Differential Effects of Apoptotic Versus Lysed Cells on Production of : Role of Proteases1

Valerie A. Fadok,2 Donna L. Bratton, Lindsay Guthrie, and Peter M. Henson

Granulocytes undergoing apoptosis are recognized and removed by phagocytes before their lysis. The release of their formidable arsenal of proteases and other toxic intracellular contents into tissues can create significant damage, prolonging the inflammatory response. Binding and/or uptake of apoptotic cells by inhibits release of proinflammatory cytokines by mechanisms that involve anti-inflammatory mediators, including TGF-␤. To model the direct effects of necrotic cells on macrophage production, we added lysed or apoptotic neutrophils and to mouse and human macrophages in the absence of serum to avoid complement activation. The results confirmed the ability of lysed neutrophils, but not lymphocytes, to significantly stimulate production of macrophage-inflammatory protein 2 or IL-8, TNF-␣, and IL-10. Concomitantly, induction of TGF-␤1by lysed neutrophils was significantly lower than that observed for apoptotic cells. The addition of selected serine protease inhibitors and anti-human elastase Ab markedly reduced the proinflammatory effects, the lysed neutrophils then behaving as an anti- inflammatory stimulus similar to intact apoptotic cells. Separation of lysed neutrophils into membrane and soluble fractions showed that the neutrophil membranes behaved like apoptotic cells. Thus, the cytokine response seen when macrophages were exposed to lysed neutrophils was largely due to liberated proteases. Therefore, we suggest that anti-inflammatory signals can be given by PtdSer-containing cell membranes, whether from early apoptotic, late apoptotic, or lysed cells, but can be overcome by proteases liberated during lysis. Therefore, the outcome of an inflammatory reaction and the potential immunogenicity of Ags within the damaged cell will be determined by which signals predominate. The Journal of , 2001, 166: 6847–6854.

poptotic cell recognition and removal by phagocytes is human monocytes following interaction with apoptotic peripheral critical for the restoration and/or maintenance of normal lymphocytes (6). In fact, Freire-de-Lima and coworkers have A tissue structure and function. Macrophages engulf apo- shown that uptake of apoptotic cells by macrophages fuels the ptotic cells before they lyse, thus preventing release into the tissue growth of trypanosomes in Chagas’ disease, in a TGF-␤- and of potentially toxic and immunogenic intracellular substances. In PGE2-dependent manner, thereby promoting disease progression addition, the binding and/or uptake of apoptotic cells not only fails (7). PGE2 has been reported to be released following the binding to induce macrophage secretion of inflammatory mediators (1, 2), and uptake of apoptotic cells by HMDM (3). By contrast, Uch- but actually inhibits their proinflammatory cytokine production imura, Kurosaka, and coworkers have suggested that uptake of following stimulation (3, 4). Human monocyte-derived macro- apoptotic cells is proinflammatory (8, 9). As phagocytic targets, phages (HMDM)3 and J774 mouse macrophages were found to they used apoptotic CTLL2 cells cultured in the absence of IL-2 produce TGF-␤1 following exposure to apoptotic cells (3, 4), and for 12 or 28 h. Both populations contained high levels of necrotic this could be mimicked by PtdSer-containing liposomes and a cells, as indicated by trypan blue and propidium iodide positivity, mAb against a newly described PtdSer receptor (5). By incubating which might explain the proinflammatory effects they observed. macrophages so treated with anti-TGF-␤ Abs, the inflammatory It is commonly believed that necrotic cells are proinflammatory. response to LPS was restored, suggesting that TGF-␤1 induced by Certainly most pathological lesions characterized by necrosis are also apoptotic cells was a major cause of the anti-inflammatory effect characterized by inflammation. Stern and colleagues showed that fol- observed. Anti-IL-10 Abs were not effective, and, in fact, IL-10 lowing association with necrotic (postapoptotic) eosinophils, unstimu- production was down-regulated during exposure to apoptotic cells lated HMDM produced equivalent amounts of thromboxane B2 and in the absence of serum (3), although Voll et al. reported that IL-10 GM-CSF to those that had ingested opsonized eosinophils or opso- appeared to play a role in the regulation of cytokine production by nized zymosan; these results contrasted sharply with the lack of in- duction by apoptotic eosinophils (2). Therefore, it was important to

Program in Cell Biology, Department of Pediatrics, National Jewish Medical and determine whether necrotic cells had similar effects on stimulated Research Center, Denver, CO 80206 macrophage production of cytokines, and whether the type of apo- Received for publication March 24, 2000. Accepted for publication March 19, 2001. ptotic cell affected the macrophage cytokine response. Therefore, we The costs of publication of this article were defrayed in part by the payment of page compared the direct effects of binding and/or uptake of apoptotic vs charges. This article must therefore be hereby marked advertisement in accordance lysed neutrophils and lymphocytes on cytokine production by un- with 18 U.S.C. Section 1734 solely to indicate this fact. stimulated or zymosan-stimulated macrophages. The experiments 1 This work was funded by the National Institutes of Health (GM 60449, GM 48211, were performed in the absence of serum to rule out the potential and HL 60980) and by the Cancer Center at University of Colorado Cancer Center (Grant P30 CA 46934). contribution of complement fixation following exposure to intracel- 2 Address correspondence and reprint requests to Dr. Valerie A. Fadok, National lular organelles (10, 11). Jewish Medical and Research Center, D509, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: HMDM, human monocyte-derived macro- phage(s); MIP2, macrophage-inflammatory protein 2; BMDM, bone marrow- derived macrophage(s); AEBSF, 4-(2-aminoethyl) benzenesulfonylfluoride; HMDM were cultured as described previously (3, 12). In brief, mononu- TMB, tetramethylbenzidine. clear cells were isolated from the blood of normal donors, and plated at 4

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 6848 LYSED NEUTROPHILS STIMULATE MACROPHAGE TNF-␣, , AND IL-10 million/well in 24-well tissue culture plates. Lymphocytes were removed obtained from Calbiochem) were added to determine their direct effects on following incubation on tissue culture plates for1hinDMEM (Irvine macrophage cytokine production. These concentrations were determined Scientific, San Diego, CA). This method yielded 1.46 Ϯ 0.5 macrophages by lack of effects on macrophage viability and peak of cytokine production. per well. After washing, the macrophages were cultured for 7 days in Supernatants from duplicate wells were pooled, centrifuged to remove cel- X-Vivo (BioWhittaker, Walkersville, MD) containing 10% human serum lular debris, and stored at Ϫ70°C until analyzed. As a control, apoptotic or pooled from five donors; the medium was changed once after the first 3 lysed cells were cultured for the same time periods in X-Vivo medium days of culture. Bone marrow cells were obtained from C3H/HeJ mice and without macrophages to determine whether they produced cytokines. Nei- cultured as described (13) in DMEM containing 10% heat-killed FCS and ther apoptotic neutrophils nor apoptotic Jurkat T cells produced significant 10% L cell-conditioned medium as a source of M-CSF. Bone marrow- levels of any of the cytokines tested. derived macrophages (BMDM) were used 5–7 days after isolation and culture. Analysis of cytokines Induction of apoptosis Cytokines assessed included TGF-␤1, TNF-␣, IL-10, and either MIP2 for mouse or IL-8 for human cells. Matched Ab pairs for the ELISAs were Human neutrophils and the human line Jurkat were used as apoptotic purchased from R&D Systems (Minneapolis, MN). For TGF-␤1, superna- targets. Neutrophils were induced to undergo apoptosis by exposure to UV tants were activated with HCl before analysis. Cytokines levels were de- irradiation for 5 min followed by culture for 3 h, as described previously tected following incubation with the biotinylated secondary Abs by incu- (3). Jurkat T cells were irradiated for 10 min and cultured for 3 h. Apo- bation with avidin-conjugated HRP, then tetramethylbenzidine and H O ptosis was assessed by morphological examination of cytocentrifuged cells 2 2 as substrate. The plates were read in a Bio-Tek EL309 ELISA reader and by flow cytometry, using FITC-conjugated annexin V. For neutrophils, (Biotek Instruments, Winooska, VT), and results were analyzed using the the average percent apoptosis was 74.3 Ϯ 5.4 (SEM); for Jurkats, 75.3 Ϯ log/log curve fit option from Delta Soft 3 (BioMetallics, Princeton, NJ). 4.2 (SEM); trypan blue positivity was Ͻ4% for either population; pro- pidium iodide positivity was Ͻ6%. Results of annexin V positivity were not significantly different from apoptosis as assessed by morphology (data Measurement of elastase not shown). For late apoptotic cells, human neutrophils were irradiated as Freshly isolated neutrophils were suspended at 10 million per ml in X- above, but cultured for 24 h; this yielded populations of cells that were 95% Vivo, then immediately centrifuged, separated into pellet and supernatant, annexin V positive, 45% trypan blue positive, and 65% propidium iodide and frozen at Ϫ70°C. Early and late apoptotic neutrophils were prepared as positive. described above except that they were cultured at 10 million per ml in Lysis X-Vivo medium, then centrifuged. The samples were frozen as pellets, and the supernatant was derived from culture. Lysed neutrophils were prepared Human neutrophils and Jurkat T cells were rapidly frozen as cell pellets on by suspending neutrophils at 10 million per ml in X-Vivo and freezing at dry ice, then stored at Ϫ70°C. They were thawed at room temperature Ϫ70°C. On the day of the analysis, the pellets and supernatants were without washing, and suspended in X-Vivo without serum before use. The thawed. The necrotic cell preparations were thawed, then centrifuged to average percent lysis, assessed by trypan blue positivity, for neutrophils separate the material into pellet and supernatant. All cell pellets were sol- was 92.6 Ϯ 2.4% (SEM) and for Jurkat T cells 95.6 Ϯ 1.4% (SEM). ubilized in lysis buffer containing 50 mM Tris (pH 7.4), 1% Triton X-100, Staining with FITC-conjugated annexin V and propidium iodide revealed 0.25% deoxycholate, 150 mM NaCl, and 1 mM EGTA. For elastase anal- that 100% of each population were positive for both. One cycle of freeze/ ysis, 20 ␮l of solubilized pellet or supernatant was added in triplicate to thaw was used to give intact but trypan blue-positive bodies. For prepara- 96-well ELISA plates, followed by 55 ␮l elastase reaction buffer (0.1 M tion of membrane and cytosol fractions, the cells were subjected to five HEPES, 0.5 M NaCl, 10% DMSO pH 7.5). Then, 150 ␮l of 0.2 M Elastase freeze-thaw cycles, homogenized in X-Vivo medium, and centrifuged at substrate I (methoxysuccinyl-ala-ala-pro-val-p-nitroanilide; Calbiochem) 100,000 ϫ g. The membrane pellets or the membrane-free supernatant in elastase reaction buffer was added, and the samples were incubated at were added to macrophages as cell equivalents to compare them to the 37°C for 1 h. Elastase levels were determined by measuring absorbance at intact but leaky lysed cells. Membrane and cytosolic fractions were also 410 nm, using serial dilutions of porcine elastase (Calbiochem) as stan- made from apoptotic and viable granulocytes by homogenizing them in dards. Specificity was determined by incubating the samples and standards X-Vivo, and by ultracentrifugation. in the presence or absence of Elastase Inhibitor III (methoxysuccinyl-ala- ala-pro-val-chloromethylketone (Calbiochem). Macrophage cytokine production Macrophages were cultured in 24-well plates and each condition was run Statistical analysis in duplicate. Five million apoptotic or necrotic (lysed) cells were added per For Figs. 1–3 and 6–7, data were evaluated by ANOVA and the Tukey well of macrophages for 1.5 h, then washed out. Fresh X-Vivo medium Kramer method. For Figs. 4 and 5, ANOVA and Dunnett’s method were without serum was added, and supernatants were collected 24 h later. Rep- used, designating macrophages treated with lysed neutrophils as the control licate wells were used to assess uptake of the phagocytic targets. For neu- for Fig. 4, and macrophages without target cells added as the control for trophils, the monolayers were stained for myeloperoxidase as previously Fig. 5. Significance was set at p Ͻ 0.05. described and shown; only those macrophages that had engulfed neutro- phils were scored as positive for staining (3). Uptake of apoptotic neutro- phils was associated with intracellular staining of discrete intact bodies, Results whereas uptake of necrotic (lysed) cells was associated with paler staining We first studied the effects of the binding and/or uptake of apo- intracellular bodies and with diffuse staining of the macrophages. Uptake ptotic vs lysed human neutrophils on HMDM. The percent mac- of lymphocytes was assessed by staining the macrophage monolayers with Ϯ a modified Wright’s Giemsa stain. For some experiments, the lysed neu- rophages positive for uptake of apoptotic neutrophils was 49 trophils were incubated with the macrophages in the presence or absence of 8.5% and for uptake of lysed neutrophils was 57.5 Ϯ 10.5%. There 100 ␮M PMSF (Sigma, St. Louis, MO), 500 ␮M 4-(2-aminoethyl) ben- appeared to be no measurable cytokine production by the apoptotic zenesulfonylfluoride (AEBSF) (Calbiochem, La Jolla, CA), 5 ␮g/ml apro- or lysed cells when incubated without macrophages (data not tinin, 0.25 ␮g/ml leupeptin, 1 ␮g/ml E-64, 40 ␮g/ml bestatin, 0.7 ␮g/ml pepstatin, and 15 ␮g/ml calpain I inhibitor (Boehringer Mannheim, Indi- shown); thus the cytokine levels shown here appear to reflect mac- anapolis, IN), anti-human neutrophil elastase, anti-human cathepsin G, and rophage production. As shown in Fig. 1, exposure to apoptotic isotype controls for 24 h before collection of supernatants for evaluation of human neutrophils in the absence of serum had the expected ef- cytokine concentrations. Preliminary dose responses were performed to fects: increased production of TGF-␤1, with no stimulation of IL- insure a lack of toxicity to the macrophage monolayers. Viability at the ␣ Յ 10, TNF- , or a chemokine (IL-8), and suppression of IL-8, concentrations used was verified by trypan blue concentration ( 4% pos- ␣ itive cells) and by preservation of the stimulatory response to zymosan TNF- , and IL-10 when macrophages were costimulated with zy- (assessed by measurement of macrophage-inflammatory protein 2 (MIP2), mosan. In contrast, exposure to lysed neutrophils induced signif- TNF-␣, IL-10, and TGF-␤). Anti-human elastase and anti-human cathepsin icantly lower levels of TGF-␤1 than apoptotic cells, and stimulated G Abs were purified sheep IgG purchased from Cortex Biochem (San production of IL-10, TNF-␣, and IL-8 ( p Ͻ 0.05). Although lysed Leandro, CA), and were used at 50 ␮g/ml. Zymosan (Sigma) was used as a stimulus for cytokine production; HMDM were stimulated with 25 ␮g/ml neutrophils stimulated proinflammatory cytokine production, the and BMDM with 75 ␮g/ml. In some experiments, 0.01 U/ml purified hu- levels were lower than those induced by zymosan. When the mac- man neutrophil elastase and 5 U/ml purified human cathepsin G (both rophages were stimulated with zymosan to produce inflammatory The Journal of Immunology 6849

FIGURE 1. Lysed neutrophils stimulate chemokine, TNF-␣, and IL-10 production by human macrophages. Apoptotic or lysed human neutrophils were coincubated with HMDM in the presence or absence of zymosan (25 ␮g/ml) for 24 h. Supernatants were collected and cytokines evaluated by -Statistically significant difference from mac ,ء .(ELISA. n ϭ 15 (Ϯ SEM rophages without addition of cells or macrophages incubated with apopto- statistically significant difference from macrophages ,ءء ;tic neutrophils incubated with apoptotic cells (p Ͻ 0.05). In all cases, results from mac- rophages stimulated with apoptotic cells are significantly different from macrophages without neutrophils added. cytokines, both apoptotic and lysed neutrophils were able to sig- nificantly suppress TNF-␣ and IL-10 production, although apopto- tic cells were significantly more effective ( p Ͻ 0.05). Similar ef- fects were seen when mouse BMDM were used (Fig. 2). Percent uptake of apoptotic cells by BMDM was 45 Ϯ 7.8% and of lysed cells 52.9 Ϯ 11.2%. If the serine protease inhibitor PMSF was added, the proinflammatory response of the unstimulated macro- phage to lysed neutrophils was significantly inhibited (Fig. 2A), as was the production of IL-10 ( p Ͻ 0.05). When PMSF was added to the lysed neutrophils coincubated with zymosan-stimulated macrophages, the lysed neutrophils behaved like apoptotic cells (Fig. 2B). Treatment with PMSF did not alter the effects of apo- ptotic cells or zymosan on macrophage cytokine production. Lysed FIGURE 2. Lysed neutrophils have similar effects on mouse macro- phages as on human macrophages, but lysed Jurkat T cells behave like Jurkat T cells had the same effects on macrophage cytokine pro- apoptotic cells. Addition of PMSF abrogates the effects of lysed neutro- duction as apoptotic cells (Fig. 2). Both populations of the lym- phils. A, Apoptotic or lysed human neutrophils or Jurkat T cells were fed ␣ phocytes inhibited stimulated production of MIP2, TNF- , and to mouse BMDM for 1.5 h then washed out. Medium was added, and IL-10, while stimulating TGF-␤ production. These data suggested supernatants were collected for evaluation of cytokine production by ,ءء ;(Significantly different from all other samples (p Ͻ 0.05 ,ء .that serine proteases liberated from the granulocyte were critical to ELISA the induction of a proinflammatory response by lysed neutrophils. significantly different from macrophages incubated with apoptotic cells and Lysed neutrophils stimulated low levels of and from macrophages incubated with lysed cells in the presence of PMSF. TNF-␣, but also exhibited some inhibitory effects on zymosan in- Macrophages incubated with either apoptotic or lysed Jurkat T cells ␤ duction of these cytokines. The effects of the lysed neutrophils showed statistically significant increased TGF- production. B, Apoptotic were significantly different from those of the apoptotic cells ( p Ͻ or lysed human neutrophils or Jurkat T cells were added to BMDM as described in A; however, after washing, fresh medium containing 75 ␮g/ml 0.05). This information, with the observation that PMSF treatment zymosan was added, and supernatants were collected 24 h later for cyto- Statistically different ,ء .(inhibited the proinflammatory effects of the lysed cells, suggested kine measurement by ELISA. n ϭ 18 (ϮSEM the hypothesis that the proinflammatory effects of serine proteases from zymosan-treated macrophages and from zymosan-treated macro- could be opposed by the anti-inflammatory effects of the PtdSer- phages preincubated with apoptotic cells. Macrophages incubated with ei- containing lysed cell membranes. To determine whether this was ther apoptotic or lysed Jurkat cells showed statistically significant in- true, lysed neutrophils were homogenized and centrifuged to ob- creased TGF-␤ production. 6850 LYSED NEUTROPHILS STIMULATE MACROPHAGE TNF-␣, CHEMOKINE, AND IL-10 tain a crude membrane fraction and a cytosolic fraction; each of cells are similar to early apoptotic cells in that they induce TGF-␤ these were added to mouse macrophages in the presence or ab- secretion and do not significantly induce MIP2, TNF-␣, and IL-10. sence of zymosan to determine their effects on cytokine produc- It was important to determine whether neutrophil elastase could tion. We also used membranes and cytosol from lysed Jurkat cells. induce the same pattern of cytokine production as lysed neutro- As shown in Fig. 3, the cytosolic fraction from neutrophils con- phils. Therefore, HMDM and mouse BMDM were exposed to pu- tained the activity that stimulated MIP2, TNF-␣, and IL-10. The rified human neutrophil elastase and human cathepsin G. As shown membranes were anti-inflammatory as they induced the secretion in Fig. 6, neutrophil elastase at 0.01 U/ml was a potent stimulator of TGF-␤, and inhibited zymosan-induced MIP2, TNF-␣, and IL- of mouse macrophage TNF-␣, MIP2, and IL-10 production; ca- 10. We also used membrane and cytosolic fractions from viable thepsin G virtually no activity, requiring 5 U/ml to see a small and apoptotic neutrophils; as for neutrophils lysed by freeze-thaw amount of IL-10 production only. Although not shown, the effects cycles, the cytosolic fraction stimulated MIP2, TNF-␣, and IL-10, on HMDMs were identical. whereas the membranes stimulated TGF-␤ (data not shown). Last, elastase concentrations in the pellet and medium from In the next set of experiments, we used a variety of protease early and late apoptotic neutrophils were compared with those inhibitors, as well as Abs against human neutrophil elastase and from freshly isolated and lysed neutrophils. Fig. 7 shows that early human cathepsin G, to determine their effects on lysed neutrophil- and late apoptotic neutrophils released virtually no elastase into induced cytokine production. In Fig. 4, we show that the serine their medium. Lysed neutrophils, as expected, released a large protease inhibitors PMSF and AEBSF were strong inhibitors of the amount of elastase, and the proportion remaining in the cell pellet proinflammatory response. The serine protease inhibitors aprotinin was significantly lower than that in the pellets from the apoptotic and leupeptin were weak inhibitors. Inhibitors of cysteine pro- cells ( p Ͻ 0.05). Lysed Jurkat T cells, as expected, were negative teases (E-64), metalloproteases (bestatin), aspartic proteases (pep- for elastase (data not shown). statin), and calpain had no effect. Anti-human elastase Ab (50 ␮g/ ml) was a strong inhibitor, but anti-human cathepsin G (50 ␮g/ml) Discussion: was only a weak inhibitor; increasing the concentration of the lat- The purpose of the experiments presented here was to determine ter did not improve the inhibition, and the isotype control (sheep whether lysed cells, used as a model for necrotic cells, could di- IgG) had no effect. Neither of these Abs at the concentration used rectly stimulate macrophages to release proinflammatory media- stimulated cytokine production from control HMDM (data not tors in the absence of serum. We found that only lysed neutrophils shown). These results support the interpretation that neutrophil could do so, and that the activity could be attributed to released elastase plays a major role in the stimulation of macrophage cy- neutrophil elastase. The binding and/or uptake of apoptotic neu- tokine production. trophils and lymphocytes by either mouse or human primary mac- Next, the production of cytokines from mouse BMDM exposed rophages suppressed zymosan-induced production of proinflam- to early apoptotic neutrophils (cultured for 3 h after UV irradiation, matory cytokines as expected, exemplified by decreased levels of trypan blue positivity Յ4%), late apoptotic neutrophils (cultured TNF-␣, IL-10, and either MIP-2 or IL-8, as we have shown pre- for 24 h after UV irradiation, trypan blue positivity 45%), and viously (3, 4). This down-regulation results in large part from se- lysed neutrophils was assessed. As shown in Fig. 5, late apoptotic cretion of TGF-␤1, at least part of which is bioactive (3, 4), and the

FIGURE 3. Supernatants from lysed neutrophils stimulate MIP2, TNF-␣, and IL-10 from mouse BMDM, whereas their membranes inhibit production. Lysed cells were homogenized and centrifuged to separate the membranes from supernatant. Each fraction was subsequently added to mouse BMDM in the presence or absence of 75 ␮g/ml zymosan. The conditioned medium from the macrophages was collected 24 h later, and cytokines were measured by ELISA. n ϭ 18 (ϮSEM). Suppression of cytokine production was statistically significant for lysed neutrophil membranes and lysed Jurkat T cell membranes (p Ͻ 0.05). Only lysed neutrophils and the supernatant fraction from these cells were able to significantly stimulate production of MIP2, TNF-␣, and IL-10 from unstimulated (open bar) macrophages, and unseparated lysed neutrophils significantly inhibited zymosan-stimulated MIP2, TNF-␣, and IL-10 (p Ͻ 0.05). Apoptotic cells and membranes from either lysed neutrophils or lysed Jurkat T cells significantly stimulated TGF-␤ whether macrophages were treated with zymosan or not (p Ͻ 0.05). The Journal of Immunology 6851

FIGURE 4. Serine protease inhibitors and anti-human elastase Ab inhibit the ability of lysed neutrophils to stimulate MIP2, TNF-␣, and IL-10 from mouse and human macrophages. Statistically significant differences (p Ͻ 0.05) are illustrated by solid black bars. n ϭ 10 (ϮSEM). Not shown are the isotype controls for the anti-protease Abs, which had no inhibitory effect. None of the inhibitors shown inhibited zymosan stimulation of MIP2, TNF-␣, or IL-10, suggesting that the protease inhibitors and Abs were not toxic. effects of apoptotic cells can be mimicked by treatment of macro- not proinflammatory (14). The response to lysed cells in the un- phages with PtdSer-containing liposomes or a mAb against a Ptd- stimulated macrophages was expected (2) in that they induced Ser receptor (5). Thus, uptake of apoptotic cells before their lysis TNF-␣ and chemokine production; however, the levels were low not only prevents the release of potentially toxic or immunogenic relative to those seen with a strong proinflammatory stimulus such intracellular contents but also induces an anti-inflammatory phe- as zymosan or LPS (the latter not shown). Lysed cells also con- notype in the macrophage. We also determined that late apoptotic sistently induced IL-10 production, in contrast to apoptotic cells. neutrophils, even though becoming permeable to propidium iodide When macrophages were stimulated with zymosan, they pro- and trypan blue, behave more like early apoptotic cells (Fig. 5) duced robust levels of MIP-2 or IL-8, TNF-␣, and IL-10. Inter- because they induced production of TGF-␤ but did not signifi- estingly, lysed neutrophils were found not to have additive effects cantly stimulate TNF-␣, IL-10, or the chemokine MIP2. Further- in the absence of serum; rather, they partially (but significantly, more, late apoptotic cells released very little elastase into their p Ͻ 0.05) inhibited zymosan-stimulated production of TNF-␣ and culture medium (Fig. 7). These results are in keeping with the IL-10, although having no effect on the chemokines MIP-2 or IL-8, earlier observations of Ren and Savill, who observed that apoptotic both of which are CXC chemokines chemotactic for neutrophils. neutrophils beginning to become permeable to trypan blue were Lysed neutrophils also induced significantly less TGF-␤1 than did

FIGURE 5. Late apoptotic neutrophils and early apoptotic neutrophils stimulate TGF-␤ secretion, and fail to stimulate MIP2, TNF-␣, or IL-10. Early and late apoptotic neutrophils, and lysed neutrophils, were prepared as described in Materials and Meth- ods, and added to mouse BMDM. Supernatants were collected and evaluated for cytokine production by significantly different ,ء ;(ELISA. n ϭ 5(ϮSEM (p Յ 0.05) from macrophages incubated in the ab- sence of added cells. 6852 LYSED NEUTROPHILS STIMULATE MACROPHAGE TNF-␣, CHEMOKINE, AND IL-10

in the soluble fraction. Furthermore, purified neutrophil elastase directly stimulated IL-10, TNF-␣, and chemokine production, and only lysed neutrophils released elastase into the medium. How neutrophil elastase directly promotes macrophage proin- flammatory cytokine production is not yet known. It may bind directly to protease-activated receptors. For example, Ishihara and coworkers recently demonstrated specific binding of neutrophil elastase to macrophages; binding was accompanied by enhanced production of chemokines, which was inhibited by PMSF (15). In vivo, the proinflammatory actions of the proteases likely result not only from their actions on macrophages but on other cell types and on extracellular matrix. Alternatively, or in addition, the proteases may cleave a critical membrane signal or signals that mediate the FIGURE 6. Purified neutrophil elastase stimulates the release of down-regulation of proinflammatory cytokines associated with the TNF-␣, IL-8, and IL-10 from HMDM. Elastase was used at 0.01 U/ml; uptake of apoptotic cells. In support of this notion is our observa- cathepsin G was used at 5 U/ml. Proteases and zymosan (positive control) tion that the PtdSer receptor can be cleaved off the cell surface by were added to macrophages in X-Vivo medium without serum, and super- trypsin (5); in addition, we have preliminary data to suggest that natants were collected for cytokine measurements 24 h later. n ϭ 5 neutrophil elastase may cleave it as well (W. Vandivier, V.A.F., Ϯ ( SEM). and P.M.H., unpublished data). In fact, the predicted extracellular domain of the PtdSerR has several potential cleavage sites for neutrophil elastase, given that the preference for P1 is AϾVϾTϾI early or late apoptotic cells. Thus, based on these results alone, the (16). This receptor, when stimulated by apoptotic cells, phosphati- presence of lysed (fully necrotic) cells in vivo would likely cause dylserine-containing membranes, or a stimulatory mAb, induces continuing influx of neutrophils, particularly following macro- ␤ phage activation. The net effect would be prolongation of an in- the release of TGF- and the down-regulation of proinflammatory flammatory response. cytokines (5). Our studies suggest that the cellular carcass of a cell As expected, lysed neutrophils appeared to be more potent in- progressing to the late stages of apoptosis can retain anti- ducers of macrophage cytokine production in the absence of serum inflammatory activity. With regard to the observations of Ren and than were lysed Jurkat T cells, which behaved, for the most part, Savill that neutrophils that have progressed from early to late ap- like apoptotic cells. Given that granulocytes produce high levels of optosis (i.e., becoming permeable to trypan blue) fail to stimulate several different types of proteases, it seemed reasonable to sug- macrophage cytokine production (14), we suggest that the anti- gest that the differing effects of lysed neutrophils compared with inflammatory signal of the exposed phosphatidylserine on the late lysed lymphocytes were related to protease release. The serine apoptotic cell predominates, as the cells have not released pro- protease inhibitors PMSF and AEBSF significantly decreased the teases to cleave the PtdSer receptor. In any event, we suggest that proinflammatory effects of lysed neutrophils on macrophage cyto- activated, necrotic, or lysed cells can, by releasing proteases, over- kine production, while leading to an up-regulation of TGF-␤1, whelm the anti-inflammatory effects of macrophage PtdSerR in- permitting them to have anti-inflammatory effects similar to those teractions with PtdSer-expressing apoptotic cells or membranous induced by apoptotic cells. The strong inhibitory effect of the anti- debris, and that prolonged inflammation would be the predicted elastase Ab supports the notion that neutrophil elastase is a major end stage of cell death in the absence of clearance by phagocytes. contributor to the induction of proinflammatory cytokines and IL- In vivo, endogenous anti-proteases also contribute to the complex- 10. This interpretation is supported by the observations that if ity of the final response. lysed neutrophils were homogenized and separated into membrane One of the interesting observations from this study was that and soluble fractions, the effects of the membranes were indistin- lysed cells induced release of IL-10, which could be attributed to guishable from apoptotic cells, which express phosphatidylserine the effect of serine proteases, particularly neutrophil elastase. Oth- on their outer leaflets, and that the proinflammatory signal resided ers have observed that IL-10 is up-regulated early and concomi- tantly with TNF-␣ and chemokines in a variety of inflammatory conditions (17–21). Although IL-10 is not believed to affect the constitutive rate of neutrophil apoptosis, it enhances apoptosis of neutrophils at inflammatory sites, promotes the survival of mac- rophages, and enhances macrophage removal of apoptotic neutro- phils (22–26). Thus, the protease-mediated release of IL-10 in in- flammatory sites may represent a protective mechanism designed to promote resolution of inflammation by enhancing neutrophil apoptosis. Certainly, in its absence, inflammation is more severe in a number of models (27–29). Both TGF-␤ (resulting from stimulation with early or late apo- ptotic cells) and IL-10 (resulting from stimulation with lysed cells) FIGURE 7. Early and late apoptotic neutrophils fail to release elastase have anti-inflammatory activities, including the down-regulation into the medium. Elastase assays were performed as described in Materials of inflammatory mediator production by macrophages (30–35). and Methods. n ϭ 3(ϮSEM). Elastase levels in the supernatant from lysed However, in our experiments, down-regulation of chemokines and cells were significantly elevated and elastase levels in the pellet from lysed ␣ ␤ cells were significantly reduced compared with those from freshly isolated TNF- was associated with TGF- production; the pattern of or apoptotic neutrophils (p Ͻ 0.05). Although not shown, all the elastase IL-10 secretion in the absence of serum was similar to that for activity represented in this figure was completely inhibited by elastase proinflammatory cytokines. Both TGF-␤ and IL-10 can be immu- Inhibitor III (see Materials and Methods). nosuppressive by exerting effects on both lymphocytes and APCs The Journal of Immunology 6853

(36–40). However, TGF-␤ has been shown to inhibit full matu- In conclusion, it seems reasonable to hypothesize that apoptotic ration of dendritic cells, even in the presence of inflammatory stim- neutrophils are anti-inflammatory for two reasons: first, they fail to uli such as TNF-␣, effectively preventing the development of po- release their serine proteases even when they begin to become tentially harmful immune responses from a resolving inflammatory leaky (as assessed by uptake of propidium iodide and trypan blue), site (41). TGF-␤ also mediates bystander suppression associated and because the phosphatidylserine exposed on their surfaces is with physiological self-tolerance in vivo, thereby preventing the significantly anti-inflammatory. One can predict that the outcome activation of autoreactive lymphocytes (42). These activities may of inflammation will be determined by the balance between proin- be critical when apoptotic cells are removed during physiological flammatory signals generated by the release of granulocyte pro- cell death or with apoptotic cell uptake associated with resolution teases and anti-inflammatory signals generated by exposure to of inflammation. phosphatidylserine-containing membranes associated with apopto- Exposure to necrotic vs apoptotic cells also has differential ef- tic cells or membranous cellular debris. The balance between pro- fects on maturation of dendritic cells for Ag presentation to lym- teases and anti-proteases will also contribute significantly to phocytes. Sauter et al. recently showed that exposure of dendritic whether an inflammatory lesion resolves or not. Importantly, the cells to necrotic tumor cells or to supernatants derived from these anti-inflammatory potential of apoptotic neutrophils appears to be cells enabled them to mature into fully functional APCs (43). They maintained even through the late stages, providing multiple op- also found differences in cell type in that only necrotic tumor cells, portunities for resolution of inflammation. but not necrotic primary cells, could induce this effect. They could not define the active factor; however, released intracellular pro- Note added in proof. Cocco and Ucker very recently showed that teases may be worth evaluating in this system as well. the inhibitory effects of apoptotic cells on macrophage cytokine In summary, we have learned that exposure to apoptotic cells, production were dominant to the proinflammatory effects of ne- whether neutrophils or lymphocytes, inhibited macrophage proin- crotic cells (45). Jaffray and coworkers recently showed that pan- flammatory cytokine by a mechanism involving TGF-␤ (data pre- creatic elastase induces macrophages to produce TNF-␣ in a NF- sented herein and in Refs. 4, 5). In contrast, lysed cells, particularly ␬B-dependent manner, supporting the hypothesis of surface granulocytes, stimulated production of proinflammatory cytokines receptors for elastase on macrophages (46). and chemokines, as well as IL-10. Necrotic cells partially down- regulated zymosan-induced production of TNF-␣ and IL-10, but had no effect on chemokines. In the presence of a serine protease Acknowledgments inhibitor, necrotic cells behaved like apoptotic cells, inhibiting Particular thanks are due to Jay Westcott for his invaluable help with and proinflammatory cytokines (including chemokines), and increas- provision of the cytokine ELISA, Azzedine Dakhama for his assistance ␤ ing release of TGF- 1. 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