Potential Role in Malaria Infection Hemozoin-Inducible Proinflammatory Events

Hemozoin-Inducible Proinflammatory Events In Vivo: Potential Role in Malaria Infection Maritza Jaramillo, Isabelle Plante, Nathalie Ouellet, Karen Vandal, Philippe A. Tessier and Martin Olivier This information is current as of September 24, 2021. J Immunol 2004; 172:3101-3110; ; doi: 10.4049/jimmunol.172.5.3101 http://www.jimmunol.org/content/172/5/3101 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Hemozoin-Inducible Proinﬂammatory Events In Vivo: Potential Role in Malaria Infection1

Maritza Jaramillo,* Isabelle Plante,* Nathalie Ouellet,* Karen Vandal,* Philippe A. Tessier,* and Martin Olivier2*†

During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), ␤-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, Downloaded from evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1␣/CCL3, MIP-1␤/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/ CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1␤ and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria. The Journal of Immunology, 2004, 172: http://www.jimmunol.org/ 3101Ð3110.

he human malarial parasite Plasmodium falciparum is re- including severe anemia, cerebral malaria (CM),3 cytoadherence, sponsible for 300–500 million clinical cases and 1–3 mil- and tissue cell damage (3, 7). In addition to proinflammatory cy- T lion deaths annually, mostly of children (1). Severe dis- tokines, increased serum concentrations of strong leukocyte che- ease and high mortality associated with malaria mainly have been moattractants and activators; myeloid-related proteins (MRPs), attributed to parasitic virulence factors (e.g., cytoadherence, roset- S100A8, S100A9, and S100A8/A9 (8, 9); and chemokines, IL-8 and macrophage-inflammatory protein (MIP)-1␣/CC chemokine ting, multiplication rate); however, different lines of evidence sug- by guest on September 24, 2021 gest that the host’s immunological response could also contribute ligand (CCL) 3 (10), have been observed in patients suffering from ␣ to malaria pathophysiology (2). Elevated serum concentrations of acute P. falciparum malaria. Furthermore, elevated MIP-1 , IL-1, IL-6, IFN-␥, and TNF-␣ have been detected in P. falciparum monocyte chemoattractant protein (MCP)-1/CCL2, and IL-8 levels were recently found during placental malaria (11). malaria patients, and their levels correlated with disease severity In addition to data from P. falciparum-infected patients, cyto- (3–6). Even though these cytokines appear to play a pivotal role in kine (e.g., TNF-␣, IFN-␥, IL-6) and chemokine (e.g., MCP-1, IFN- parasite control, it has been proposed that an exacerbated inflam- ␥-inducible protein-10 (IP-10), RANTES) induction has also been matory response could also participate in malaria pathogenesis, reported in experimental models of murine malaria (12–14), in which it appears to be involved in both protective immunity and pathogenesis. Moreover, in vitro studies have provided evidence of leukocyte activation during malaria infection. In response to Plas- *Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Quebec, modium, macrophages (M␾) have been found to secrete TNF-␣, Pavillon Centre Hospitalier de l’Universite´ Laval, and De´partement de Biologie IL-1␤, IL-6, and IL-8 (15), and dendritic cells to synthesize IL-6 Me´dicale, Faculte´deMe´decine, Universite´ Laval, Ste-Foy, Quebec, Canada; and †Research Institute of the McGill University Health Centre, Centre for the Study of and IL-12 (16). Host Resistance, Departments of Medicine, Microbiology, and Immunology, McGill Even though proinflammatory cytokine and chemokine produc- University, Montreal, Quebec, Canada tion during malaria are well documented, the direct contribution of Received for publication September 8, 2003. Accepted for publication December specific parasite components to such induction has not yet been 22, 2003. fully unraveled. Because the classic malaria paroxysm character- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ized by fever and chills is associated with schizogeny (3), the with 18 U.S.C. Section 1734 solely to indicate this fact. ability of parasite metabolites GPI and hemozoin (HZ), released 1 This work was supported by grants from the Canadian Institutes in Health Research during this process, to stimulate the host immune response has to M.O., who is member of a Canadian Institutes in Health Research Group in Host- been investigated. In this regard, Schofield and Hackett (17) dem- Pathogen Interactions. M.O. is a recipient of a Canadian Institutes in Health Research ␣ Investigator Award and is a Burroughs Welcome Fund awardee in Molecular Para- onstrated that the P. falciparum GPI moiety induces both TNF- sitology. P.A.T. is a recipient of a scholarship award from Fonds de la Recherche en Sante´du Quebec. M.J. is a recipient of a Ministe`redel’E´ ducation du Quebec Ph.D. studentship. 3 Abbreviations used in this paper: CM, cerebral malaria; CCL, CC chemokine ligand; 2 Address correspondence and reprint requests to Dr. Martin Olivier, McGill Univer- HZ, hemozoin; IP-10, IFN-␥-inducible protein-10; MCP, monocyte chemoattractant sity, Department of Microbiology and Immunology, 3775 University Street, Duff protein; mCR, murine chemokine receptor; MIP, macrophage-inflammatory protein; Medical Building (Room 600), Montreal, Quebec, Canada H3A 2B4. E-mail address: MRP, myeloid-related protein; M␾, macrophage; N␾, neutrophil; RPA, RNase pro- [email protected] tection assay; sHZ, synthetic HZ.

and IL-1 in M␾, and when administered to mice, the parasite GPI Heme quantitation leads to cytokine release, transient pyrexia, and hypoglycemia. Total heme content was determined, as described by Sullivan et al. (27), by HZ or malarial pigment is a polymer of heme produced by the depolymerizing heme polymer in 1 ml of 20 mM NaOH/2% SDS, incu- parasite during hemoglobin digestion inside the host RBC (18). bating the suspension at room temperature for 2 h, and then reading the OD This pigment is released along with merozoites as the RBC bursts at 400 nm (Beckman DGB UV/visible spectrophotometer (Beckman ␮ and is avidly phagocytosed by circulating monocytes, neutrophils Coulter, Fullerton, CA)). A total of 25 g of sHZ equals 26 nmol of heme content. (N␾), and resident M␾ (reviewed by Arese and Schwarzer (19)). Formerly, HZ was considered only as a waste product of the par- Air pouch experiments asite resulting from heme detoxification; however, several lines of Air pouches were raised on the dorsum of BALB/c mice by s.c. injection evidence strongly suggest that it could play an important role in of 3 ml of sterile air on days 0 and 3, as we previously described (28). On cytokine production and parasite cytoadherence during malaria in- day 7, mice were injected with either 5–50 ␮g/ml sHZ in 1 ml of endo- fection. In vitro studies showed that either Plasmodium HZ or toxin-free PBS for6hor50␮g/ml sHZ over a 24-h period. It should be synthetic HZ (sHZ), which is structurally identical with the native noted that the amounts of injected sHZ are not solubilized, but are the suspension of insoluble material. Control group mice were treated for 6 h pigment (20), induces the release of various proinflammatory me- with either 1 ml of endotoxin-free PBS or 10 ␮g/ml LPS, as negative and diators, including IL-1␤, TNF-␣ (21), MIP-1␣, and MIP-1␤/CCL4 positive controls, respectively. At specific times after intrapouch inocula- (22), in either murine M␾ or human monocytes, as well as adhe- tion (0, 1, 3, 6, 12, 24 h), five animals per experimental group were lethally sion molecule expression and IL-6 production in human endothe- exposed to CO2 and the pouches were washed with a total of 5 ml of lial cells (23). These findings have been supported by those of endotoxin-free PBS/1 mM of EDTA. Recruited leukocytes in the pouch exudates were counted directly with a hemacytometer following acetic blue Biswas et al. (24), who reported the presence of Abs against P. staining. Differential counts of leukocyte subpopulations were performed Downloaded from falciparum HZ among complicated malaria patients, which had on cytospin preparations stained with Wright-Giemsa (Diff-Quik; Baxter inhibitory effects on HZ-mediated TNF-␣ and IL-1␤ production by Healthcare, Deerfield, IL). Then exudates from five animals per experi- ϫ human monocytes. Moreover, we recently found that both P. fal- mental group were pooled and centrifuged at 1200 rpm 10 min at room ␥ ␾ temperature. Total RNA was isolated from recruited cells with TRIzol ciparum HZ and sHZ increase IFN- -mediated M NO generation reagent (Life Technologies), according to the manufacturer’s protocol, for through specific signaling pathways (25). Finally, in vivo studies further cytokine, chemokine, and chemokine receptor mRNA analysis. Pro-

revealed that sHZ has the ability to regulate body temperature in teins in the collected supernatants were precipitated with acetone (1:4 v/v) http://www.jimmunol.org/ rats (22), suggesting that this parasite metabolite could contribute at Ϫ20°C overnight. Following centrifugation at 3000 rpm ϫ 10 min at ␮ Ϫ to fever during acute malaria. 4°C, pellets were resuspended in 100 l of PBS and were kept at 20°C for further ELISA analysis. In the present study, we sought to investigate the proinflammatory properties of sHZ in vivo. Using the murine air pouch model Intravenous experiments in BALB/c mice, we demonstrate that sHZ injection causes a signif- BALB/c mice were injected i.v. into the tail vein with 200, 750, or 1500 ␮g icant leukocyte recruitment in the pouch lumen (mainly N␾ and of sHZ in 100 ␮l of endotoxin-free PBS (suspension of insoluble material). monocytes), which is accompanied by increased levels of various che- Negative control mice were either left untreated or injected with 100 ␮lof mokines (MIP-1␣, MIP-1␤, MIP-2/CXC ligand 2, and MCP-1), CCR endotoxin-free PBS. As positive controls, mice were inoculated with 10 ␮g and CXCR (CCR1, CCR2, CCR5, CXCR2, and CXCR4), proin- of LPS. After specific times (6 h for dose-response experiments; and 1, 3, by guest on September 24, 2021 6, 12, or 24 h for kinetic analyses), mice were lethally exposed to CO . ␣ ␤ 2 flammatory cytokines (IL-1 , IL-1 , and IL-6), and murine MRPs Sections of liver tissue were ground in TRIzol to isolate total RNA for (S100A8, S100A9, and S100A8/A9). In addition, i.v. sHZ inocu- subsequent RNase protection assays (RPA). lation revealed that the malarial pigment leads to a dose-dependent mRNA up-regulation of liver chemokine (MIP-1␣, MIP-1␤, RNase protection assays (RPA) MIP-2, and MCP-1) and cytokine (IL-1␤ and IL-12) expression. mRNA expression studies were performed using a RPA kit (Riboquant; Taken together, our data suggest that HZ could play an important BD PharMingen, San Diego, CA), as we described previously (28). One of ␣ 32 role in malaria immunopathology related to proinflammatory me- the various multiprobe templates was labeled with [ - P]dUTP using T7 RNA polymerase. A total of 3 ϫ 105 cpm of labeled probe was allowed to diator overproduction. hybridize with 10 ␮g of total RNA, isolated as described above, for 16 h at 56°C. mRNA probe hybrids were treated with RNase A and phenol- chloroform extracted. Protected hybrids were resolved on a 5% denaturing Materials and Methods polyacrylamide sequencing gel and exposed to radiographic film overnight Materials at Ϫ80°C. Laser densitometry was performed using an ␣ Imager 2000 digital imaging and analysis system (Alpha Innotech, San Leandro, CA). Hemin chloride and LPS (Escherichia coli, serotype 0111:B4) were pur- ␣ 32 The Multiprobe templates (BD PharMingen) used in this study were as chased from Sigma-Aldrich (St. Louis, MO). Isotope [ - P]dUTP (3000 follows: mouse chemokine-5, for the murine chemokines lymphotactin, Ci/mmol) was purchased from PerkinElmer (Wellesley, MA). Six- to RANTES, eotaxin, MIP-1␣, MIP-1␤, MIP-2, IP-10, MCP-1, and TCA-3; 8-wk-old female BALB/c mice, 20–30 g body weight, were obtained from murine chemokine receptor 5 (mCR5), for the murine chemokine receptors Charles River Breeding Laboratories (St. Colomban, Quebec, Canada). CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5; mCR6, for CXCR2, CXCR4, and BLR-1; and a custom template for murine cytokines IL-4, ␤-hematin (sHZ) preparation IL-12p40, IL-10, IL-1␣, IL-1␤, IL-2, IL-6, and IFN-␥. In addition, the template sets included housekeeping genes mL-32 and/or GAPDH. The method for ␤-hematin synthesis described by Egan et al. (26) was adapted, as we previously described (25). Briefly, 45 mg of hemin chloride Determination of chemokine and cytokine protein expression (Sigma-Aldrich) was solubilized in 4.5 ml of 1 N NaOH and neutralized with 450 ␮l of 1 N HCl. Then 10.2 ml of 1 M sodium acetate, pH 4.8, was Sandwich ELISAs were established for MIP-1␣, MIP-2, MCP-1, IL-1␤, added and the suspension was stirred with a magnet for 2–3hat60°C. and IL-6 to detect these chemokines and proinflammatory cytokines in the Following addition of 1/100 vol of 10% SDS and 14,000 ϫ g centrifuga- supernatant of pouch exudates. Briefly, 96-well plates (Immunoplate; tion for 15 min, the pellet was sonicated at lowest setting in 100 mM of Nunc, Naperville, IL) were coated with anti-MIP-1␣, anti-MIP-2, anti- sodium bicarbonate, pH 9.0, 0.5% SDS, and again centrifuged. The pellet MCP-1, anti-IL-1␤, or anti-IL-6 (R&D Systems, Minneapolis, MN) in 100 was then washed three to four times in 2% SDS and then in water to wash ␮l/well of PBS, pH 7.4. Plates were incubated for 16 h at 4°C and were out SDS. The pigment was dried at 37°C overnight, resuspended in endo- blocked with PBS/5% BSA. Next, 50 ␮l of PBS/2% BSA was added to toxin-free PBS (Life Technologies, Rockville, MD) at a final concentration each well, followed by the addition of 50 ␮l/well of standards (R&D Sys- of 2.5 mg/ml, and kept at Ϫ20°C until further use. Absence of contami- tems) or samples diluted in PBS/2% BSA and incubation for2hatroom nation by endotoxins was confirmed by performing the Limulus amebocyte temperature. The biotinylated polyclonal goat anti-chemokine/cytokine Ab lysate test, E-toxate kit (Sigma-Aldrich). (100 ␮l/well) was added and incubated for1hinPBS/2% BSA. Bound The Journal of Immunology 3103

FIGURE 1. sHZ induces leukocyte recruitment in vivo. A, Number of leukocytes accumulating in the air pouch of BALB/c mice in response to p Ͻ 0.05, sHZ or LPS Downloaded from ,ء .(increasing doses of sHZ (5–50 ␮g/ml), LPS (10 ␮g/ml), or endotoxin-free PBS (1 ml) after 6 h. PBS (Ⅺ), LPS ( ), sHZ (f vs PBS. B, Kinetics of total leukocyte recruitment in response to sHZ (50 ␮g/ml) over a 24-h period (1, 3, 6, 12, 24 h). Six-hour LPS- and PBS-treated p Ͻ 0.05, sHZ vs PBS. Total cell counting was performed directly by using a ,ء .mice were used as positive and negative control groups, respectively hemacytometer. Results represent mean ϩ SEM of ﬁve mice. Data are representative of one of three independent experiments.

biotinylated anti-chemokine/cytokine Ab was detected by streptavidin-per- lected and the total number of leukocytes accumulating in response oxidase conjugate and further addition of 100 ␮l 3,3Ј,5,5Ј-tetramethyl- to the various stimuli was determined. As shown in Fig. 1A, sHZ http://www.jimmunol.org/ benzidine substrate (Research Diagnostics, Flanders, NJ). The limit of de- led to a significant and dose-dependent leukocyte recruitment, tection for MIP-1␣, MIP-2, MCP-1, IL-1␤, and IL-6 was 20–500 pg/ml. reaching up to a 15-fold increase over negative control mice when MRP secretion in the air pouch 50 ␮g/ml sHZ was inoculated. Of interest, these values were found For murine S100A8, S100A9, and S100A8/A9, ELISAs were performed, to be very close to those obtained when LPS was administered, as we previously described (29). Briefly, Costar high binding 96-well further indicating that the malarial pigment is a strong leukocyte plates (Corning Glass, Corning, NY) were coated overnight at 4oC with recruiter. In addition, time course experiments revealed that the 100 ␮l/well of purified rabbit IgG against S100A8 or S100A9 (for S100A9 sHZ-inducible leukocyte accumulation in the air pouches was tran- and S100A8/A9) diluted to 1 ␮g/ml in 0.1 M of carbonate buffer, pH 9.6.

sient over a 24-h period, being maximal at 6 h, still significant by guest on September 24, 2021 The wells were blocked with PBS/0.1% Tween 20/2% BSA (150 ␮l/well) for 30 min at room temperature. Then the samples and standards (100 ␮l) following a 12-h stimulation, and declining thereafter (Fig. 1B). By were added, and after a 45-min period at room temperature, the plates were performing differential cell counts of leukocyte subpopulations, we incubated with rat IgG (100 ␮l/well) against S100A8 (for S100A8 and S100A8/A9) or S100A9 diluted in PBS/0.1% Tween 20/2% BSA (1/10,000) for 45 min. To reveal the immune complex, 100 ␮l/well of peroxidase-conjugated goat anti-rat IgG (H ϩ L) (minimum cross-reaction to rabbit serum proteins) (1/10,000) was added and incubated for 45 min. Next, 100 ␮l/well of 3,3Ј,5,5Ј-tetramethylbenzidine substrate (Research Diagnostics) was added, according to the manufacturer’s instructions, and the ODs were read at 500 nm. The lower limit of quantification was determined as 4 ng/ml for both S100A8 and S100A9, and 10 ng/ml for S100A8/S100A9. All ELISAs were tested using excess amounts of the other S100 proteins and were shown to be specific under the conditions reported in this work. Statistical analysis Statistically significant differences between groups were determined by ANOVA module of Statview ϩ SE Software (Abacus Concepts, Berkeley, CA) and the Fisher least significant difference test. Values of p Ͻ 0.05 were deemed statistically significant. All data are presented as mean ϩ SEM. Results sHZ induces leukocyte recruitment in the air pouch To investigate the ability of sHZ to act as a proinflammatory agent in vivo, the air pouch model was chosen. As it has been shown by others (30) and by us (28), this provides a suitable isolated envi- ronment to study the proinflammatory properties of a given stim- FIGURE 2. Leukocyte subtypes accumulating in the air pouch in re- ulus. To this end, air pouches were raised on the dorsum of sponse to sHZ. Number of monocytes, neutrophils, and lymphocytes re- BALB/c mice, and 7 days later they were inoculated with increas- cruited in the pouch exudates in response to sHZ (50 ␮g/ml) at different ␮ ing doses of sHZ (5–50 g/ml), which we previously reported to time points: 1, 3, 6, 12, and 24 h. Differential cell counts were performed be effective in vitro (25). As positive and negative control groups, on Wright-Giemsa-stained cytospin preparations. Results represent mean p Ͻ 0.05, sHZ 6/12 h vs sHZ 1 h. Data are ,ء .mice were injected with LPS (10 ␮g/ml) and endotoxin-free PBS, ϩ SEM of five mice respectively. Following a 6-h treatment, pouch exudates were col- representative of one of three separate experiments. 3104 IN VIVO MODULATION OF PROINFLAMMATORY MEDIATORS BY HZ found that most of the infiltrated cells in response to sHZ were N␾, sHZ-mediated chemokine up-regulation in the air pouch which corresponded to 88 and 73% of the total cell numbers Chemokines are small peptides leading to activation and recruit- present at 6 and 12 h, respectively. However, following a 12-h ment of selected leukocyte populations to the sites of infection. treatment, sHZ also induced a significant rise in the number of These proteins are known to play important roles in protozoan monocytes, which accounted for 21% of the total recruited leuko- parasite infections by affecting interactions between parasites and cytes (Fig. 2). their host cells, as well as by influencing the immune system, sHZ increases cytokine mRNA and protein expression in vivo thereby further inducing inflammatory diseases (31). Therefore, we sought to examine the induction patterns of chemokine tran- To establish whether sHZ-mediated leukocyte accumulation cor- scripts in leukocytes from pooled exudates of sHZ-stimulated mice related with proinflammatory cytokine up-regulation, BALB/c (1–12 h). In correlation with the leukocyte populations migrating mice were inoculated with 50 ␮g/ml sHZ over a 24-h period, and into the pouches, RPA analyses revealed a significant and time- pouch exudates from each experimental group were pooled. Then dependent mRNA up-regulation of N␾ (MIP-2 (32)) and mono- total RNA from the recruited leukocytes was extracted, and cyto- cyte (MIP-1␣, MIP-1␤, and MCP-1 (31)) chemoattractants and kine mRNA levels were monitored using a cytokine multiprobe activators (Fig. 4A). Maximal induction for MIP-1␣, MIP-1␤, and RPA system. As shown in Fig. 3A, sHZ led to a rapid and transient accumulation of various cytokine transcripts. Whereas IL-1␣ MIP-2 was observed at 3 h poststimulation, reaching a 16-, 32-, mRNA up-regulation was maximal after 6 h (14-fold increase over and 188-fold increase over negative control mice, respectively. PBS control mice), both IL-1␤ and IL-6 gene expression peaked Even though a slight rise in MCP-1 mRNA levels was detected at already at 3 h posttreatment, reaching a 9- and 20-fold increase 3 h, maximal messenger accumulation (26-fold over PBS control) Downloaded from over negative control, respectively. In contrast, sHZ was unable to was found only after6hofsHZinoculation. Even though multiple modulate IL-4, IL-10, IL-2, IL-12, and IFN-␥ mRNA levels (data chemokine induction was observed in sHZ-injected mice, such not shown). These data indicate that a Th2 response (IL-4, IL-10) event was found to be a specific one. In fact, the malarial pig- is not favored by the malarial pigment and that sHZ-dependent ment did not exert any effect on various of the chemokine tran- proinflammatory cytokine expression is not an unspecific event. In scripts evaluated (lymphotactin, RANTES, eotaxin, IP-10, and addition, ELISAs confirmed that the noticed sHZ-inducible cytokine TCA-3) (data not shown). In line with our data obtained by http://www.jimmunol.org/ transcription was accompanied by protein expression. As depicted in RPA, maximal MIP-1␣, MIP-2, and MCP-1 secretion was de- Fig. 3B, supernatants from pooled exudates of 3- and 6-h-treated mice tected in supernatants of pooled exudates from 3- and 6-h- contained elevated levels of IL-1␤ and IL-6; however, cytokine se- treated mice, following a similar kinetics to that of chemokine cretion was maximal only after 12 h of sHZ inoculation. mRNA induction (Fig. 4B). by guest on September 24, 2021

FIGURE 3. sHZ increases cytokine mRNA and protein expression in vivo. A, Kinetic analyses of sHZ-inducible IL-1␣, IL-1␤, and IL-6 mRNA expression in vivo. BALB/c mice were injected in the air pouches with 50 ␮g/ml sHZ. At various times (1, 3, 6, 12, and 24 h) following inoculation, exudates from each experimental group were pooled and total RNA was extracted from the leukocytes recruited. Cytokine mRNA levels were monitored using a cytokine multiprobe RPA system (left panel). Densitometric quantiﬁcation of cytokine mRNA expression over negative control after normalization to GAPDH (right panel). B, IL-1␤ and IL-6 secretion in the pouches of BALB/c mice following sHZ stimulation (50 ␮g/ml) over a 24-h period. After sHZ inoculation, exudates from each group of mice were pooled and the resulting supernatants were subjected to ELISA. Negative control mice were treated with PBS for 6 h. PBS (Ⅺ), sHZ (f). Data represent cytokine mRNA and protein induction in pooled samples (ﬁve mice) from each experimental group. Results are representative of one of two independent experiments. The Journal of Immunology 3105 Downloaded from http://www.jimmunol.org/

FIGURE 4. sHZ-mediated chemokine up-regulation in the air pouch. A, Time course of chemokine mRNA expression in the air pouch was monitored following sHZ (50 ␮g/ml) inoculation over a 12-h period. Exudates from each experimental group were pooled, and total RNA was extracted from the leukocytes recruited. Samples were submitted to RPA using the mouse chemokine-5 multiprobe template (left panel). Integrated density values of chemokine mRNA levels normalized to GAPDH (right panel). B, Kinetic analyses of MIP1␣, MIP-2, and MCP-1 protein expression in the air pouches of BALB/c mice in response to sHZ (50 ␮g/ml). Supernatants of pooled exudates from 1- to 24-h-treated mice were subjected to ELISA. As a negative control group, mice were inoculated with PBS for 6 h. PBS (Ⅺ), sHZ (f). Data represent chemokine mRNA and protein expression in pooled samples (ﬁve mice) from each experimental group. Results are representative of one of two separate experiments. by guest on September 24, 2021

FIGURE 5. sHZ increases chemokine receptor mRNA levels in leukocytes recruited in the air pouch. To monitor gene expression of CCR (A) and CXCR (B), mice were injected with 50 ␮g/ml sHZ for 1–24 h or 12 h, respectively. Exudates from each experimental group were pooled and total RNA was extracted from the leukocytes recruited. mCR5 and mCR6 multiprobe templates were used to evaluate the induction patterns of the various receptor transcripts by RPA (left panels). Densitometric quantification of CCR and CXCR mRNA expression over negative control after normalization to GAPDH (right panels). Negative control mice were treated with PBS for 6 h. PBS (Ⅺ), sHZ (f). Data represent CCR and CXCR mRNA levels in pooled samples (five mice) from each experimental group. Results are representative of one of two independent experiments. 3106 IN VIVO MODULATION OF PROINFLAMMATORY MEDIATORS BY HZ sHZ increases chemokine receptor mRNA levels in leukocytes recruited in the air pouch Chemokines exert their effects by binding to a family of specific seven-transmembrane G protein-coupled receptors (33). Because both CC (MIP-1␣, MIP-1␤, and MCP-1) and CXC (MIP-2) chemokine production was detected in response to sHZ, we next investigated whether CCR and CXCR gene expression was up-reg- ulated in leukocytes from pooled exudates of sHZ-injected mice. In line with our data regarding chemokine mRNA induction, sHZ was found to selectively modulate CCR and CXCR expression. Although no changes were observed in CCR1b, CCR3, and CCR4 transcripts (data not shown), a rapid and significant increase of CCR1 mRNA levels occurred upon sHZ inoculation (Fig. 5A), reaching maximal messenger accumulation already after1h(6- fold increase over negative control mice). To a lesser extent, CCR2 and CCR5 mRNA up-regulation was also detected at 3- to 6-h poststimulation (2-fold increase over PBS control mice). Similarly, CXCR mRNA expression was also enhanced by sHZ. As depicted in Fig. 5B, CXCR2 and CXCR4 transcripts, but not BRL-1 (data Downloaded from not shown), were transiently increased, with the highest mRNA accumulation (3-fold increase) occurring upon a 3-h treatment and a gradual decrease thereafter.

MRP secretion is induced by sHZ in vivo http://www.jimmunol.org/ S100A8 and S100A9 are calcium-binding proteins that belong to a subset of the S100 family known as MRPs because their expression is almost completely restricted to cells of myeloid origin (34). S100A8 and S100A9 associate noncovalently to form homodimers and the heterodimer S100A8/A9 (35). S100A8, S100A9, and S100A8/A9 have been shown by others and by us to induce chemotaxis for N␾ as well as N␾ adhesion to ﬁbrinogen (36–38), and to play an essential role in N␾ migration in response to LPS (29).

Importantly, MRP secretion has been documented in various in- by guest on September 24, 2021 fectious and inflammatory conditions, including malaria (8, 9). Based on this previous evidence and because our first set of experiments indicated that most of the leukocytes recruited in response to sHZ corresponded to a N␾ population, we set out to elucidate whether this proinflammatory event was, at least in part, due to the production of MRPs. We found that sHZ induced secretion of three different murine MRPs, S100A8, S100A9, and S100A8/S100A9, in pooled air pouch exudates of BALB/c mice (Fig. 6). Of interest, and in contrast to our observations regarding another powerful N␾ chemoattractant, MIP-2, MRP production was sustained up to 12 h following sHZ inoculation, further suggesting an important role for these proteins in the noticed sHZ- inducible N␾ accumulation. sHZ induces liver chemokine and cytokine mRNA expression After having demonstrated that sHZ led to the production of proinflammatory mediators in vivo, we next sought to evaluate the potential role of sHZ as an immunomodulator during malaria infection. To mimic HZ release into the bloodstream at the end of the erythrocytic life cycle of the parasite, increasing doses of sHZ (200–1500 ␮g) were injected i.v. into the tail vein of BALB/c FIGURE 6. MRP secretion is induced by sHZ in vivo. Production of mice. sHZ effects were monitored in the liver because its accumu- S100A8, S100A9, and S100A8/S100A9 was measured in the air pouches lation in this organ has been associated with infection chronicity in of BALB/c mice treated either with sHZ (50 ␮g/ml) over a 24-h period or humans (39), and with cumulative parasite burden and duration of with PBS for 6 h. At various times after inoculation (1, 3, 6, 12, and 24 h), infection in experimental malaria (40). The various concentrations pouch exudates were pooled and the resulting supernatants were subjected were chosen based on previous studies performed by Sherry et al. to ELISA. PBS (Ⅺ), sHZ (f). Results represent MRP secretion in pooled (22), who, by reference to standard hematological measurements, samples (five mice) from each experimental group. Data are representative estimated that as much as 200 ␮mol of HZ (ϳ3 ␮mol/Kg) is re- of one of two separate experiments. leased into the circulation of a 70 kg P. falciparum-infected human patient who has a 1% synchronized parasitemia. According to our The Journal of Immunology 3107 Downloaded from http://www.jimmunol.org/

FIGURE 7. sHZ induces liver chemokine and cytokine mRNA expression. BALB/c mice were injected i.v. in the tail vein either with PBS or with ␮ increasing doses of sHZ (200–1500 g). After a 6-h treatment, mice were lethally exposed to CO2, and their livers were dissected and ground. Total RNA extraction was followed by RPA analyses to monitor chemokine (A) and cytokine (B) mRNA expression in liver. Data presented in left panels correspond to one animal representative of each experimental group. Graphs in right panels represent integrated density values of mean chemokine and cytokine mRNA .p Ͻ 0.05, sHZ vs PBS ,ء .(levels normalized to GAPDH. Results represent mean ϩ SEM of ﬁve mice. PBS (Ⅺ), sHZ (f by guest on September 24, 2021

measurements, 25 ␮g of sHZ is equal to 26 nmol of heme content; Discussion therefore, the doses used in this set of experiments, 200, 750, and The pathogenic manifestations during a malaria crisis occur after ␮ ϳ ϳ ϳ ␮ 1500 g( 8, 30, and 60 mol heme/Kg, respectively), are schizogeny and have been attributed to proinflammatory cytokines equivalent to the amounts of HZ, which would be found ranging released in response to malaria parasites and their products (7). ϳ ϳ ϳ from mild to severe malaria: 3, 10, and 20% parasitemia. As Several in vitro studies have reported cytokine and chemokine pro- shown in Fig. 7A, a significant increase of various liver chemokine duction in response to HZ (21–24), and our data indicated that both ␮ transcripts was already detectable when 200 g of sHZ was in- P. falciparum HZ and sHZ induce mRNA expression of the same jected, whereas maximal messenger accumulation was observed chemokines in M␾ (M. Jaramillo and M. Olivier, manuscript in upon stimulation with intermediate and high doses of sHZ (750– preparation); however, the in vivo proinflammatory properties of 1500 ␮g): a 28-fold increase over PBS control was observed for the malarial pigment remained unexplored. In the present study, MIP-1␣, a 5-fold for MIP-1␤, a 26-fold for MIP-2, and a 70-fold we demonstrate that sHZ leads to leukocyte recruitment in vivo for MCP-1. In addition, kinetic analyses performed by injecting and induces the release of various chemokines, chemokine recep- the lowest dose of sHZ (200 ␮g) over a 24-h period revealed that tors, proinflammatory cytokines, and MRPs, supporting the idea the sHZ-inducible liver chemokine mRNA up-regulation occurred that this parasite metabolite is likely to play a key role in malaria at early times of injection, already detectable after 1 h and maximal at 6 h (data not shown). Of interest, significant mRNA levels of immunopathology. MIP-1␣, MIP-1␤, and MIP-2 were still found at 24 h posttreat- Analysis of the inflammatory cells accumulating in response to ␾ ment. In parallel, our data indicating proinflammatory sHZ-medi- sHZ revealed a high increase of N early after stimulation accom- ated chemokine induction in the air pouch prompted us to inves- panied by a significant rise in monocyte numbers at later times. tigate the ability of sHZ to modulate liver cytokine expression. As These findings are in agreement with previous studies reporting a depicted in Fig. 7B, 200-1500 ␮g of i.v. injected sHZ resulted in transient increase in the activity and number of circulating phago- substantial mRNA accumulation of two strong immunomodula- cytes in response to schizogeny (3), and suggest that HZ could be, tors, IL-1␤ (41) and IL-12 (42), reaching a 2- and 22-fold increase at least in part, responsible for such augmentation following par- over negative control mice, respectively. Altogether, this last set of asite release into the bloodstream. Even though autopsy studies of experiments indicates that at doses that could be achievable during P. falciparum-infected patients have yielded evidence of parasite malaria infection (ϳ3% parasitemia), HZ is already able to elicit a clearance by activated M␾ and N␾ (43, 44), neutrophilia and proinflammatory response in the host, and suggests that at higher monocytosis occurring in malaria have also been associated with parasitemias HZ is likely to contribute to an exacerbated inflam- poor prognosis of the disease (45). Therefore, by inducing leuko- matory immune response. cyte recruitment into the sites of sequestration of infected RBC, 3108 IN VIVO MODULATION OF PROINFLAMMATORY MEDIATORS BY HZ sHZ could contribute to reduce parasite burden, but could also Even though liver chemokine induction has only been reported in favor an exacerbated phagocyte response, resulting in tissue dam- murine malaria (14, 22), MIP-1␣, MIP-1␤, and MCP-1 have been age and microvascular flow disturbance. detected during human hepatic disease, and their expression has The presence of increased leukocyte numbers in response to been associated with monocyte infiltration and hepatic injury (re- sHZ was paralleled by its ability to induce the expression of var- viewed by Simpson et al. (51)). During CM, HZ-dependent CC ious proinflammatory mediators with chemoattractant properties. chemokine modulation could participate in the observed monocyte Consistent with elevated N␾ numbers, increased levels of MIP-2, infiltration (56) and adhesion (57) in cerebral microvessels, a powerful N␾ chemoattractant and activator (32), were detected thereby favoring microvascular damage (58). In the case of pla- in the exudates. Of interest, a significant and dose-dependent up- cental malaria, by increasing CC chemokine levels, HZ might be regulation of MIP-2 transcripts was also observed in liver of i.v. implicated in poor birth outcomes because monocyte infiltration at injected mice. Even though the proinflammatory role of MIP-2 has this location constitutes a key risk factor for low birth weight (59). not been explored in murine models of malaria, increased hepatic Furthermore, given that malaria and HIV-1 coinfections are com- N␾ accumulation has been reported in mice following systemic mon in pregnant women in subSaharan Africa (60), HZ has the administration of high doses of MIP-2 (46), and immunoneutral- potential to facilitate HIV-1 replication and vertical transmission ization of MIP-2 was shown to decrease N␾ influx into the liver by creating a reservoir for M␾-tropic HIV-1. Moreover, by in- and reduce hepatic injury (47). In light of these studies, it is con- creasing both MIP-1␣ and MIP-1␤, which are potent pyrogenic ceivable that by increasing MIP-2 expression, HZ could contribute molecules (61), HZ may play a role in fever episodes occurring to favor peripheral N␾ recruitment and subsequent liver injury. In during acute malaria. This hypothesis is indeed supported by a humans, serum levels of IL-8, a potent N␾ chemoattractant and previous in vivo study demonstrating the ability of sHZ to regulate Downloaded from activator (48), increase during severe malaria (49). Interestingly, body temperature in rats (22). In addition to its capacity to evoke IL-8 expression was reported only in HZ-laden M␾ in placental fever, MIP-1␣ also exerts a marked suppressive effect on hemo- malaria (50). Even though no murine homologue has been de- poietic stem cell proliferation (62); therefore, it can be envisaged scribed for IL-8, this human chemokine most closely resembles that HZ-mediated MIP-1␣ expression might contribute to the ane- murine MIP-2 (51); therefore, HZ-mediated MIP-2 and IL-8 up- mia characteristic of malaria infection. Further studies are needed

regulation may have some common biological effects during to demonstrate the physiological contribution of HZ to fever epi- http://www.jimmunol.org/ malaria. sodes and susceptibility to anemia, as well as to the metabolic and In addition to MIP-2, sHZ-inducible MRP S100A8, S100A9, morphological alterations in liver, brain, and placenta related to and S100A8/A9 synthesis is also likely to account for the noticed malaria. N␾ infiltration in the air pouches. Murine S100A8 (36) as well as Chemokine actions depend on the expression of chemokine re- human S100A8, S100A9, and S100A8/A9 cause N␾ chemotaxis ceptors that bind and respond to such ligands, bringing about cell in vivo and induce N␾ adhesion to fibrinogen in vitro (37, 38). activation via intracellular signaling mechanisms (33). Analysis of Moreover, we recently demonstrated that S100A8 and S100A9 chemokine receptor up-regulation in cells from air pouch exudates play important roles in N␾ accumulation to extravascular sites revealed that sHZ increased mRNA levels of CCR1, CCR2, CCR5, (52). In P. falciparum-infected children, serum levels of CXCR2, and CXCR4, whose induction patterns correlated with the by guest on September 24, 2021 S100A8/A9 were shown to be significantly related to parasitemia leukocyte populations recruited. Whereas CCR2 and CCR5 are and fever (9) and, during CM, analysis of cellular activation in the expressed by monocytes, CCR1, CXCR2, and CXCR4 are known brain revealed S100A8 and S100A9 expression in microglial cells to be present in both monocytes and N␾ (63). In addition, a rise in (8). In line with these findings, Chen et al. (53) found that N␾ play these receptor transcripts in the recruited cells could explain, at a critical role in the development of experimental CM. Based on least in part, their infiltration in the pouches via their response to these data and knowing that HZ accumulation also occurs in the sHZ-inducible chemokine secretion. In fact, MCP-1 is a known brain (3), it is possible that HZ-dependent MRP secretion and sub- ligand for CCR2, MIP-2 for CXCR2, and MIP-1␣ for CCR1 and sequent N␾ infiltration contribute to both acute malaria and CM. CCR5, whereas MIP-1␤ binds exclusively CCR5 (63). Current In concert with our results showing monocyte infiltration in the data indicate the involvement of chemokine receptors in the patho- air pouches, sHZ induced the expression of MIP-1␣, MIP-1␤, and genesis of a number of relevant acute and chronic inflammatory MCP-1, three CC chemokines that are potent monocyte chemoat- diseases (33). In CM, an important role for CCR5 has been tractants and activators (31), and the same pattern of chemokine strongly suggested by Belnoue et al. (64, 65), who recently showed induction was detected in the liver of i.v. sHZ-injected mice. This that CCR5 and not CCR2 expression, on both brain-sequestered is in agreement with data from experimental malaria showing in- leukocytes and nonhemopoietic cells, is required for experimental creased mRNA levels of MIP-1␣ and MIP-1␤ in liver of infected CM development. Such effect has been attributed to increased mice (22), as well as MCP-1 mRNA up-regulation in brain and brain sequestration of cytotoxic CD8ϩ T cells, a leukocyte subset liver of mice suffering of CM (14). In humans, elevated serum found to be critical for CM progression (66, 67). Moreover, Tka- concentrations of MIP-1␣ were detected during acute P. falcipa- chuk et al. (68) found that P. falciparum infection enhances CCR5 rum malaria (10), and more recently, Abrams et al. (11) found a mRNA expression on placental M␾ of pregnant women. CCR5 is correlation between placental monocyte recruitment and elevated an important coreceptor required for efficient HIV-1 cell entry (69) MIP-1␣ and MCP-1 expression in P. falciparum-infected pregnant and has been identified as the main secondary receptor implicated women. Of interest, in this study, production of MIP-1␤ and in HIV-1 vertical transmission (70). In addition to CCR5, it was MCP-1 was observed in some of the HZ-laden maternal M␾. Be- ascertained that CCR2 (71) and CXCR4 (72) could also serve as cause of their biological activities, through the induction of CC cofactors to permit HIV-1 entry. Therefore, the potential of HZ chemokines, HZ may be involved in various aspects of malaria favoring CM through CCR5 induction in the brain, as well as immunopathology. On one hand, HZ would contribute to increase HIV-1 infection and intrauterine transmission during malaria and monocyte numbers in the sites of parasitation and, depending on HIV-1 coinfections via CCR2, CCR5, and CXCR4 up-regulation, the specific location, exert different effects. In the liver, HZ-me- deserves further investigation. diated CC chemokine expression is likely to be involved in hepatic In addition to chemokine modulation, proinflammatory cytokine damage observed both in human (54) and murine (55) malaria. expression in response to sHZ was also observed. In the air The Journal of Immunology 3109 pouches, IL-1␣, IL-1␤, and IL-6 levels were increased and, in the Acknowledgments liver, a significant rise in IL-1␤ transcripts occurred in a dose- We thank Dr. David Sullivan (The Johns Hopkins University, Baltimore, dependent manner. IL-1 and IL-6 share multiple functions, includ- MD) for his helpful advice regarding ␤-hematin synthesis and heme ing N␾ and M␾ activation (73, 74) and the induction of hepatocyte quantitation. acute-phase responses such as fever and leukocytosis (75, 76). Im- portantly, IL-1 and IL-6 can stimulate chemokine secretion (30, References 77); thus, their release may offer a means of locally amplifying the 1. Breman, J. G., A. Egan, and G. T. Keusch. 2001. The intolerable burden of immunological response. Consistent with these findings, extensive malaria: a new look at the numbers. Am. J. Trop. Med. Hyg. 64(Suppl. 1–2):iv. data have documented high IL-1 and IL-6 levels in both human 2. Miller, L. H., D. I. Baruch, K. Marsh, and O. K. Doumbo. 2002. The pathogenic basis of malaria. Nature 415:673. (3–5) and murine malaria (12, 13), which have been implicated in 3. Urquhart, A. D. 1994. Putative pathophysiological interactions of cytokines and both antiparasitic activity and disease severity. Similarly, in vitro phagocytic cells in severe human falciparum malaria. Clin. Infect. Dis. 19:117. 4. Wenisch, C., K. F. Linnau, S. Looaresuwan, and H. Rumpold. 1999. Plasma studies have revealed IL-1 and IL-6 induction in response to P. levels of the IL-6 cytokine family in persons with severe Plasmodium falciparum falciparum (15) and to HZ (21, 23, 24). Based on this evidence, malaria. J. Infect. Dis. 179:747. our data regarding IL-1 and IL-6 in vivo up-regulation further 5. Day, N. P., T. T. Hien, T. Schollaardt, P. P. Loc, L. V. Chuong, T. T. Chau, N. T. Mai, N. H. Phu, D. X. Sinh, N. J. White, and M. Ho. 1999. The prognostic support a role for HZ in malaria immunopathology related to and pathophysiologic role of pro- and antiinflammatory cytokines in severe ma- proinflammatory cytokine overproduction. laria. J. Infect. Dis. 180:1288. Finally, a significant increase of IL-12 mRNA levels was found 6. Wenisch, C., B. Parschalk, E. Narzt, S. Looareesuwan, and W. Graninger. 1995. Elevated serum levels of IL-10 and IFN-␥ in patients with acute Plasmodium in liver of HZ i.v. injected mice. IL-12 is a potent immunomodu- falciparum malaria. Clin. Immunol. Immunopathol. 74:115. latory cytokine (42) that seems to play a dual role in malaria. IL-12 7. Malaguarnera, L., and S. Musumeci. 2002. The immune response to Plasmodium Downloaded from has been shown to induce protective immunity against blood stage falciparum malaria. Lancet Infect. Dis. 2:472. 8. Schluesener, H. J., P. G. Kremsner, and R. Meyermann. 1998. Widespread ex- in the murine model (78); however, it has been proposed that pression of MRP8 and MRP14 in human cerebral malaria by microglial cells. raised concentrations of IL-12 could also be associated with bone Acta Neuropathol. 96:575. marrow dyserythropoiesis (78) and with the acute symptoms of 9. Bordmann, G., G. Burmeister, S. Saladin, H. Urassa, S. Mwankyusye, N. Weiss, and M. Tanner. 1997. MRP 8/14 as marker for Plasmodium falciparum-induced malaria via IFN-␥-mediated IL-1 induction (79). Therefore, if a malaria episodes in individuals in a holoendemic area. Clin. Diagn. Lab. Immu- direct effect of HZ on IL-12 regulation can be established, con- nol. 4:435. http://www.jimmunol.org/ 10. Burgmann, H., U. Hollenstein, C. Wenisch, F. Thalhammer, S. Looareesuwan, trolling this pathway of cellular modulation may allow ameliora- and W. Graninger. 1995. Serum concentrations of MIP-1␣ and IL-8 in patients tion of morbidity and mortality during malaria infection. suffering from acute Plasmodium falciparum malaria. Clin. Immunol. Immuno- Although the cellular source of HZ-inducible chemokine, cyto- pathol. 76:32. 11. Abrams, E. T., H. Brown, S. W. Chensue, G. D. Turner, E. Tadesse, V. M. Lema, kine, and MRP expression remains to be determined in vivo, in M. E. Molyneux, R. Rochford, S. R. Meshnick, and S. J. Rogerson. 2003. 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Cytokine and chemokine re- HZ in these cells has been associated with cellular activation and sponses in a cerebral malaria-susceptible or -resistant strain of mice to Plasmo- disease severity (19). In addition, evidence from autopsy and in dium berghei ANKA infection: early chemokine expression in the brain. Int. Immunol. 15:633. vivo histological studies suggests that in P. falciparum malaria, the 15. Wahlgren, M., J. S. Abrams, V. Fernandez, M. T. Bejarano, M. Azuma, M. Torii, main sources of cytokines are likely to be activated tissue M␾ in M. Aikawa, and R. J. Howard. 1995. Adhesion of Plasmodium falciparum-in- liver (Kupffer cells), spleen, lungs (alveolar M␾), and brain (mi- fected erythrocytes to human cells and secretion of cytokines (IL-1-␤, IL-1RA, IL-6, IL-8, IL-10, TGF␤, TNF␣, G-CSF, GM-CSF). Scand. J. Immunol. 42:626. croglial cells), as well as PBMC (3). Because erythrocytic schizog- 16. Seixas, E., C. Cross, S. Quin, and J. Langhorne. 2001. Direct activation of den- eny and subsequent HZ release occur in the microvasculature dritic cells by the malaria parasite Plasmodium chabaudi chabaudi. Eur. J. Im- where parasitized erythrocytes are sequestered, and both HZ and munol. 31:2970. 17. Schofield, L., and F. Hackett. 1993. Signal transduction in host cells by a gly- leukocyte accumulation have been reported in these organs, it is cosylphosphatidylinositol toxin of malaria parasites. J. Exp. Med. 177:145. possible that the HZ-inducible immunological response would be 18. Francis, S. E., D. J. J. Sullivan, and D. E. Goldberg. 1997. Hemoglobin metab- olism in the malaria parasite Plasmodium falciparum. Annu. Rev. Microbiol. directed against the sites of maximal parasitation, and the intensity 51:97. and nature of this response may therefore contribute to the patho- 19. Arese, P., and E. Schwarzer. 1997. Malarial pigment (hemozoin): a very active physiology of malaria. Given the differences between human and ‘inert’ substance. 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