Hemozoin Induces Macrophage Chemokine Expression through -Dependent and -Independent Mechanisms This information is current as of October 7, 2021. Maritza Jaramillo, Marianne Godbout and Martin Olivier J Immunol 2005; 174:475-484; ; doi: 10.4049/jimmunol.174.1.475 http://www.jimmunol.org/content/174/1/475 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Hemozoin Induces Macrophage Chemokine Expression through Oxidative Stress-Dependent and -Independent Mechanisms1

Maritza Jaramillo,† Marianne Godbout,* and Martin Olivier2†*

Chemokine production has been associated with the immunopathology related to . Previous findings indicated that he- mozoin (HZ), a parasite metabolite released during schizogeny, might be an important source of these proinflammatory mediators. In this study we investigated the molecular mechanisms underlying HZ-inducible macrophage (M␾) chemokine mRNA expres- sion. We found that both falciparum HZ and synthetic HZ increase mRNA levels of various chemokine transcripts (MIP-1␣/CCL3, MIP-1␤/CCL4, MIP-2/CXCL2, and MCP-1/CCL2) in murine B10R M␾. The cellular response to HZ involved ERK1/2 phosphorylation, NF-␬B activation, reactive species (ROS) generation, and ROS-dependent protein-tyrosine phosphatase down-regulation. Selective inhibition of either I␬B␣ or the ERK1/2 pathway abolished both NF-␬B activation and Downloaded from chemokine up-regulation. Similarly, blockage of HZ-inducible M␾ ROS with superoxide dismutase suppressed chemokine in- duction, strongly reduced NF-␬B activation, and restored HZ-mediated M␾ protein-tyrosine phosphatase inactivation. In con- trast, superoxide dismutase had no effect on EKR1/2 phosphorylation by HZ. Collectively, these data indicate that HZ triggers ROS-dependent and -independent signals, leading to increased chemokine mRNA expression in M␾. Overall, our findings may help to better understand the molecular mechanisms through which parasite components, such as HZ, modulate the immune response during malaria infection. The Journal of Immunology, 2005, 174: 475–484. http://www.jimmunol.org/

lasmodium falciparum (Pf),3 the etiological agent of the MIP-1␣/CCL3 and IL-8/CXCL8 were detected during acute infec- most lethal form of human malaria, is responsible for tion (4). In placental malaria, increased MIP-1␣, IL-8, and MCP- P 300–500 million clinical cases and 1–3 million deaths 1/CCL2 mRNA expression was associated with placental mono- annually (1). Accumulating evidence indicates that in addition to cyte recruitment (5), a risk factor for low birth weight (6). In parasite virulence factors, the host’s immunological response (e.g., addition, experimental models of cerebral malaria have underlined proinflammatory cytokines and chemokines) also participates in the importance of the neutrophil (N␾) population in the develop- malaria pathophysiology, including tissue cell damage, microvas- ment of this pathology (7) and have shown the ability of both N␾ cular obstruction, severe anemia, and cerebral malaria (2). Because and macrophages (M␾) to express elevated chemokine mRNA lev- by guest on October 7, 2021 of their ability to induce activation and recruitment of specific els (8). leukocyte subsets to the sites of infection, chemokines influence In an attempt to identify the parasite components that are re- the immune system, thereby further inducing inflammatory condi- sponsible for the activation of the host immune response during tions (3). In Pf malaria-infected patients, elevated serum levels of malaria, we (9, 10) and others (11–13) have focused on evaluating the proinflammatory properties of hemozoin (HZ) or malarial pig- ment. HZ crystals result from aggregated , which is produced *Research Institute of McGill University Health Center, Center for the Study of Host Resistance, Departments of Medicine, Microbiology, and Immunology, McGill Uni- during digestion inside the host RBC (14). This pig- versity, Montre´al, Que´bec, Canada; and †Centre de Recherche en Infectiologie, Cen- ment is released after schizogeny, along with merozoites, as the tre Hospitalier Universitaire de Que´bec, Pavillon Centre Hospitalier de l’Universite´ ␾ Laval, and De´partement de Biologie Me´dicale, Faculte´deMe´decine, Universite´La- RBC bursts and is phagocytosed by circulating monocytes, N , val, Ste-Foy, Que´bec, Canada and resident M␾ (reviewed by Arese and Schwarzer (15)). Both Pf Received for publication February 6, 2004. Accepted for publication October HZ and synthetic HZ (sHZ), which is structurally identical with the 22, 2004. native pigment (16), induced the release of MIP-1␣ and MIP-1␤/ The costs of publication of this article were defrayed in part by the payment of page CCL4 as well as of TNF-␣ and IL-1␤ in murine M␾ and human charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. PBMC (11, 12). These observations were also supported by our in vivo data indicating that sHZ leads to liver chemokine and cyto- 1 This work was supported by grants from the Canadian Institutes in Health Research (CIHR; to M.O.). M.O. is member of a CIHR Group in Host-Pathogen Interactions, kine mRNA up-regulation in i.v. injected mice and to significant a recipient of a CIHR Investigator Award, and a Burroughs Wellcome Fund Awardee leukocyte recruitment and proinflammatory mediator release in the in Molecular Parasitology. M.J. is a recipient of a Ministe`redel’E´ ducation du Que´bec Ph.D. studentship. M.G. is a recipient of a Fonds pour la Formation de Chercheurs et air pouch lumen of mice inoculated intradermally (10). In line with l’Aide a`la Recherche M.Sc. studentship. these findings, Biswas and colleagues (13) reported the presence of 2 Address correspondence and reprint requests to Dr. Martin Olivier, Department of Abs against Pf HZ among complicated malaria patients, which had Microbiology and Immunology, McGill University, 3775 University Street, Duff inhibitory effects on HZ-dependent monocyte cytokine production. Medical Building (Room 600), Montre´al, Que´bec, Canada H3A 2B4. E-mail address: [email protected] Although these studies strongly suggest that HZ is likely to play a key role in proinflammatory mediator overproduction, the mech- 3 Abbreviations used in this paper: Pf, ; HZ, hemozoin; IKK, I␬B kinase; M␾, macrophage; N␾, neutrophil; PTK, protein-tyrosine kinase; PTP, anism by which such regulation occurs remains unexplored. In the . protein-tyrosine phosphatase; O2, superoxide anion; Poly B, polymyxin B; pNPP, present study we demonstrate that both Pf HZ and sHZ increase para-nitrophenyl phosphate; redox, reduction-oxidation; RPA, RNase protection as- ␣ ␤ say; ROS, ; sHZ, synthetic hemozoin or ␤-hematin; SOD, mRNA levels of various chemokines (MIP-1 , MIP-1 , MIP-2, superoxide dismutase. and MCP-1) in murine M␾ through a mechanism that involves

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 476 HZ-INDUCIBLE M␾ CHEMOKINE MODULATION

activation of oxidative stress-dependent and -independent path- study (ϳ26–29 nmol/ml heme content) could be easily released by a pa- ways. A better understanding of the transductional signals through tient with a mild parasitemia load. which HZ modulates the host immune response might be helpful in RNase protection assay (RPA) defining specific therapeutic targets to tame proinflammatory me- diator overproduction during malaria infection. mRNA expression studies were performed using an RPA kit (RiboQuant; BD Pharmingen), as we described previously (22). Total RNA was isolated from stimulated cells with TRIzol reagent (Invitrogen Life Technologies) Materials and Methods according to the manufacturer’s protocol. One of the multiprobe templates Materials was labeled with [␣-32P]dUTP using T7 RNA polymerase. Then, 3 ϫ 105 cpm of labeled probe was allowed to hybridize with 10 ␮g of total RNA for chloride, polymyxin B (Poly B) sulfate, LPS (Escherichia coli, 16 h at 56°C. mRNA probe hybrids were treated with RNase A and phenol- serotype 0111:B4), 3% H2O2 (v/v), and superoxide dismutase (SOD) were chloroform-extracted. Protected hybrids were resolved on a 5% denaturing purchased from Sigma-Aldrich. Isotopes [␣-32P]dUTP (3000 Ci/mmol) polyacrylamide sequencing gel and exposed to radiographic film overnight and [␥-32P]dATP (3000 Ci/mmol) were obtained from PerkinElmer. BAY at Ϫ80°C. Laser densitometry was performed using an ␣Imager 2000 dig- 11-7082 was purchased from BIOMOL. Apigenin and PD 98059 were ital imaging and analysis system (␣Innotech). The multiprobe templates obtained from Calbiochem. (BD Pharmingen) used in this study were mCK-5, for the murine chemo- kines lymphotactin, RANTES, eotaxin, MIP-1␣, MIP-1␤, MIP-2, inducing Cell and culture conditions protein-10, MCP-1, and TCA-3, and a custom template for murine cyto- ␣ ␤ ␥ ␾ kines IL-4, IL-12p40, IL-10, IL-1 , IL-1 , IL-2, IL-6, and IFN- . The The murine M cell line B10R, derived from the bone marrow of template sets included housekeeping genes ml-32 and/or GAPDH. B10A.Bcgr (B10R) mice (17), was provided by Dr. D. Radzioch (McGill University, Montre´al, Canada). Cells were maintained in DMEM (Invitro- EMSA gen Life Technologies) supplemented with 10% heat-inactivated FBS (Hy- Downloaded from Clone) plus 100 ␮g/ml streptomycin and 2 mM L-glutamine at 37°C and Cell stimulation was terminated by the addition of ice-cold PBS, nuclear

5% CO2. extracts were prepared according to the microscale protocol, and EMSA was performed using 6 ␮g of nuclear proteins, as we described previously Cell viability assays (22). Briefly, nuclear extracts were incubated for 20 min at room temper- ature in 1.0 ␮l of binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenil)-2-(4-sulfophe- 10% Ficoll, 250 mM KCl, 10 mM DTT, 5 mM EDTA, and 250 mM NaCl), nyl)-2H-tetrazolium (inner salt) assays for cell viability were performed 2 ␮g of poly(dI-dC) and 10 ␮g of nuclease-free BSA (fraction V; Sigma- and indicated no cytotoxic or cytostatic effect from the various specific ␥ 32 Aldrich) containing 1.0 ng of [ - P]dATP radiolabeled dsDNA oligonu- http://www.jimmunol.org/ ␾ inhibitors at the concentrations used (data not shown). Briefly, B10R M cleotide. This mixture was incubated for 20 min at room temperature, and ϫ 4 were seeded in 96-well plates (3 10 cells/well) and were stimulated for the reaction was stopped using 5 ␮l of 0.2 M EDTA. DNA-protein com- ␮ 3 h with increasing concentrations of the various inhibitors (100 l). Then, plexes were resolved from free-labeled DNA by electrophoresis in native ␮ cells were incubated with 10 l of a 20/1 solution of 2 mg/ml (3-(4,5- 4% (w/v) polyacrylamide gels containing 50 mM Tris-HCl (pH 8.5), 200 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenil)-2-(4-sulfophenyl)-2H- mM glycine, and 1 mM EDTA. The gels were subsequently dried and tetrazolium (inner salt; Promega) and 3 mM phenazine methosulfate (Sig- autoradiographed. The dsDNA oligonucleotide containing a consensus ma-Aldrich) for 1 h, and OD was read at 492 nm. binding site for NF-␬B/c-Rel homodimeric and heterodimeric complexes Ј Ј ␤ (5 -AGTTGAGGGGACTTTCCCAGGC-3 ) was obtained from Santa -Hematin (sHZ) preparation Cruz Biotechnology. The oligonucleotides containing NF-␬B-binding sites ␤-Hematin was synthesized as previously described (9). Briefly, 45 mg of of the murine chemokine promoters were synthesized in our laboratory as ␬ Ј Ј by guest on October 7, 2021 hemin chloride (Sigma-Aldrich) was solubilized in 4.5 ml of 1 N NaOH follows: NF- B/MIP-2, 5 -GAGCTCAGGGAATTTCCCTGGTCC-3 ␬ Ј Ј and neutralized with 450 ␮l of 1 N HCl. Then, 10.2 ml of 1 M sodium (23); and NF- B/MCP-1, 5 -AAGGGTCTGGGAACTTCCAATACTGC-3 Ј acetate, pH 4.8, was added, and the suspension was stirred with a magnet (24). The nonspecific probe Oct-2A (5 -GGAGTATCCAGCTCCGTAG Ϫ Ј for 2–3 h at 60°C. After addition of 1/100 volume of 10% SDS and CATGCAAATCCTCTGG 3 ), which was used to confirm the specificity of 14,000 ϫ g centrifugation for 15 min, the pellet was sonicated at the lowest the DNA/nuclear protein reaction, was also synthesized in our laboratory. Cold setting in 100 mM sodium bicarbonate (pH 9.0)/0.5% SDS and again cen- competitor assays were conducted by adding a 100-fold molar excess of ho- trifuged. The pellet was then washed three or four times in 2% SDS and mologous unlabeled oligonucleotides of the various labeled dsDNA probes. then in water to wash out the SDS. The pigment was dried at 37°C over- Supershift assays were performed by preincubation of nuclear extracts with 2 ␮ night, resuspended in PBS-endotoxin free (Life Technologies) at a final g of polyclonal Abs against p65 (Rel A) or p50 (Santa Cruz Biotechnology) concentration of 2.5 mg/ml, and kept at Ϫ20°C. Pf HZ was extracted as in the presence of all components of the binding reaction described above for described previously (9) and was provided by Dr. D. C. Gowda (Pennsyl- 1 h at 4°C. vania State University College of Medicine, Hershey, PA). To assess the Western blotting level of endotoxins in the HZ preparations, the Limulus amebocyte lysate test (E-toxate kit; Sigma-Aldrich) was performed, and no endotoxin con- Cells were collected after stimulation, lysed in cold buffer containing 20 tamination was detected. mM Tris-HCl (pH 8.0), 0.14 M NaCl, 10% glycerol (v/v), 1% Igepal (v/v), 25 ␮M nitrophenyl guanidinobenzoate, 10 ␮M sodium fluoride, 1 mM Heme quantitation sodium orthovanadate, 25 ␮g/ml leupeptin, and aprotinin. The lysates (20 ␮g/lane) were subjected to SDS-PAGE, and the separated proteins were The total heme content was determined, as described by Sullivan et al. (18), transferred onto a polyvinylidene difluoride membrane (Millipore). After a by adding 20 mM NaOH/2% SDS to the HZ preparations, incubating the 1-h blocking period in TBST containing 5% milk, the membranes were suspension at room temperature for 2 h, and then reading the OD at 400 nm incubated overnight in TBST/5% BSA at 4°C with one of the following (DGB UV/visible spectrophotometer; Beckman Coulter). Twenty-five mi- rabbit polyclonal Abs (New England Biolabs): phospho-ERK1/2 (Thr202/ crograms of sHZ equals 26 nmol of heme content, and 25 ␮gofPf HZ Tyr204), ERK1/2, phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-I␬B␣ equals 29 nmol of heme content. It should be noted that the concentrations (Ser32), and I␬B␣. Proteins were then detected with an anti-rabbit, HRP- of HZ used to stimulate M␾ (25–75 ␮g/ml) were chosen based on reported conjugated, goat Ab (Affini-Pure; Jackson ImmunoResearch Laboratories) estimates of the HZ concentrations encountered during malaria infection and subsequent visualization by ECL (ECL Western blotting detection sys- (12, 19, 20) as well as on previous studies in which these amounts of HZ tem; Amersham Biosciences). were shown to be effective both in vitro and in vivo (12, 21). Briefly, by reference to standard hematological measurements and assuming that 1) a Phosphatase activity 70-kg adult has a 1% synchronized Pf parasitemia (a relatively mild par- asite load), 2) 50% of the hemoglobin in an infected cell is degraded, and After stimulation, M␾ were washed in PBS and lysed in a buffer containing 3) all heme in the degraded hemoglobin is converted into HZ, it has been 50 mM Tris-HCl (pH 7.0), 0.1 mM EDTA, 0.1 mM EGTA, 0.1% 2-ME estimated that as much as 200 ␮mol of HZ (ϳ3 ␮mol/kg) is released into (v/v), 1% Igepal (v/v), 25 ␮g/ml aprotinin, and 25 ␮g/ml leupeptin. Cel- the circulation of a Pf-infected human patient at schizogeny (12). In addi- lular protein-tyrosine phosphatase (PTP) activity was determined by eval- tion, it was calculated that 106 Pf trophozoite-infected RBC contain be- uating the capacity of cell lysates to hydrolyze para-nitrophenyl phosphate tween 339 and 652 ng of HZ (equal to 0.5–1.0 nmol of heme content) (19, (pNPP; Roche), as we previously described (25). Briefly, 20 ␮g of total 20). According to these data, the concentrations of HZ used in the current proteins were incubated with 180 ␮l of a reaction mixture containing 50 The Journal of Immunology 477

mM HEPES (pH 7.5), 0.1% 2-ME (v/v), and 10 mM pNPP. After a 10- to tion occurred very rapidly (0.5–1 h poststimulation), peaked after 60-min incubation at 37°C, PTP activity was monitored, reading the OD at 2 h, and transiently decreased over an 8-h period (Fig. 1B). Sub- 405 nm (DGB UV/visible spectrophotometer; Beckman Coulter). sequent experiments presented in this section are those after cell Oxidative stress generation stimulation with sHZ; however, all of them were performed in parallel with Pf HZ, and the results were the same as those ob- To monitor reactive oxygen species (ROS) generation, M␾ were seeded in 96-well plates (3 ϫ 104 cells/well), and after two washes with PBS, they tained with sHZ. Ј Ј were incubated for 30 min with H2DCFDA, 2 ,7 -dichlorofluorescein di- acetate (Calbiochem), a commonly used fluorogenic probe, diluted to a final concentration of 100 ␮M. Cell washing was followed by a 1-h ex- Synthetic HZ-dependent chemokine mRNA up-regulation is not

posure to either H2O2 or sHZ with or without SOD, and fluorescence was due to LPS contamination read at 485/530 nm using an LS50 luminescence spectrometer (PerkinElmer). To demonstrate that chemokine expression in response to sHZ was not due to the presence of LPS, cells were incubated for2hin Statistical analysis culture medium (DMEM/10% FBS) containing either LPS (100 ␮ ␮ Statistically significant differences between groups were determined by ng/ml) or sHZ (25 g/ml) and treated, or not, with 5–10 g/ml ANOVA module of StatView Plus SE software (Abacus Concepts) and Poly B for 30 min before cell stimulation. Because Poly B binds to Fisher’s least significant difference test. A value of p Ͻ 0.05 was consid- LPS and blocks its activity (26), this compound has the ability to ered statistically significant. All data are presented as the mean Ϯ SEM. inhibit LPS-inducible M␾ activation. As shown in Fig. 2A, Poly B did not exert any inhibitory effect on sHZ-dependent chemokine Results modulation; however, this compound dramatically reduced the

␾ Downloaded from Pf HZ and sHZ increase M chemokine mRNA expression chemokine increase in response to LPS, reaching 100% of inhibi- M␾ have been identified as one of the main sources of proinflam- tion in the presence of 10 ␮g/ml Poly B. These data are in line with matory mediators in response to Pf infection (2). Thus, we initially those of our previous study showing that the synergistic effect of established whether HZ induced chemokine mRNA expression in sHZ on IFN-␥-mediated NO production was not reduced in the the murine M␾ cell line B10R. As depicted in Fig. 1A, when cells presence of Poly B and was not affected in TLR4-deleted M␾- were stimulated for 2 h with 10–75 ␮g/ml of either Pf HZ or its derived from LPS-unresponsive mice. In contrast, the ability of synthetic form, sHZ, a concentration-dependent increase in various LPS to induce NO synthesis was suppressed upon Poly B pretreat- http://www.jimmunol.org/ chemokine transcripts was detected. Maximal values over the neg- ment and was abolished in TLR4-deleted M␾ (9). In addition, we ative control were obtained for MIP-1␤ (3- and 4-fold), MIP-1␣ found that although LPS, at very low concentrations (0.1 and 1 (2- and 3-fold), MIP-2 (140- and 170-fold), and MCP-1 (3- and ng/ml), led to the up-regulation of various proinflammatory cyto- 4-fold) when 75 ␮g/ml Pf HZ or sHZ was added, respectively. In kine transcripts (IL-12, IL-1␣, IL-1␤, and IL-6), sHZ was unable addition, time-course experiments performed with an intermediate to do so even when added at 25 ␮g/ml (Fig. 2B). This set of concentration of sHZ (25 ␮g/ml) revealed that chemokine induc- experiments indicates the absence of LPS in the sHZ preparations by guest on October 7, 2021

FIGURE 1. PfHZ and sHZ increase chemokine mRNA levels in murine M␾. Cells were treated with 10–75 ␮g/ml PfHZ or sHZ for 2 h (A) or with 25 ␮g/ml sHZ over an 8-h period (B), and chemokine mRNA expression was monitored using an mCK5 multiprobe RPA system (left panels). Densitometric quantification of chemokine mRNA levels over the negative control values after normalization to GAPDH (right panels). f, PfHZ; u, sHZ. The results shown are representative of one of three independent experiments. 478 HZ-INDUCIBLE M␾ CHEMOKINE MODULATION

FIGURE 2. Synthetic HZ-dependent chemokine mRNA up-regulation is not due to LPS contamination. A, DMEM/10% FBS medium containing 100 ng/ml LPS or 25 ␮g/ml sHZ was incubated, or not, with 5–10 ␮g/ml ␾

Poly B for 30 min at 37°C. Then B10R M were stimulated with one of Downloaded from the various conditioned media for 2 h, and chemokine mRNA levels were monitored as described in Fig. 1. B, Cells were treated with increasing concentrations of either LPS (0.1–100 ng/ml) or sHZ (1–25 ␮g/ml) for 2 h. After total RNA extraction, samples were hybridized with a cytokine mul- tiprobe and subjected to RPA. The results shown are representative of one of three separate experiments. FIGURE 3. Kinetic analyses of sHZ-inducible MEK1/2 and ERK1/2 phosphorylation. After cell lysis, total proteins from either untreated or http://www.jimmunol.org/ sHZ-stimulated M␾ (0.5–4 h) were subjected to Western blotting. MEK1/2 and suggests that the two agonists exert different biological effects (A) and ERK1/2 (B) phosphorylation status was revealed with phospho- on M␾. MEK1/2 and phospho-ERK1/2 Abs, respectively. Equal protein levels were verified using MEK1/2 and ERK1/2 Abs. The results shown are rep- Activation of the ERK1/2 pathway is required for sHZ-inducible resentative of one of three independent experiments. chemokine modulation The role of ERK1/2 MAPK on chemokine induction is well doc- the nature of the sHZ-induced NF-␬B complex, supershift assays umented (22, 27). Therefore, we tested the ability of sHZ to acti- were performed using Abs directed at p50 and p65, two ubiquitous by guest on October 7, 2021 vate this signaling cascade. A transient phosphorylation of the im- members of the NF-␬B family. As illustrated in Fig. 5B, the com- mediate ERK1/2 upstream activator MEK1/2 (Fig. 3A) and of plex binding was diminished and partially supershifted in the pres- ERK1/2 (Fig. 3B) occurred after cell exposure to sHZ (25 ␮g/ml). ence of an anti-p50 Ab and was almost completely abrogated by an Both MEK1/2 and ERK1/2 phosphorylation were detected at 30 anti-p65 Ab. These data indicated that sHZ activates DNA binding min poststimulation and remained sustained for up to 2 h. Whereas of both p50 and p65 NF-␬B subunits in M␾. To address the ques- MEK1/2 phosphorylation declined thereafter, that of ERK1/2 was tion of whether sHZ not only led to NF-␬B nuclear translocation still detectable after 4 h. To investigate the involvement of the but also to its binding to one or more chemokine genes, nuclear ERK1/2 pathway in sHZ-mediated chemokine up-regulation, cells extracts from sHZ-stimulated cells were incubated with oligonu- were incubated for 1 h with increasing concentrations of specific cleotides containing specific sequences of the NF-␬B-binding sites inhibitors directed against MEK1/2 (PD 98059) and ERK1/2 (api- present in the murine MIP-2 and MCP-1 promoters. We observed genin) before sHZ stimulation (2 h). As shown in Fig. 4A,5␮M that after 1 h, sHZ led to maximal NF-␬B binding to both sites. PD 98059 inhibited the expression of all four chemokines. In cor- Whereas NF-␬B binding to the MIP-2 sequence was sustained for relation with these observations, cell exposure to 20 ␮M apigenin up to 4 h, NF-␬B binding to the MCP-1 promoter was transient, resulted in a marked reduction of their chemokine transcripts (Fig. but still detectable at 4 h poststimulation (Fig. 5C). Given that 4B; ϳ45% for MIP-1␤, ϳ50% for MIP-1␣, ϳ60% for MIP-2, and specific blockage of the ERK1/2 pathway abolished chemokine ϳ40% MCP-1), whereas maximal inhibitor concentrations (40 expression, we next determined its involvement in sHZ-inducible ␮M) completely abrogated chemokine expression. These results I␬B␣ phosphorylation and subsequent NF-␬B nuclear transloca- indicate that ERK1/2-dependent signals are necessary for M␾ che- tion. As illustrated in Fig. 6A, sHZ led to a rapid phosphorylation mokine modulation in response to the malarial pigment. of I␬B␣ (15 min poststimulation). This intracellular event ap- peared to be under the control of MEK1/2-ERK1/2-dependent sig- ␬ Involvement of the transcription factor NF- B on sHZ- nals, because specific inhibitors directed against these MAPKs ab- dependent chemokine induction rogated the effect of sHZ on I␬B␣. Similarly, cell exposure to NF-␬B proteins control the expression of multiple genes involved either apigenin or PD 98059 caused a concentration-dependent in inflammatory processes such as those encoding chemokines diminution of NF-␬B nuclear translocation (Fig. 6B). These data (28). Of interest, NF-␬B binding sites have been found in the pro- prompted us to further evaluate the role of NF-␬B in chemokine moter regions of several murine chemokine genes, including those modulation. As a control experiment, M␾ were incubated for 1 h of MIP-2 (23) and MCP-1 (24). Based on these observations, we with BAY 11-7082 (1 or 5 ␮M), an inhibitor of I␬B␣ phosphor- sought to establish whether this NF was activated in M␾ by sHZ. ylation (29), before sHZ stimulation, and NF-␬B nuclear translo- As shown in Fig. 5A, NF-␬B nuclear translocation was maximal at cation was monitored by EMSA (Fig. 6C). As expected, cells in- 1 h poststimulation and then decreased over a 4-h period. To define cubated with BAY 11-7082 showed a concentration-dependent The Journal of Immunology 479 Downloaded from http://www.jimmunol.org/

FIGURE 4. The ERK1/2 pathway is involved in chemokine mRNA induction in response to sHZ. M␾ were incubated for 1 h with increasing con-

centrations of either PD 98059 (A) or apigenin (B) before sHZ stimulation (25 ␮g/ml for 2 h), and their effects on chemokine mRNA expression were by guest on October 7, 2021 evaluated by RPA (left panels). Integrated density values of chemokine mRNA levels were normalized to GAPDH (right panels). The results shown are representative of one of three separate experiments. Ⅺ, Nil, untreated; f, sHZ with or without PD 98059 or apigenin.

reduction in the binding of the NF-␬B complex. We were thus dated whether sHZ-mediated ERK1/2 phosphorylation, NF-␬B ac- interested in examining the effect of this inhibitor on chemokine tivation, and chemokine modulation were due to ROS generation. induction. To this end, after cell stimulation, as described in the Whereas SOD pretreatment (300 U/ml) did not affect ERK1/2 previous experiment, total RNA was extracted and subjected to phosphorylation (data not shown), NF-␬B nuclear translocation in RPA analysis. As illustrated in Fig. 6D,M␾ exposure to 5 ␮M response to sHZ was strongly reduced in the presence of this en- BAY 11-7082 abrogated the expression of all four transcripts. zyme (Fig. 7C). Importantly, cell incubation with 300 U/ml SOD These data suggest that chemokine modulation in response to sHZ abrogated the expression of all chemokine transcripts (Fig. 7D). involves ERK1/2-dependent NF-␬B activation. This set of experiments demonstrated that the malarial pigment leads to a mild oxidative stress in M␾, which seems to be in part Chemokine mRNA up-regulation is mediated through sHZ- responsible for NF-␬B activation and is required for sHZ-inducible inducible ROS generation chemokine mRNA up-regulation. M␾ from Pf-infected mice were shown to release oxygen metab- ␾ olites (30) and in vitro oxidative killing of Pf was described in M PTP inactivation in response to sHZ is ROS dependent peritoneal M␾ (31). Similarly, in response to Pf HZ (32) or sHZ Intracellular signaling is regulated by the equilibrium between pro- (33), peritoneal M␾ produced ROS. Therefore, we assessed tein-tyrosine kinase (PTK) and PTP activity. Knowing that PTP ␾ . whether sHZ generated ROS in B10R M (Fig. 7A). When cells inactivation occurs in response to ROS, including O2 (35, 36) and ␮ were stimulated with 10–50 g/ml sHZ, a concentration-depen- H2O2 (37), the ability of sHZ to reduce PTP activity was examined dent induction of ROS was detected, which was statistically sig- as a possible mechanism explaining M␾ signal transduction acti- nificant compared with that in untreated M␾ (OD values) and was vation. To this end, time-course experiments were conducted in the equivalent to 30% of the oxidative stress generated upon admin- presence of sHZ (25 ␮g/ml) from 5 min to 4 h, and M␾ PTP ␮ istration of exogenous H2O2 (500 M). In addition, we found that activity was monitored. As shown in Fig. 8A, sHZ rapidly down- most of the ROS produced by sHZ-stimulated M␾ corresponded to regulated M␾ PTP activity, leading to a significant diminution . ϳ superoxide anion (O2). As shown in Fig. 7B, in the presence of ( 40% reduction compared with basal activity) after a 30-min . ϳ increasing concentrations of SOD, an that detoxifies O2 treatment and a more dramatic decrease ( 55%) after 2 h. Next, (34), sHZ-inducible ROS were significantly reduced, reaching we evaluated whether the effect of sHZ on M␾ PTP activity was 91% inhibition when 300 U/ml SOD was added. Next, we eluci- due to its ability to induce ROS. When cells were treated with 480 HZ-INDUCIBLE M␾ CHEMOKINE MODULATION Downloaded from http://www.jimmunol.org/

FIGURE 5. Synthetic HZ leads to NF-␬B nuclear translocation and binding to the murine MIP-2 and MCP-1 promoters. A, Nuclear extracts from M␾, either left untreated or stimulated with 25 ␮g/ml sHZ for different time periods (0–4 h), were incubated with a ␥-32P-labeled NF-␬B probe and subjected to EMSA. B, For supershift assays, nuclear extracts from cells stimulated with 25 ␮g/ml sHZ (1 h) were incubated, or not, with specific Abs against the p50 and p65 NF-␬B isoforms for 1 h before EMSA. C, EMSA analyses were performed as described in A, but nuclear proteins were incubated with one of the NF-␬B probes specific for the murine MIP-2 (upper panel) and MCP-1 (lower panel) promoters. Binding specificity was tested by adding to nuclear by guest on October 7, 2021 extracts from 1-h treated cells a 100-fold molar excess of either a cold NF-␬B consensus, NF-␬B/MIP-2, or NF-␬B/MCP-1 oligonucleotide (100ϫ spec.) or a nonspecific Oct-2A probe (100ϫ non-spec.). The results shown are representative of one of three independent experiments.

75–300 U/ml SOD before sHZ stimulation, M␾ PTP activity was rapid chemokine production. In this context, HZ could stimulate restored in a concentration-dependent manner, reaching up to 93% the immunological response at sites of parasite sequestration by of basal PTP activity in the presence of 300 U/ml SOD (Fig. 8B). inducing potent chemoattractants and activators. However, at Collectively, these data indicate that M␾ PTP inactivation in re- higher parasitemias and subsequently higher HZ concentrations, sponse to sHZ is an early event that seems to be dependent on the malarial pigment might also contribute to an exacerbated oxidative stress. proinflammatory response (e.g., leukocytosis and high ROS pro- duction), causing tissue damage and microvascular flow Discussion disturbance. ␾ In Pf malaria, M have been proposed as one of the main sources Our results, demonstrating the capacity of sHZ to trigger M␾ of proinflammatory mediators (2); therefore, it is of paramount oxidative stress, are in line with previous studies reporting the importance to identify the parasite components participating in same phenomenon after M␾ treatment with Pf HZ (32) or sHZ chemokine induction and to define the underlying regulatory (33). However, these studies associated HZ-inducible ROS with mechanisms. In this regard, Sherry et al. (12) reported that both Pf depression of M␾ functions. Human monocyte-derived M␾ fed HZ and sHZ lead to the release of chemokines MIP-1␣ and with Pf HZ displayed a long-lasting oxidative burst, but were un- MIP-1␤ in murine M␾ and human PBMC. More recently, we dem- able to repeat the phagocyte cycle. Moreover, their ability to gen- onstrated that sHZ also induces chemokine transcription and se- cretion in vivo (10). Confirming and extending these previous find- erate oxidative stress in response to PMA was irreversibly sup- ings, we show that either Pf HZ or sHZ increases mRNA levels of pressed (32). Similarly, the ability of sHZ to reduce LPS-mediated ␾ MIP-1␣, MIP-1␤, MIP-2, and MCP-1 in B10R murine M␾ cytokine and NO production in M was attributed to sHZ-induc- through a mechanism that is oxidative stress and ERK1/2 depen- ible oxidative stress (33). In contrast, we found that ROS appear to dent and involves PTP down-regulation and NF-␬B activation. be one of the mechanisms through which the malarial pigment Chemokine up-regulation was detected at early times of stimula- leads to M␾ chemokine induction. These inconsistencies probably tion and occurred in a concentration-dependent manner. These data stem from the fact that, as discussed by others (33) and by us (9), suggest that upon its release into the bloodstream, HZ might in- the effects of HZ most likely depend on cell susceptibility to ox- teract with resident M␾ and circulating monocytes and lead to idative stress, which can vary according to the cell type as well as The Journal of Immunology 481 Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021

FIGURE 6. Role of NF-␬BinM␾ sHZ-dependent chemokine mRNA up-regulation. Total proteins from untreated or sHZ-stimulated M␾ (15 min), pretreated, or not, with apigenin or PD98059 (␮M), were subjected to Western blotting, and I␬B␣ phosphorylation was monitored with a phospho-I␬B␣ Ab. I␬B␣ protein levels were monitored using an I␬B␣ Ab (A). Labeled NF-␬B probe was incubated with nuclear extracts from either untreated or sHZ-stimulated cells (25 ␮g/ml for 1 h), pretreated, or not, with increasing concentrations (micromolar) of apigenin, PD 98059 (B), or BAY 11-7082 (C), and EMSA was performed. Binding specificity was tested by adding to nuclear extracts from 1-h treated cells a 100-fold molar excess of cold NF-␬B oligonucleotide or a nonspecific Oct-2A probe. D, After a 1-h exposure to BAY 11-7082, M␾ were additionally stimulated with 25 ␮g/ml sHZ for 2 h. Then, total RNA was extracted, and changes in chemokine mRNA levels were monitored by RPA. Ⅺ, Nil, untreated; f, sHZ with or without BAY 11-7082. These results are representative of one of three separate experiments.

its tissue source. Whereas HZ was shown to exert a down-regu- pression of various chemokine transcripts and enhanced their pro- lating effect on NO production by peritoneal M␾, it did not mod- duction in response to infection (41). In parallel, we detected a ulate this activity in microglial cells (33), and it was able to in- greater chemokine induction in M␾ deficient in Src homology re- crease it in bone marrow-derived M␾ (9). In this context, it is gion 2 domain-containing phosphatase-1, one of the major M␾ conceivable that rather than a unique effect, HZ might lead to a PTPs (42), compared with wild-type M␾ (our unpublished obser- localized negative or positive ROS-mediated regulation of M␾ vations). In light of these findings, it seems plausible to propose function depending on their antioxidant defenses. ROS-mediated PTP inactivation as a possible mechanism for HZ- In addition to its capacity to activate ROS, sHZ caused a marked inducible M␾ chemokine modulation. Even though the M␾ PTP(s) ␾ reduction of M PTP activity. Moreover, in the presence of SOD, targeted by sHZ remains to be identified, a general explanation that an enzyme involved in O. catabolism (34), basal PTP activity was 2 could account for our results is that upon HZ stimulation, the in- almost completely restored, indicating that this down-regulatory tracellular equilibrium, in which basal PTP activity predominates event depends mostly on sHZ-inducible O. generation. Consistent 2 over that of PTK, would be altered toward a higher PTK activity with this, PTPs possess reduction-oxidation (redox)-sensitive cys- due to an increase in ROS and subsequent reduction of PTP ac- teine residues in their active sites (38), and their activity is inhib- . tivity via their redox-sensitive residues. ited in the presence of either O2 (35, 36) or H2O2 (37). Of interest, our in vitro data revealed that peroxovanadium (bpV (phen)), a Current data provide evidence that a mild oxidative stress can potent and selective PTP inhibitor (39), suppressed basal M␾ act as a second messenger leading to redox-responsive transcrip- phosphatase activity (40) and increased chemokine mRNA levels tion factor activation and subsequent chemokine gene expression in B10R M␾ (our unpublished observations). Moreover, we dem- (22, 43). In agreement with these studies, we found that chemokine onstrated that PTP inhibition strongly up-regulated the in vivo ex- modulation in response to sHZ required nuclear translocation and 482 HZ-INDUCIBLE M␾ CHEMOKINE MODULATION Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021

FIGURE 7. HZ-mediated chemokine induction is dependent on ROS generation. A, Cells were stimulated with either 10–50 ␮g/ml sHZ or with 500 ␮M f u H2O2 for 1 h, and ROS generation was measured by fluorescence at 532 nm. , sHZ; ,H2O2. The results shown are representative of one of three Ͻ ␾ ء Ϯ ϭ independent experiments performed in triplicate (mean SEM; n 3). , p 0.05, sHZ or H2O2 vs Nil (OD measurements). B,M , pretreated, or not, with SOD (0–300 U/ml) for 1 h, were incubated with 25 ␮g/ml sHZ, and ROS generation was monitored after 1 h. The data presented are representative p Ͻ 0.05, sHZ plus SOD vs sHZ (OD measurements). C, Cells were ,ء .(of one of three separate experiments performed in triplicate (mean Ϯ SEM; n ϭ 3 stimulated as described in B, but this time nuclear proteins were extracted, and after incubation with a radiolabeled NF-␬B consensus probe, they were subjected to EMSA. D, After M␾ stimulation with 25 ␮g/ml sHZ (2 h) in either the absence or then presence of SOD (0–300 U/ml), total RNA was extracted, and chemokine mRNA modulation was evaluated by RPA. Ⅺ, Nil, untreated; f, sHZ with or without SOD. The results shown are representative of three independent experiments. binding of the redox-sensitive NF-␬B to murine chemokine pro- IKK␤, and the p65 NF-␬B subunit (50). Thus, according to this moters. Importantly, blockage of oxidative stress with SOD sig- evidence and the knowledge that IKKs phosphorylate p65 (51), it nificantly reduced NF-␬B translocation, indicating a key role for O is plausible that IKK activation is required for sHZ-inducible p65 . 2 generation in this signaling event. Our previous work (44) dem- nuclear translocation. onstrated the induction of NF-␬B activity in response to specific Blockage of ROS with SOD markedly down-regulated NF-␬B PTP inhibitors, and others reported that SOD pretreatment abro- nuclear translocation in response to the malarial pigment, but did gated M␾ I␬B␣ tyrosine phosphorylation (45) and NF-␬B activa- not abolish it, suggesting that ROS-independent mechanisms are tion (46) by silica with or without pervanadate. Therefore, it can be also involved. Analysis of possible alternative second messengers ␬ . envisaged that NF- B induction by sHZ involves O2-dependent revealed that sHZ leads to rapid phosphorylation of ERK1/2 PTP inactivation and subsequent PTK up-regulation. This is in MAPK. Because this event was not affected by SOD administra- concert with the demonstration that hypoxia/reoxygenation acti- tion (data not shown), these data indicate that ERK1/2 activation is ␬ ␬ ␣ . vates NF- B via tyrosine phosphorylation of I B (47), which is not dependent on O2 generation. Consistent with previous studies mediated by the PTK c-Src (48) and is dependent on the PTK Syk suggesting the involvement of MEK/ERK1/2 in NF-␬B activation ␬ ␣ in response to H2O2 (49). Even though ROS do not phosphorylate (52–54) and chemokine modulation (22, 27), sHZ-inducible I B I␬B␣ on its serine residues, they induce I␬B kinase (IKK) activa- serine phosphorylation, NF-␬B nuclear translocation, as well as tion as well as p65 serine phosphorylation and nuclear transloca- chemokine up-regulation were impaired in the presence of specific tion (49). Moreover, it was recently reported that SOD treatment inhibitors of the ERK1/2 pathway. These results are perfectly in . ␣ reversed increased O2 levels and prevented the activation of IKK , line with our published data showing that the up-regulating effect The Journal of Immunology 483

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