with DNA. fuged through Ultrafree-MC Amicon polyvinylidene fluoride (PVDF) filters The immunodeficiency of NE and MPO knockout mice with a 0.65-µm pore size (Millipore) to separate the soluble flowthrough, containing the cytoplasm and the granules, from the nuclear material and may be due in part to a defect in NET formation. Our findings bound proteins, which are retained by the filter. The filters were washed may help explain the nonredundancy of NE with other related three times in cell lysis buffer, and the nuclear material was eluted with serine proteases. Mutations in NE are associated with neutro- 120 µl of 1× SDS sample buffer. 20 µl of 6× SDS sample buffer was added to the 100 µl FT. Before the initiation of the reactions, a 3-µl aliquot was penias that are treatable with granulocyte colony-stimulating removed and placed on diagnostic slides, and 0.3 µl of 10× Sytox was factor (G-CSF; Ancliff et al., 2001; Aprikyan and Dale, 2001). added to measure nuclear decondensation by microscopy at the indicated Patients treated with G-CSF display neutrophils devoid of time points. granular proteins (Horwitz et al., 2007). In spite of having near Quantitation of chromatin decondensation and NET formation normal neutrophil counts, death from sepsis still poses a high Sytox images of unfixed neutrophils or nuclei were analyzed using ImageJ risk in patients treated with G-CSF. With G-CSF treatment, image processing software. The area of Sytox signal for 300–500 cells per sample was individually measured. For quantitation of nuclear deconden- mortality caused by sepsis has decreased from 6% to 0.9% per sation, we plotted the mean DNA area derived from each nucleus (circles) year (Rosenberg et al., 2006). The remaining risk in neutropenic and the standard deviation of the values (bars) denoting the range of areas patients with mutations in NE may be due in part to a defect in for each condition. For NET formation, the distribution of the number of cells across the range of nuclear area was obtained using the frequency NET formation. Because the molecules involved in NET for- function in Excel (Microsoft). The data were converted to a percentage of mation play important roles in other neutrophil antimicrobial Sytox-positive cells by dividing the Sytox-positive counts by the total num- mechanisms, it is difficult to dissect the contribution of NETs ber of cells as determined from corresponding phase-contrast images, and plotted as the percentage of all cells that were positive for Sytox for each in immune defense. Notably, microbes have evolved virulence DNA area range. factors to degrade NETs (Beiter et al., 2006; Buchanan et al., 2006; Walker et al., 2007; Lauth et al., 2009). Therefore, it NET formation We seeded 5 × 10 neutrophils per well in 24-well plates, in HBSS(+) could be informative to document the types of microbes that (including calcium and magnesium), supplemented with 10% FCS. Cells infect patients with different immunodeficiencies, and associate were allowed to settle onto uncoated plates for 1 h before stimulation with them to their susceptibility to NETs. 100 nM PMA. Sytox green (1:15,000) was added and NETs were visu- alized by fluorescence microscopy. Wherever indicated, cells were pre - The advances presented here uncover a novel mechanism treated with 5 µM NEi (GW311616A; Sigma-Aldrich) or CGi (219372; for NET formation that may help to better understand and treat EMD) for 1 h before stimulation. Stimulation with C. albicans was per- human immunodeficiency, sepsis, and autoimmune disease. formed in HBSS(+) medium supplemented with 10% human serum at an MOI of 10. C. albicans were grown overnight at 30°C in YPD media and subcultured to reach an exponential phase. Sytox images were taken 3 h Materials and methods after infection. Cell isolation and NET formation Histone degradation during NET formation Blood was drawn from healthy volunteers, and neutrophils were isolated At each time point, the medium was removed and 5 × 10 neutrophils were over a Histopaque 1119 bed and a discontinuous Percoll gradient as de- resuspended in 300 µl of 1× SDS loading buffer. Samples were resolved scribed previously (Fuchs et al., 2007). Cells were stored in HBSS() (with- by SDS-PAGE electrophoresis, transferred to PVDF, blocked in 5% BSA, out calcium or magnesium) before experiments. PBMCs were isolated from and labeled with primary antibodies and secondary horseradish peroxidase– the same preparation. conjugated antibodies (see Antibodies for Western immunoblotting). Extracellular NE was measured by removing half the volume of Preparation of extracts, nuclei, and granular fractionation media. NE levels were quantitated by ELISA. MNase (2 U/ml) was pres- Extracts were prepared from 5 × 10 neutrophils/ml, which were lysed ent throughout the time course to solubilize any extracellular NE bound by douncing in 20 mM Hepes, pH 7.4, 100 mM KCl, 100 mM sucrose, to DNA. 100 mM NaCl, 3 mM MgCl , 1 mM EGTA, protease inhibitor cocktail pellets (Roche), and 0.1 mM PMSF. LSS was prepared by removal of Antibodies for Western immunoblotting nuclei by centrifugation at 300 g for 10 min. HSS was prepared by 1:1,000 rabbit anti-H2A (2578, recognizes the C terminus; Cell Signaling centrifugation of LSS at 100,000 g for 1 h. For granule preparations, Technology), 1:5,000 anti-H2B (07-371, recognizes aa 118–126; Milli- cells were lysed by nitrogen cavitation followed by light douncing. pore), 1:10,000 anti-H3 (07-690, recognizes the C terminus; Millipore), The granule fractionation was performed by centrifugation (37,000 g, 1:5,000 anti-H4 (04-858, epitope mapped to aa 25–28; Millipore), and 20 min) of LSS prepared in 20 mM Hepes, pH 7.4, 100 mM KCl, 100 mM the pan-histone antibody MAB052 (1:500; Millipore) for analysis of H1. sucrose, 3 mM NaCl, 3 mM MgCl , and 1 mM EGTA, over a discon- 2 Rabbit anti-NE (ab21595; Abcam) was used at 1:200, or rabbit anti-NE tinuous (1.050, 1.090, and 1.120 g/ml) Percoll gradient as described (EMD) was used at 1:500. 1:10,000 anti-MPO (A0398; Dako), 1:500 previously (Kjeldsen et al., 1994; Lominadze et al., 2005). Gradient anti-gelatinase (Dako), and 1:500 anti-lactoferrin (Sigma-Aldrich) were also fractions were isolated and centrifuged at 100,000 g for 1 h to remove used. Secondary antibodies conjugated to horseradish peroxidase (Jackson the residual Percoll. Granule fractions were resuspended into the original ImmunoResearch Laboratories, Inc.) were used at a 1:20,000 dilution. volume of LSS. HL-60 lysates and nuclei were prepared from HL-60 cells differentiated with 5 µM retinoic acid for 96 h. Intact nuclei were isolated Immunostaining and microscopy as described by Celis (1998). Cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 3% BSA, and stained with the following primary anti- Cell-free nuclear decondensation assay bodies: 1:500 mouse anti-NE (in house), 1:200 rabbit anti-NE (EMD), Reactions of 10 µl of LSS extract at 10 mg/ml total protein (derived from 1:200 rabbit anti-MPO (Dako), 1:200 EPC rabbit anti-PR3, 1:200 mouse 3–5 × 10 neutrophils/ml), the equivalent amount of HSP, or isolated gran- anti-CD63 (CBL 553; Millipore),and DRAQ5 (Biostatus Limited). Second- ules, were mixed with 10 nuclei and Sytox. Before incubation, 3-µl ali- ary antibodies conjugated with Cy2 and Cy3 (Invitrogen) were used. Con- quots were transferred onto 12-well, 5-mm diagnostic slides (Menzel-Glaser) focal images were obtained using a confocal fluorescence microscope and covered with 20 × 50 mm coverslips. Reactions were performed in a (TCS-SP; Leica) and a Fluotar 100× objective lens; images were captured 688 JCB • VOLUME 191 • NUMBER 3 • 2010 using Leica software. Epifluorescence images were obtained using an epi - of 1 mM H O . Where indicated (Fig. S3), 0.3 mg/ml of plasmid DNA or 2 2 fluorescence microscope (Axioplan; Carl Zeiss, Inc.) and 10×, 20×, or 2 × 10 /ml nuclei derived from RA-differentiated HL-60 cells were added 40× objective lenses. Images were captured using a ProgRes camera and to protease activity reactions. NE ELISA was performed using the Human software (Jenoptik). Elastase ELISA test kit (Hycult biotechnology). Statistical analysis Luminol assay Raw measurements were analyzed in Graph Pad Prism 5 software using For ROS production, 10 neutrophils, either untreated or treated with 5 µM Kruskal-Wallis analysis of variance and Dunn’s multiple comparisons test NEi, 5 µM CGi, or 10 µM of the NADPH inhibitor DPI, were activated with for pairwise comparisons (***, P < 0.0001). All experiments have been 100 nM PMA. ROS was measured by monitoring luminol fluorescence repeated at least three times unless indicated. (50 µM) in the presence of 1.2 U/ml horseradish peroxidase. Online supplemental material Cytotoxicity assay Fig. S1 shows the specificity of the NE inhibitor. Fig. S2 shows that histone Cells were either untreated or treated with NEi at the indicated concentra- degradation mediates NE-mediated nuclear decondensation. Fig. S3 tions. After 5 h of incubation, the medium was collected, and LDH release shows that DNA and nuclear binding inhibits NE activity. Fig. S4 shows was measured using the LDH cytotoxicity detection kit (Takara Bio, Inc.) and that azurophilic granules do not fuse with the nuclear membrane during normalized to the total LDH of an equivalent number of lysed neutrophils. NET formation. Fig. S5 shows that mouse peritoneal neutrophils do not form NETs in response to PMA. Online supplemental material is available Mouse lung infections at http://www.jcb.org/cgi/content/full/jcb.201006052/DC1. Five WT 129S2 mice and five NE (129S2 NE KO) knockout mice were infected with 2 × 10 Klebsiella pneumonia (KP52145; Benghezal et al., We thank Volker Brinkmann for obtaining scanning electron micrograph 2007). The bacteria were picked from an overnight plate and diluted into images of mouse lungs, Kaaweh Molawi and Juana de Diego for useful com- PBS. Mice were anesthetized and infected intranasally with 50 µl of PBS ments on the manuscript, and Marc Monestier for his generous gift of the solution containing bacteria. 48 h later, mice were sacrificed, and lungs antibody against the histone-DNA complex. V. Papayannopoulos designed were removed and fixed with 2% PFA overnight. Lungs were embedded the project and all the experiments. V. Papayannopoulos performed all experi- into paraffin and sectioned into 5-µm sections. Paraffin was removed with ments with the exception of experiments performed by K.D. Metzler and where Neo Clear (Merck) and samples were rehydrated in ethanol. Samples assisted by A. Hakkim. V. Papayannopoulos wrote the manuscript. K.D. Metzler were incubated in PBS containing 0.075% Tween 20 for 30 min at 50°C performed all human neutrophil immunofluorescence stains and imaging to expose the antigens, blocked with 5% FCS/5% donkey serum, and (Fig. 4), the nuclear digestion with purified NE (Fig. 1, H and I), the blots of stained with rabbit anti-MPO (1:50; Dako) and a mouse Fabs against granule fractions (Fig. 1 D), and the NE ELISA (Fig. 4 B). A. Hakkim developed a H2A–H2B–DNA complex (a gift from Marc Monestier, Department of the mouse infection model and performed the mouse lung infections and lung Microbiology and Immunology, School of Medicine, Temple University, isolations. A. Hakkim and V. Papayannopoulos adjusted and optimized the Philadelphia, Pennsylvania; Losman et al., 1992) fused to ATT0 550. Lung sublethal dose of K. pneumoniae for knockout animals. V. Papayannopoulos inflammation and neutrophil recruitment were evaluated by hematoxylin performed lung processing, staining, and image analysis of lung sections. and eosin stains. The experiment was repeated twice with five and seven A. Zychlinsky advised and coordinated the project. A. Zychlinksy supervised mice per group, respectively. the writing of the manuscript. For scanning electron microscopy, paraffin-embedded samples This work was funded by the Max Planck Society. V. Papayannopoulos were rehydrated, postx fi ed with glutaraldehyde, contrasted using repeated was supported by an EMBO Long term fellowship. None of the authors have changes of 0.5% OsO and 0.05% tannic acid, dehydrated in a graded any commercial or other conflict of interest. ethanol series, critical-point dried, and coated with 5 nm platinum/carbon. 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