of December 13, 2018.This information is current as

BκMAPK, and NF-Involvement of Phosphoinositide 3-Kinase, Heme Inhibits Human Neutrophil Apoptosis:

Freitas, Christina Barja-Fidalgo and Aurélio V. Graça-SouzaMaria Augusta Arruda, Adriano G. Rossi, Marta S. de

http://www.jimmunol.org/content/173/3/2023doi: 10.4049/jimmunol.173.3.2023

2004; 173:2023-2030; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Heme Inhibits Human Neutrophil Apoptosis: Involvement ofPhosphoinositide 3-Kinase, MAPK, and NF-�B

Maria Augusta Arruda,* Adriano G. Rossi, ‡ Marta S. de Freitas,* Christina Barja-Fidalgo,1*and Aurelio V. Graca-Souza†

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemiareperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflamma-tion. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen speciesgeneration, and IL-8 expression. In this study, we show that heme (1–50�M) delays human neutrophil spontaneous apoptosis invitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo proteinsynthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting theinvolvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in themodulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Fu-thermore, inhibition of NF- �B translocation reversed heme antiapoptotic effect. NF-�B (p65 subunit) nuclear translocation andI�B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulatingsignaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play animportant role in the development of chronic inflammation by interfering with the longevity of neutrophils. The Journal ofImmunology, 2004, 173: 2023–2030.

S evere hemolysis occurring during pathological states suchas sickle cell disease, ischemia reperfusion, and malariaresults in high levels of free heme (up to 20 �M). Under

these conditions, the physiological mechanisms of removing freeheme from the circulation, especially its binding to hemopexin,collapse, allowing nonspecific heme uptake and heme-catalyzedoxidation reactions (1, 2). We have recently reported that heme isa proinflammatory molecule able to induce neutrophil migration invivo and in vitro (3). Interaction of free heme with human neu-trophils leads to actin cytoskeleton reorganization and reactive oxy-gen species (ROS)2 generation through the induction of proteinkinase C (PKC) activity, and also increases IL-8 expression (3).These findings attested to a prominent role for free heme in thedevelopment of inflammation associated with hemolytic diseases.In agreement, it has been shown that increased levels of heme inplasma are accompanied by a rise in cytokine and chemokine con-centrations, as well as enhanced leukocyte function (4–8), eventsoften associated with an inflammatory response that usually de-velops into chronic inflammation (9, 10).

Neutrophil apoptosis and subsequent clearance by phagocytesare critical to the resolution of acute inflammation (11, 12). Theseterminally differentiated cells constitute the first line of host de-fense against invading microorganism, being promptly recruited toinflamed loci in response to infection or tissue injury. Once acti-vated, neutrophils are able to phagocytose, to release granular lyticenzymes and antimicrobial polypeptides into the phagolysosome,and to generate large amounts of ROS as well as reactive nitrogenspecies (13). Under normal conditions, neutrophils have a veryshort t1/2, being committed to programmed cell death (apoptosis).During this process, cell membrane integrity is maintained, avoid-ing the release of proinflammatory and potentially cytotoxic agentsand the subsequent amplification of the inflammatory response.Apoptotic neutrophils also express surface markers that allow theirrecognition and nonphlogistic ingestion by professional phago-cytes such as macrophages, or potential phagocytes such as fibro-blasts and mesangial cells (14).

Culturing peripheral blood neutrophils in vitro reproducibly re-sults in spontaneous apoptosis of �50% of the cells within 24 h.These apoptotic cells exhibit the classical features associated withthis phenomenon, such as cytoplasmic condensation, phosphati-dylserine exposure on the outer leaflet of the plasma membrane,and internucleosomal DNA cleavage, followed by chromatin con-densation. Ultimately, it is the activation of caspases, a family ofredox-sensitive cysteine proteases, that coordinates the structuraldismantling of the cell (15).

Agents that promote neutrophil responsiveness, such as IL-8,GM-CSF, LPS, and leukotriene B4, also delay human neutrophilapoptosis (16–19). These stimuli promote neutrophil survival bymodulating intracellular signaling pathways, including the MAPK,especially ERK and PI3K/Akt pathways (20, 21). Evidence hasshown that activation of NF-�B pathway has a protective effect inseveral cell types, regulating the expression of antiapoptotic genes(22). In human neutrophils, NF-�B activation seems to regulatespontaneous apoptosis and the antiapoptotic effect of TNF-�, a

*Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado doRio de Janeiro, and †Departamento de Bioquímica Médica, Instituto de Ciencias Bi-omédicas/Centro de Ciencias da Saúde, Universidade Federal do Rio de Janeiro, Riode Janeiro, Brazil; and ‡Centre for Inflammation Research, Respiratory MedicineUnit, University of Edinburgh Medical School, Edinburgh, United Kingdom.

Received for publication January 20, 2004. Accepted for publication May 19, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Christina Barja-Fidalgo, Depar-tamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio deJaneiro, Av 28 de setembro 87-Vila Izabel, Rio de Janeiro, RJ, 20551-030 Brazil.E-mail address: barja-fidalgo@uerj.br2 Abbreviations used in this paper: ROS, reactive oxygen species; BIM, bis-indoyl-maleimide IV; CO, carbon monoxide; DPI, diphenyleneiodonium; HO, heme oxy-genase; PDTC, pyrrolidine dithiocarbamate; PKC, protein kinase C; SnPPIX, tin pro-toporphyrin IX.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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cytokine that exerts dual effects upon these cells. The modulationof all those pathways most likely regulates the balance betweenpro- and antiapoptotic proteins to influence neutrophil survival,especially the members of Bcl-2 family, which comprises bothpro- and antiapoptotic members (23).

In the present study, we demonstrate that heme is able to pro-long neutrophil life span by inhibiting apoptosis by a mechanismdependent on heme oxygenase (HO) activity and ROS generation.This effect depends on de novo protein synthesis and seems to bemediated by MAPK and PI3K/Akt pathways and involves NF-�Bactivation, indicating that heme may control neutrophil apoptosisthrough activation of these survival pathways. Our data support arole for heme as a proinflammatory mediator during hemolyticstates, suggesting that this molecule is important in the develop-ment of chronic inflammation associated with hemolysis andhemoglobinemia.

Materials and MethodsReagents

Cycloheximide, diphenyleneiodonium (DPI), pyrrolidine dithiocarbamate(PDTC), and apocynin (acetovanillone) were purchased from Sigma-Aldrich (St. Louis, MO). LY294002, bis-indoylmaleimide IV (BIM), andPD98059 were from Calbiochem (San Diego, CA). Biliverdin and bilirubinwere from Valeant Pharmaceuticals (Costa Mesa, CA). Tin protoporfirinIX (SnPPIX) was from Porphyrin Products (Logan, UT). Human rIL-8 wasa gift from F. Cunha (Faculdade de Medicina de Ribeirao Preto-Univer-sidade de Sao Paulo, Sao Paulo, Brazil), and anti-IL-8 mAb was donatedby P. Bozza (Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil).

Heme

Hemin (cell culture grade; Sigma-Aldrich) stock solutions were made inDMSO (culture grade; Sigma-Aldrich) and diluted in sterile PBS imme-diately before use. The final concentration of DMSO was kept lower than0.01% for all assays.

Neutrophil isolation and culture

Neutrophils were isolated from EDTA (0.5%)-treated peripheral venousblood of healthy human volunteers by a method of dextran sedimentationand density gradient centrifugation, as previously described (24). Residualerythrocytes were removed by hypotonic lysis. Isolated neutrophils (5 �106/ml) were incubated in DMEM supplemented with 10% heat-inacti-vated FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in ahumidified atmosphere containing 5% CO2 for 20 h, unless otherwise in-dicated. Under all experimental conditions, �99% of cells were viable, asassessed by trypan blue dye exclusion.

Assessment of neutrophil apoptosis

Morphology. Cells were cytocentrifuged, stained with Diff-Quik, andcounted under light microscopy (�1000) to determine the proportion ofcells showing characteristic apoptotic morphology. At least 400 cells werecounted per slide. The results were expressed as mean � SD.

Annexin V-binding assay

To measure phosphatidylserine exposure on apoptotic cell surface, a flowcytometric assay using annexin V binding (annexin V-FLUOS; Roche Mo-lecular Biochemicals, Mannheim, Germany) was performed. A workingsolution of annexin V-FLUOS was made from stock annexin V-FLUOS(0.1 �g/ml) diluted 1/3000 in HBSS supplemented with 2.5 mM CaCl2.Neutrophils (20 �l of 5 � 106/ml) were added to 200 �l of a workingsolution of annexin V before being assessed on a FACSCalibur flow cy-tometer (BD Biosciences, San Jose, CA) and analyzed on associatedCellQuest (BD Biosciences) software. All experiments were performed atleast three times.

DNA electrophoresis

DNA fragmentation was analyzed, as previously described (25). Briefly,neutrophils (5 � 106 cells/ml) were taken after 20 h and lysed with 500 �lof lysing buffer (0.2% Triton X-100; 100 and 1 mM EDTA, pH 7.4). Celllysates were then centrifuged at 13,000 � g, and the supernatants (con-taining fragmented DNA) were separated from the pellet. The supernatantsobtained were treated with 50 �l of 5 M NaCl and 500 �l of isopropanol

and left for 12 h at �70°C. DNA pellets were washed with 70% ethanol,air dried, and resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH7.4). Fragmented DNA was separated on a 1% agarose gel electrophoresiscontaining 1 �g/ml ethidium bromide. The products of DNA fragmentationwere visualized and documented under UV light.

Subcellular localization

Cells were cytocentrifuged and fixed with paraformaldehyde (4%), andthen permeabilized with 0.5% Triton X-100 in PBS for 20 min. The slideswere incubated with rabbit polyclonal anti-ERK-2 Ab (Santa Cruz Bio-technology, Santa Cruz, CA; 1/200) at 4°C overnight, incubated at roomtemperature for 1 h with biotin-conjugated goat anti-rabbit IgG (Santa CruzBiotechnology; 1/1000), and finally incubated with FITC-conjugatedstreptavidin for 1 h at room temperature. Slides were then mounted usingN-propylgalate solution before examination under an Olympus BX40F4microscope (Melville, NY) equipped for epifluorescence. Images were an-alyzed using Adobe Photoshop software (Adobe Systems, San Jose, CA).

Preparation of cell extracts

To obtain the whole cell extracts to analyze I�B� degradation, an indicatorof NF-�B pathway activation, neutrophils (5 � 106 cells/ml) were resus-pended in lysis buffer (50 mM HEPES, pH 6.4, 1 mM MgCl2, 10 mMEDTA, 1% Triton X-100, 1 �g/ml DNase, 0.5 �g/ml RNase) containingthe following protease inhibitors: 1 mM PMSF, 1 mM benzamidine, 1 �Mleupeptin, and 1 �M soybean trypsin inhibitor (Sigma-Aldrich).

Preparation of nuclear extracts

For the analysis of NF-�B nuclear translocation, neutrophils (5 � 106

cells/ml) were incubated with heme (3 �M) for 1 or 2 h at 37°C in a 5%CO2 atmosphere. Nuclear extracts were obtained, as described earlier (26).Briefly, cells were lysed in ice-cold buffer A (10 mM HEPES, pH 7.9, 10mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mMPMSF), and after a 15-min incubation on ice, Nonidet P-40 was added toa final concentration of 0.5% (v/v). Nuclei were collected by centrifugation(1,810 � g; 5 min at 4°C). The nuclear pellet was suspended in ice-coldbuffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mMEGTA, 1 mM DTT, 1 mM PMSF, 1 �g/ml pepstatin, 1 �g/ml leupeptin,and 20% (v/v) glycerol) and incubated for 30 min. Nuclear proteins werecollected in the supernatant after centrifugation (12,000 � g; 10 min at4°C), and the immunoblotting for nuclear NF-�B and histone (H3) contentwas performed, as described below.

Immunoprecipitation

Neutrophils (5 � 106 cells/ml) were incubated with heme (3 �M) for 5, 15,and 30 min at 37°C in a 5% CO2 atmosphere in the absence or the presenceof LY290042 (3 �M) or BIM (10 nM). Cells were lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, Triton X-100(1%, v/v), glycerol (10%, v/v), aprotinin (10 �g/�l), leupeptin (10 �g/�l),pepstatin (2 �g/�l), and 1 mM PMSF. Lysates (2 �g of protein/�l) wereincubated overnight at 4°C with polyclonal anti-Akt1 (1/200; Santa CruzBiotechnology) Ab. After this time, protein A/G-agarose (20 �l/mg pro-tein; Santa Cruz Biotechnology) was added, and samples were incubated at4°C in a rotatory shaker for 2 h. The content of total and phosphorylatedAkt on serine residues was analyzed by Western blot, as described below.

Western blot analysis

The total protein content in the cell extracts was determined by Bradford’smethod (27). Cell lysates were denatured in sample buffer (50 mM Tris-HCl, pH 6.8, 1% SDS, 5% 2-ME, 10% glycerol, and 0.001% bromphenolblue) and heated in a boiling water bath for 3 min. Samples (30 �g of totalprotein) were resolved by 12% SDS-PAGE, and proteins were transferredto polyvinylidene difluoride membranes (Hybond-P; Amersham Bio-sciences). Rainbow markers (Amersham Biosciences, Uppsala, Sweden)were run in parallel to estimate molecular weights. Membranes wereblocked with Tween TBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1%Tween 20) containing 1% BSA and probed with polyclonal anti-Bcl-xL

(Santa Cruz Biotechnology; 1/500), polyclonal anti-Bad (Santa Cruz Bio-technology; 1/500), monoclonal anti-phosphoserine (Sigma-Aldrich;1/1000), polyclonal anti-Akt1 Ab (Santa Cruz Biotechnology; 1/1000),polyclonal anti-NF-�B Ab (Santa Cruz Biotechnology; 1/1000), polyclonalanti-I�B Ab (Santa Cruz Biotechnology; 1/1000), or monoclonal anti-histone H3 Ab (Cell Signaling Technology, Beverly, MA; 1/1000). Afterextensive washing in Tween TBS, polyvinylidene difluoride sheets wereincubated with biotin-conjugated anti-rabbit or anti-mice IgG (Santa Cruz

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Biotechnology; 1:1000) Ab for 1 h and then incubated with HRP-conju-gated streptavidin (Caltag Laboratories, Burlingame, CA; 1/1000). Immu-noreactive proteins were visualized by 3,3�-diaminobenzidine (Sigma-Al-drich) staining. The bands were also quantified by densitometry usingScion Image Software (Scion, Frederick, MD).

Statistical analysis

Statistical significance was assessed by ANOVA, followed by Bonferroni’st test, and p � 0.05 was taken as statistically significant.

ResultsHeme delays human neutrophil apoptosis in vitro

The effect of heme in the modulation of spontaneous neutrophilapoptosis was simultaneously assessed by morphology (Fig. 1A),annexin V binding (Fig. 1B), and DNA electrophoresis (Fig. 1C).We have observed that incubation of human neutrophils with heme(3 �M) significantly delayed the apoptotic rate of these cells.Heme-mediated delay of apoptosis at 1–3 �M was similar to thatobserved with IL-8 (100 nM), a cytokine with a known ability toprotect neutrophil from apoptosis. Heme was able to delay neu-trophil apoptosis in vitro at all concentrations studied (1–50 �M;Fig. 2A), although its protective effects were less pronounced at thehighest concentration used (50 �M). Because heme is able toevoke IL-8 synthesis in human neutrophils (3), we speculatedwhether the effect of free heme on neutrophil apoptosis would bemediated by an autocrine production of this antiapoptotic chemo-kine. Pretreatment of neutrophils with anti-IL-8 Ab did not inhibitthe heme effect, indicating that heme inhibits human neutrophilapoptosis per se (Fig. 2B).

SnPPIX reverses heme effects on neutrophil apoptosis

We have previously observed that heme enhances HO-1 proteinexpression on neutrophils (A.V.G.-S., C.B.-F., and M.A.A., un-published data). To investigate the involvement of HO in the in-hibition induced by heme on neutrophil apoptosis, cells were co-incubated with SnPPIX (50 �M), a competitive inhibitor of thisenzyme (Fig. 3). HO inhibition partially reversed heme effects onneutrophil apoptosis, suggesting that heme metabolites may playan important role on neutrophil survival under these conditions.However, neither biliverdin nor bilirubin (3 �M) protected humanneutrophils from spontaneous apoptosis (51 and 53% of apoptoticcells, repectively), suggesting that other HO metabolites might beinvolved in heme effect.

Delaying of neutrophil apoptosis by heme requires ROSproduction

We have previously reported that heme evokes an oxidative burstin human neutrophils (3), which may lead to profound changes inthe redox status of the cells. These alterations are known to mod-ulate the activity of redox-sensitive proteins, including caspases

FIGURE 1. Inhibition of spontaneous apoptosis in human neutrophilsby heme. A, Morphological analysis of apoptotic neutrophils: 1) mediumalone, 0 h; 2) medium alone, 20 h; 3) 3 �M heme, 20 h; 4) 100 nM IL-8,20 h. B, Flow cytometric analysis of annexin V binding in fresh neutrophils(1), neutrophils incubated for 20 h in the absence (2) or the presence of 3�M heme (3), or 100 nM IL-8 (4). The percentage of annexin V-positivecells in each sample is indicated. This representative experiment was re-peated three times, showing similar results each time. C, Agarose gel elec-trophoresis of DNA showing an increase in the formation of oligonucleo-somal DNA fragments in spontaneous apoptosis after 20 h (lane 1, 0 h;lane 2, 20 h), which is inhibited by incubation with 3 �M heme (lane 3)and 100 nM IL-8 (lane 4). Lane M represents the m.w. marker.

FIGURE 2. Concentration-dependent effect of heme on neutrophil ap-optosis. A, Neutrophils (5 � 106/ml) were incubated in the absence or inthe presence of varying concentrations of heme (1–50 �M) or IL-8 (100nM). After 20 h, cells were cytocentrifuged, and the number of apoptoticcells was determined microscopically. B, The same protocol was per-formed with a single heme (3 �M) or IL-8 (100 nM) concentration in theabsence or the presence of anti-IL-8 mAb (7.2 �g/ml; o). Data shown arethe results (mean � SD) of three experiments, each performed in triplicate.�, Indicates a significant difference (p � 0.001) between heme- or IL-8-treated and control neutrophils. §, Indicates that this group is significantlydifferent from other treatments (p � 0.05).

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(28). In Fig. 4, we show that the incubation of cells with DPI andapocynin, two different NADPH oxidase inhibitors, reversed theinhibition of apoptosis in neutrophils treated with heme. Theseresults suggest that alterations in the redox potential of these cells,mediated by NADPH oxidase activity, might be critical for theheme-induced antiapoptotic effect.

Heme-induced delay of neutrophil apoptosis requires proteinsynthesis

The balance between levels of pro- and antiapoptotic proteins hasa pivotal role in the modulation of apoptosis. To determine therequirement of de novo protein synthesis on the heme-inducedeffect on neutrophil apoptosis, cells were pretreated with cyclo-

heximide (3.6 �M) and incubated with heme (1–10 �M; Fig. 5).As we have previously observed that cycloheximide is cytotoxic atlater time points (e.g., 20 h) (data not shown), we evaluated apo-ptosis after 5-h incubation in which there is no detectable cyto-toxicity. Cycloheximide completely abolished the survival effectinduced by heme on human neutrophil apoptosis, suggesting thatthe effects of heme rely on newly synthesized antiapoptoticproteins.

Heme induces Bad degradation and Bcl-xL expression on humanneutrophils

The balance between the expression of anti- and proapoptotic pro-teins of Bcl-2 family has been shown to be a prominent feature onthe control of apoptosis. As mature neutrophils exhibit a very shortlife span, the expression of proapoptotic Bcl-2 members is consti-tutively high, whereas antiapoptotic members’ levels are very lowor not detectable (23). Fig. 6A shows that heme induced the deg-radation of Bad, a proapoptotic Bcl-2 member, which reachedlower levels 30 min after heme treatment. In contrast, heme in-duced the synthesis of the antiapoptotic protein Bcl-xL (Fig. 6B),reinforcing the regulation of prosurvival signaling by heme.

Heme delays neutrophil apoptosis via PI3K- and ERK-dependent pathways

Because the Ras/Raf/MAPK and PI3K pathways have been re-ported to be actively involved in regulating the antiapoptotic effectof other proinflammatory agents, we investigated the participationof these signaling pathways in heme-mediated delay of neutrophilapoptosis in vitro. Pretreatment of cells with the PI3K inhibitorLY294002 (3 �M) or the MEK1/2 inhibitor PD98059 (10 �M)totally reversed the delay of apoptosis promoted by heme (3 �M)and IL-8 (100 nM; Fig. 7), suggesting a pivotal role of these sig-naling pathways in the modulation of heme effect on neutrophils.

Heme activates ERK-2 nuclear translocation

The phosphorylation of ERK-1/2 regulatory sites can drive itstranslocation to the nucleus, where ERK exerts part of its biolog-ical activity. Confirming the involvement of the ERK pathway in

FIGURE 4. Heme inhibition of neutrophil apoptosis requires NADPHoxidase activity. Neutrophils (5 � 106/ml) were cultured with or withoutheme (1–10 �M) in the absence (f) or in the presence of DPI (10 �M; o)or apocynin (10 �M; �). After 20 h, cells were cytocentrifuged, and thenumber of apoptotic cells was determined microscopically. Data shown arethe results (mean � SD) of three experiments, each performed in triplicate.�, Indicates a significant difference (p � 0.01) between heme-treated andcontrol neutrophils. #, Indicates that DPI and apocynin significantly inhib-ited the delay of apoptosis induced by heme (p � 0.05).

FIGURE 5. Inhibition of neutrophil apoptosis by heme requires proteinsynthesis. Neutrophils (5 � 106/ml) were incubated in the absence or in thepresence of heme (1–10 �M), with (p) or without (f) cycloheximide (3.6�M). After 5 h, cells were cytocentrifuged, and the number of apoptoticcells was determined microscopically. Data shown are the results (mean �SD) of three experiments, each performed in triplicate. �, Indicates a sig-nificant difference (p � 0.05) between heme-treated and control neutro-phils. #, Indicates that cycloheximide significantly inhibited the delay ofapoptosis induced by heme (p � 0.05). The results are expressed asmean � SD.

FIGURE 3. Inhibition of HO activity by SnPPIX reverses the antiapop-totic effect of heme on human neutrophils. Neutrophils (5 � 106/ml) wereincubated with indicated concentrations of heme in the absence (f) or inthe presence of SnPPIX (50 �M; p). After 20 h, cells were cytocentri-fuged, and the number of apoptotic cells was determined microscopically.Data shown are the results (mean � SD) of three experiments, each per-formed in triplicate. �, Indicates a significant difference (p � 0.001) be-tween heme-treated and control neutrophils. #, Indicates that SnPPIX sig-nificantly inhibited the delay of apoptosis induced by heme (p � 0.05).

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heme antiapoptotic effects, Fig. 8 shows that heme (3 �M), as wellas IL-8 (100 nM), promoted ERK-2 translocation to the nucleus inhuman neutrophils, after 1 h of incubation, as detected by immu-nofluorescence microscopic analysis.

Heme induces Akt activation

Akt phosphorylation on serine residues is a key event in PI3K/Aktsignaling cascade. Once activated, Akt is able to promote cell sur-vival phosphorylating proapoptotic Bcl-2 family members, espe-cially Bad, inducing their degradation by the proteasome (20). Fig.9 shows that heme (3 �M) induced Akt phosphorylation on serineresidues. This effect was highly significant after 5 min of incuba-tion, peaking after 15 min and decreasing thereafter. The timecourse of Akt phosphorylation seems to be modulated hierarchi-cally by PI3K and PKC. Although LY294002 (3 �M; p) inhibitedonly the early effect observed at 5 min after incubation with heme,BIM, a PKC inhibitor (10 nM; �), exclusively inhibited heme-induced Akt phosphorylation on later time points (15–30 min).These results strongly suggest that heme triggers Akt signaling inhuman neutrophils in a PI3K-dependent manner, but requires PKCactivity to sustain this effect.

Heme induces a redox-sensitive NF-�B activation

It is well established that NF-�B modulates prosurvival signalingpathways, inhibiting apoptosis of several cell types (29–31).

NF-�B activation requires I�B phosphorylation and degradation inthe cytoplasm and subsequent translocation of NF-�B to the nu-cleus. This process can be regulated by redox-sensitive mecha-nisms. In Fig. 10A, we show that PDTC (100 nM), an antioxidantable to inhibit NF-�B activation, abrogated the delay of neutrophilapoptosis induced by heme, suggesting that NF-�B activationmodulates heme-mediated cell survival.

To confirm that NF-�B activation occurs in neutrophils stimu-lated with heme, we evaluated the degradation of cytoplasmic I�Band the translocation of NF-�B p65 subunit to the nucleus in these

FIGURE 6. Heme induces Bad degradation and Bcl-xL synthesis. Neu-trophils (5 � 106/ml) were cultured in the absence or the presence of heme(3 �M). At the indicated time points, Bad (A) or Bcl-xL (B) protein ex-pression was accessed by Western blot analysis, as described in Materialsand Methods. Quantification of band OD is expressed in arbitrary units.

FIGURE 7. Inhibition of PI3K and ERK pathway abrogates the anti-apoptotic effect of heme on neutrophils. Neutrophils (5 � 106/ml) werecultured with heme (3 �M) or IL-8 (100 nM), in the absence (f) or in thepresence of LY294002 (3 �M; p) or PD98059 (10 �M; �). After 20 h,cells were cytocentrifuged, and the number of apoptotic cells was deter-mined microscopically. Data shown are the results (mean � SD) of threeexperiments, each performed in triplicate. �, Indicates a significant differ-ence (p � 0.01) between heme- or IL-8-treated and control neutrophils. #,Indicates that PD98059 and LY294002 significantly inhibited the delay ofapoptosis induced by IL-8 and heme (p � 0.05). The results are expressedas mean � SD.

FIGURE 8. Heme induces ERK nuclear translocation. Neutrophils (5 �106/ml) were incubated for 1 h with medium alone (control; A), IL-8 (100nM; B), or heme (3 �M; C). Cytocentrifuge preparations were fixed, andsubcellular localization of endogenous ERK-2 was detected by stainingwith anti-ERK-2 Ab. Quantification of nuclear fluorescence intensity (ar-rowheads) is shown in D. Experiments were performed three times withsimilar results.

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cells. After 1 h of incubation with 3 �M heme, the levels of I�Bsignificantly decreased in neutrophils treated with heme (Fig.10B). This is followed by a significant increase of the NF-�B p65subunit in nuclear extracts of neutrophils, which was more prom-inent after 2 h of incubation with heme and comparable to thatevoked by LPS (1 �g/ml; Fig. 10C). The translocation of NF-�Bevoked by heme and LPS was inhibited when cells were treatedwith the NADPH oxidase inhibitor DPI, providing an additionalevidence for the role of ROS on NF-�B activation.

DiscussionChronic inflammation, an important feature in hemolytic diseases,involves an intense neutrophil activation accompanied by in-creased survival rate of these cells (12), events that may lead to anundesirable persistence of inflammation. In this study, we show,for the first time, that free heme increases neutrophil survival, pro-viding evidence that this effect may contribute to the inflammatoryprocess often associated with hemolytic episodes.

It was previously reported that erythrocytes are able to inhibitapoptosis of human neutrophils even when these cells are physi-cally separated (32), suggesting the existence of a diffusible mol-ecule able to retard neutrophil apoptosis. Our findings stronglysuggest that the ability of erythrocytes to prevent neutrophil apo-ptosis may be directly linked to an increase in free heme concen-trations released by lysed RBC.

A putative mechanism by which the organism can control thedeleterious effect of large amounts of heme is through the HOactivity. HO are ubiquitous enzymes able to catalyze the initial andrate-limiting step in the oxidative degradation of heme to bilirubin,producing equimolar amounts of biliverdin, free iron (Fe), andcarbon monoxide (CO) (33). The expression of the inducible HOisoform (HO-1) is positively modulated by several inflammatorymediators and oxidative stress and also by heme itself. HO-1 hasalso been highlighted as one of the major inducible enzymes dur-ing the inflammatory response, especially during the resolutionphase of inflammation (34). Its expression is associated with the

inhibition of apoptosis of several cell types such as fibroblasts,endothelial cells, pancreatic � cells, and hepatocytes (35), andcould be related to the action of one or more catalytic by-productsgenerated by HO-1 (36). Recent reports have shown that the in-duction of HO-1 activity inhibited TNF-�-induced apoptosis inendothelial cell, via CO generation (37), whereas E-selectin andVCAM expression promoted by this stimulus is negatively mod-ulated by HO-1 activity via bilirubin and Fe (38). In agreement

FIGURE 9. Heme induces Akt phosphorylation via PI3K and PKC.Neutrophils (5 � 106/ml) were incubated with heme (3 �M) for the indi-cated time points in the absence or in the presence of LY294002 (3 �M; p)or BIM (10 nM; �). Cells were then harvested, cell extracts were immu-noprecipitated with anti-Akt, and Western blots were performed for phos-phoserine detection. Quantification of band OD is expressed in arbitraryunits.

FIGURE 10. Heme-mediated delay of apoptosis requires NF-�B acti-vation. A, Neutrophils (5 � 106/ml) were incubated with heme (3 �M) inthe absence (�) or in the presence (f) of PDTC (100 nM). After 20 h, cellswere cytocentrifuged, and the number of apoptotic cells was determinedmicroscopically. Data shown are the results (mean � SD) of three exper-iments, each performed in triplicate. �, Indicates a significant difference(p � 0.01) between heme-treated and control neutrophils. #, Indicates thatPDTC significantly inhibited the delay of apoptosis induced by heme (p �0.05). B, Western blot analysis of whole cell extracts prepared from non-stimulated or heme-stimulated (3 �M) neutrophils for the indicated timepoints. C, Western blot analysis of nuclear extracts prepared from neutro-phils stimulated or not with heme (3 �M) or LPS (1 �g/ml) for 1 or 2 h inthe absence (f) or the presence of DPI (p). Quantification of band OD isexpressed in arbitrary units.

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with these findings, we showed that HO activity is, at least in part,required for heme effects upon human neutrophils because the de-lay of neutrophil apoptosis was partially reversed by a competitiveinhibitor of this enzyme. However, neither bilverdin nor its me-tabolite, bilirubin, was able to influence on neutrophil life span,suggesting that probably CO and/or Fe might be the effector mol-ecules generated by HO-1. The role of heme and HO-1 in modu-lating neutrophil spontaneous apoptosis points to a new and in-triguing role of this system during the inflammatory response.

We have shown that heme is also able to trigger oxidative burstin neutrophils (3). This effect appears to be closely related to theantiapoptotic effect of heme because the inhibition of neutrophilapoptosis is reversed by DPI and apocynin, two NADPH oxidaseinhibitors, providing evidence that alterations in redox potentialmediate heme effects on human neutrophils. Data concerning therelationship between ROS generation, and the subsequent alter-ations in the redox potential, and the apoptotic process of differentcells have been conflicting. Several reports have shown that anti-oxidants could both elicit or delay apoptosis (39, 40). As neutro-phils can change their redox potential through their primary func-tion of killing invading microorganisms, ROS generation as wellas the presence of intracellular antioxidant molecules may interferewith the regulation of apoptosis in these cells. Although ROSand glutathione were shown to block caspase activity, both can in-hibit, as well as trigger, apoptosis in human neutrophils, depending onthe experimental conditions (41, 42). These data indicate that the reg-ulation of neutrophil survival most likely involves a delicate balancein the redox status of the cell rather than the prevalence of intracellularoxidants or antioxidants. This may probably explain the reduced in-hibition of neutrophil apoptosis when the cells were incubated with ahigher concentration of heme (50 �M; Fig. 2), which was able togenerate higher superoxide anion production (3).

Evidence has shown that the antiapoptotic effect of some agentsrequire de novo protein synthesis, although it was recently reportedthat cAMP inhibits neutrophil apoptosis via a novel, reversible,and transcriptionally independent mechanism (43). Heme inhibi-tion of neutrophil apoptosis was blocked by cycloheximide, sug-gesting that newly synthesized antiapoptotic proteins are likely tobe involved in this effect. Neutrophils constitutively express pro-apoptotic proteins, including Bax, Bid, Bak, and Bad, while theexpression of antiapoptotic Bcl members (Bcl-xL, A1, and Mcl-1)is very low or undetectable in resting cells (23). These antiapop-totic proteins are highly and transiently expressed when neutro-phils are exposed to survival factors, such as IL-8 and GM-CSF,which act through the activation of MAPK, PI3K, NF-�B, andother distinct signaling pathways (20, 21, 23). We have shown thatexposition of neutrophils to heme induced the degradation of Badreducing after to 30 min to very low levels. In contrast, hemetreatment increased the levels of Bcl-xL, corroborating to hemeantiapoptotic effects. In parallel, we have also shown that the in-hibition of ERK and PI3K pathways, which have been directlycorrelated to the expression of those proteins, successfully re-versed heme-induced delay of neutrophil apoptosis. Involvementof ERK activation in the heme-mediated antiapoptotic effects wassupported by the observation that heme induces ERK-2 nucleartranslocation, a prominent feature of ERK activation.

As an additional parameter to evaluate the involvement of PI3Kon heme antiapoptotic effect, we analyzed the phosphorylation ofAkt on serine residues. Akt is the main downstream target of PI3K,which induce Akt translocation to the plasma membrane and sub-sequent phosphorylation by the PI3K-dependent kinase. Heme-in-duced Akt phosphorylation was dependent on PI3K and PKC ac-tivation. However, while PI3K activation is an essential and earlyevent for Akt phosphorylation induced by heme, PKC appears to

be involved in the late and sustained activation of Akt, which isprobably associated with the modulation of antiapoptotic effect. Agreat deal of effort has been directed toward defining which PKCisoforms are involved in the regulation of the apoptotic process. Itis now believed that classical (�, �I, �II, and �) and atypical (�, �,and �) isoforms are associated with cell survival, whereas novelPKC isoenzymes (�, , , and �) are considered proapoptotic (44,45). Although the precise participation of different PKC memberon heme antiapoptotic effect requires further investigation, ourdata suggest a prominent role for PI3K/Akt pathway on heme an-tiapoptotic effect, which is probably up-regulated by PKC.

Many inflammatory mediators regulate gene expression in targetcells by influencing the activity of transcription factors, especiallyNF-�B, to evoke their proinflammatory response (46). NF-�Bcomprises a family of transcription factors that act to regulategenes involved in a variety of events such as inflammatory andimmune responses, apoptosis, proliferation, and differentiation(47–50). NF-�B regulation seems to be highly cell specific andredox sensitive (51–53). Moreover, Asehnoune et al. (54) haverecently shown that the antioxidants N-acetylcysteine and �-to-copherol prevented LPS-induced nuclear translocation of NF-�Bon early events in TLR4 signaling. We have observed that hemeactivates NF-�B in polymorphonuclear cells, inducing its translo-cation to the nucleus. The inhibition of the heme-induced anti-apoptotic effect by PDTC suggests that heme induces redox-sen-sitive NF-�B activation. The evidence that the inhibitor DPIimpairs both heme- and LPS-induced NF-�B nuclear translocationpoints to a prominent role of ROS generated by NADPH oxidaseon this phenomenon. Furthermore, this is the first report showingthe importance of NADPH oxidase-derived ROS in NF-�B acti-vation on human neutrophils. The involvement of NF-�B on hemecytoprotective effects reinforces the close relationship between ac-tivation status and enhanced granulocyte life span already ob-served for other antiapoptotic stimuli (52).

Taken together, our data strongly suggest that the protectiveeffect of heme on human neutrophil apoptosis is modulated byPI3K and MAPK pathways and involves NF-�B activation in anNADPH oxidase-dependent manner. The activation of those path-ways might lead to the transcription of HO-1 (55), whose activitycould modulate the heme-induced antiapoptotic effect, amongother antiapoptotic proteins, as previously discussed.

In summary, we are presenting novel data showing that freeheme is able to delay human neutrophil apoptosis in vitro at con-centrations found during hemolytic events in vivo. This effect isdependent on HO activity and ROS generation, requires de novoprotein synthesis, and is modulated through PI3K, ERK, andNF-�B pathways. Furthermore, the data suggest that heme maycontribute to the development of chronic inflammation associatedwith hemolytic episodes by delaying apoptosis and promotingfunctional longevity of neutrophils. The understanding of this pro-cess can lead to the establishment of new strategies to amelioratetissue damage associated with severe hemolysis.

AcknowledgmentsWe thank Dr. I. M. Fierro for the critical reading of this manuscript. Wealso thank Dr. F. Q. Cunha for supplying human rIL-8, and Dr. P. Bozzafor anti-IL-8 mAb, as well as Carla Gregorio and Pedro Barcellos de Souzafor excellent technical assistance.

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