The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178, 6416-6425
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vigário, A. M.
Right arrow Articles by Rénia, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vigário, A. M.
Right arrow Articles by Rénia, L.
Right arrowPubmed/NCBI databases
*Substance via MeSH

Recombinant Human IFN-{alpha} Inhibits Cerebral Malaria and Reduces Parasite Burden in Mice1

Ana Margarida Vigário*,{dagger},{ddagger},§,¶, Elodie Belnoue2,*,{dagger},{ddagger},§, Anne Charlotte Grüner*,{dagger},{ddagger},§, Marjorie Mauduit*,{dagger},{ddagger},§, Michèle Kayibanda*,{dagger},{ddagger},§, Jean-Christophe Deschemin*,{dagger},{ddagger},§, Myriam Marussig||, Georges Snounou{dagger}{dagger}, Dominique Mazier||, Ion Gresser* and Laurent Rénia3,*,{dagger},{ddagger},§

* Institut Cochin, Département d’Immunologie, Paris, France; {dagger} Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 567, Paris, France; {ddagger} Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Paris, France; § Université René Descartes, Hôpital Cochin, Paris, France; Instituto Gulbenkian de Ciências, Oeiras, Portugal; || Université Pierre et Marie Curie-Paris 6, Unité Mixte de Recherche S511, Paris, France; # Assistance Publique-Hopitaux de Paris, Groupe hospitalier Pitié-Salpétrière, Service Parasitologie Mycologie, Paris, France; ** INSERM Unité 511, Paris, France; {dagger}{dagger} Equipe Parasitologie Comparée et Modèles Expérimentaux Unite Scientifique de Museum 0307, Centre National de la Recherche Scientifique Institut Federatif de Recherche 101, Muséum National d’Histoire Naturelle, Paris, France; and * INSERM Unité 255, Institut des Cordeliers, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Most C57BL/6 mice infected i.p. with Plasmodium berghei ANKA (PbA) die between 7 and 14 days with neurologic signs, and the remainder die later (>15 days) with severe anemia. Daily i.p. injections of a recombinant human IFN-{alpha} (active on mouse cells) prevented death by cerebral malaria (87% deaths in the control mice vs 6% in IFN-{alpha}-treated mice). The mechanisms of this IFN-{alpha} protective effect were multiple. IFN-{alpha}-treated, PbA-infected mice showed 1) a marked decrease in the number of PbA parasites in the blood mediated by IFN-{gamma}, 2) less sequestered parasites in cerebral vessels, 3) reduced up-regulation of ICAM-1 expression in brain endothelial cells, 4) milder rise of blood levels of TNF, 5) increased levels of IFN-{gamma} in the blood resulting from an increased production by splenic CD8+ T cells, and 6) fewer leukocytes (especially CD8+ T cells) sequestered in cerebral vessels. On the other hand, IFN-{alpha} treatment did not affect the marked anemia observed in PbA-infected mice. Survival time in IFN-{alpha}-treated mice was further increased by performing three blood transfusions over consecutive days.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malaria infection, in both in humans and in rodent models, induces a wide range of pathologies, the most important of which are cerebral malaria (CM)4 and severe anemia. The pathogenesis of CM is complex and is likely the result of the subtle interplay between sequestration of infected RBC (iRBC) and/or leukocytes within postcapillary venules of the brain (1, 2, 3) and systemic and local cytokine production (4). Large amounts of cytokines (TNF-{alpha} and IFN-{gamma}), produced during malaria infection, can up-regulate endothelial adhesion molecules such as the ICAM-1. The increase in ICAM-1 can increase sequestration of iRBC and/or leukocytes within the microvasculature of the brain.

In the Plasmodium berghei ANKA (PbA) model of CM in mice (5), there is much less parasite sequestration in the brain than in humans (Ref. 6 and B. Lucas, unpublished results). However, we recently showed that PbA sequestration in the brain is associated with CM development and with the subsequent intravascular sequestration of leukocytes observed in the brain that occurs at the time of neurologic signs (7). Moreover, the CD8+-T cell subset of these brain-sequestered leukocytes (BSL) have been shown to be responsible for CM death (7).

The pathogenesis of anemia is also multifactorial with the rupture of iRBC (8), the accelerated removal of iRBC and normal RBC by the spleen (9, 10), and alterations of erythropoiesis (10). Cytokines, in particular TNF-{alpha}, have been implicated in the pathogenesis of severe anemia, occurring both in man and in mouse models due to inhibitory effects on erythropoiesis (11, 12, 13, 14, 15, 16).

Pharmacologic intervention to prevent CM is highly desirable. One possibility would be to interfere with the cytokine network induced by malarial infection. For example, in a mouse model of sepsis in which injection of LPS leads to mortality through induction of TNF-{alpha}, treatment with IFN-{alpha} resulted in decreased blood levels of TNF and in increase in overall survival (17).

We report here the results of our experiments showing a clear-cut protective effect of IFN-{alpha} treatment on the development of CM in PbA-infected mice without any protective effect on the development of anemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and parasite

Female 5- to 6-wk-old C57BL/6J mice were purchased from Charles River Breeding Laboratories and Harlan France. Blood stage parasites of PbA clone lines (6, 18) were stored as a stabilate (107 iRBC/ml in Alsever’s buffer) in liquid nitrogen and were derived from lines of parasites passaged in vivo in C57BL/6J mice. The batches were free of other infectious agents. All experiments and procedures conformed to the French Ministry of Agriculture Regulations for Animal Experimentations.

Infection and disease assessment

Mice were inoculated i.p. with 106 iRBC in 100 µl of Alsever’s buffer. They were considered to have CM only when they displayed neurologic signs: paralysis, deviation of the head, ataxia, and convulsions.

The percentage of parasitemia was calculated as the number of parasitized cells per 100 erythrocytes. Reticulocytes were counted every 2 days using the same blood smears as described previously (19). Hemoglobin (Hb) concentrations were determined every 2 days as described previously (20). Briefly, 2 µl of blood from the tail vein was diluted in 500 µl of Drabkin’s solution (Sigma-Aldrich), and Hb was assayed in a 96-well microtiter plate (Costar) in a volume of 100 µl by measuring A405 nm, in an ELISA reader (Bio-Tek Instruments). Values were converted to milligrams per milliliter using a standard curve of human Hb (Sigma-Aldrich) dissolved in Drabkin’s solution. Erythrocyte numbers were obtained every 2 days by counting erythrocytes using a Malassez chamber loaded with a solution of 2 µl of tail blood in 1 ml of PBS. Since infected mice develop anemia, the density of the parasites in the blood was also assessed in some experiments. This was defined as the number of parasites per microliter of blood (parasite load), which was calculated by multiplying the number of erythrocytes per the percentage of parasitemia.

IFN-{alpha} treatment

A recombinant human hybrid IFN-{alpha} (BDBB, a hybrid between {alpha}1 and {alpha}8, CIBA-GEIGY 35269), which cross-reacts with murine cells, was a gift from Dr. M. Grütter (Ciba Geigy, Basel, Switzerland). Its specific activity on mouse embryonic fibroblasts was 5.1 x 106 U/mg (21). The lyophilized IFN-{alpha} was diluted in PBS containing 0.1% (w/v) BSA (fraction V; Sigma-Aldrich). The control was 0.1% BSA in PBS (and referred as diluent). Mice were injected i.p. with 200 µl of IFN-{alpha} (4 x 105 mouse units/injection/mouse) or with the control preparation and 1 h later injected with the parasites. This dose was shown previously to be active against another malaria parasite, Plasmodium yoelii (19). Duration of IFN-{alpha} treatment varied in the experiments as indicated in the legends.

Histology and immunostaining of brain tissues

Brains from mice displaying neurologic signs were removed and fixed in Carnoy solution (ethanol/chloroform/acetic acid, 6:3:1) for 24 h, stored in butanol, and included in paraffin. Sections (5 µm) from the midbrain region of two mice per group were cut and stained with H&E. Uninfected and PbA-infected mice without signs of CM were sacrificed at the same time.

Brain tissues were also snap-frozen in isopentane cooled in liquid nitrogen and stored at –80°C. Cryostat sections (6 µm) were cut, mounted in SuperFrost Plus slides (Menzel-Glaser), and maintained at –20°C. Sections were fixed in acetone for 10 min and washed in Tris-NaCl. All the subsequent washes, and incubations were done in 4 mM Earl’s buffered salt solution-HEPES. After blockade of endogenous biotin using a biotin blocking kit (DakoCytomation), sections were incubated 2–3 h at room temperature with 2.5 µg/ml biotinylated hamster anti-mouse ICAM-1 mAb (3E2; BD Pharmingen) or with the biotinylated isotype control. Binding was detected with extravidin-FITC (Sigma-Aldrich). Slides were then mounted in PBS-glycerol (1/1). They were observed using a Leica DMLD with a x10 ocular and a x40 objective (numerical aperture: 0.7, PL Fluotor; Leica) Photographs were taken with a Pentax camera. They were scanned with an Epson apparatus and assembled with Photoshop (Adobe Systems). In one experiment, ICAM-1-positive vessels were enumerated from photographs corresponding to different microscope fields (magnification, x400) for each section from mice obtained in the different groups.

Preparation of BSL, lymph node cells, and splenocytes

Spleens and inguinal and periaortic lymph nodes were removed from sacrificed mice. Mice were then perfused intracardially with PBS to remove nonadherent RBC and leukocytes from the brain. The brains were removed and adherent leukocytes isolated as described previously (7). They were crushed in RPMI 1640 medium (Invitrogen Life Technologies). The tissue extract was then centrifuged at 400 x g for 5 min. The pellet was resuspended with 10 ml of a HEPES buffer containing 100 mM NaCl, 2 mM KCl, 0.3 mM Na2HPO4·12 H2O, and 0.01 M HEPES (Sigma-Aldrich), supplemented with 100 IU/ml penicillin/streptomycin (Invitrogen Life Technologies), 0.05% collagenase (Boehringer Mannheim), and 2 U/ml DNase (Sigma-Aldrich). The mixture was stirred at room temperature for 30 min. The tissue extract was passed through sterile gauze and centrifuged at 80 x g for 30 s to remove debris. The supernatant was deposited on a 30% Percoll gradient (Pharmacia Biotech) and centrifuged at 1400 x g for 10 min. The pellet was collected, and residual RBC were removed by hypotonic shock using ammonium chloride potassium lysis buffer. Spleens and lymph nodes were also disaggregated at room temperature, and RBC from splenic cell suspensions were lysed by hypotonic shock as above. Whole BSL, splenic, and lymph node cells were then washed and resuspended in FACS buffer (PBS containing 1% FCS and 0.01% NaN3) and counted.

Immunolabeling and flow cytometry analysis

Spleen cells and BSL were identified by their size (forward light scatter) and granulosity (side scatter), as described previously (7). Macrophages were identified as F4/80+ (biotinylated rat IgG2b mAb anti-mouse F4/80, clone C1:A3-1; Tebu). Neutrophils were identified as F4/80– and Gr-1+ (rat IgG2b mAb anti-mouse Gr-1 conjugated to FITC, clone RB6-8C5; BD Pharmingen). T cells were identified by their small size and by using the following Abs: hamster IgG mAb anti-mouse CD3 conjugated to PE (clone 17A2; BD Pharmingen), rat IgG2a mAb anti-mouse CD8{alpha} conjugated to FITC (clone 53-6.7; BD Pharmingen), rat IgG2a Ab anti-mouse CD8{alpha} conjugated to QR (clone 53-6.7; Sigma-Aldrich), and rat IgG2a mAb anti-mouse CD4 conjugated to QR (clone H129-19; Sigma-Aldrich) diluted at the appropriate concentration in FACS buffer. Five thousand cells were analyzed for each sample. The data were collected using a FACSCalibur flow cytometer analyzed using the CellQuest software (BD Biosciences).

PCR quantification of parasites in the brain of PbA-infected mice

Quantification of parasites in organs from infected animals was performed as described previously (24). Mice were perfused intracardially with PBS, and a portion of the brain was removed, frozen in liquid nitrogen, and DNA was extracted using a kit following instructions of the manufacturer (Qiagen). A first amplification of beta-actin gene fragment primers was used to verify whether all the samples contained similar amounts of mouse DNA. The DNA was then used as a template in duplicate PCR using oligonucleotides specific for the small subunit ribosomal RNA gene of the parasite (22). Negative control tubes containing no DNA were included in each run to detect contamination. A standard curve corresponding to DNA extracted from equal quantities of erythrocytes in samples containing 10-fold dilutions of a known number of P. berghei parasite nuclei was made. The PCR products were analyzed on a 2% agarose gel and quantified by scanning densitometry. Brain parasite load units correspond to the log number of parasite nuclei per microgram of brain DNA.

Detection of IFN-{gamma} and TNF

Animals were sacrificed when control PbA-infected mice displayed neurologic signs. Blood was taken from control and IFN-{alpha}-treated mice, infected or uninfected, and further processed to obtain sera. Sera were also taken 1 day postinfection from IFN-{alpha}-treated or control PbA-infected mice. In an additional experiment, splenocytes collected 6 days postinfection were plated in triplicate in 24-well tissue culture plates at a final concentration of 1 x 106/ml. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. Supernatant fluids were harvested 24 h after and assayed for IFN-{gamma} production. IFN-{gamma} was detected with double-sandwich ELISA using a capture Ab (purified anti-mouse IFN-{gamma} mAb R4-6A; BD Pharmingen) and a detection Ab (biotinylated anti-mouse IFN-{gamma} mAb XMG1.2; BD Pharmingen). Standard curves were generated using a rIFN-{gamma} (BD Pharmingen). Revelation was conducted by extravidin phosphatase alkaline (Sigma-Aldrich) for 1 h at room temperature. To measure phosphatase activity, 4-methylumbelliferyl phosphate (Sigma-Aldrich) was used as substrate, and fluorescence was read 360/460 nm using a microplate reader (Victor 1420; Wallac). Detection limit was 30 pg/ml for IFN-{gamma}. TNF (both TNF-{alpha} and lymphotoxin) was quantified using a bioassay as described previously (23). Briefly, murine WEHI 164 cl 13 cells (5 x 104 cells/well) were cultured with 1 µg/ml actinomycin D plus filtered sera to be tested in flat-bottom 96-well microtiter plates for 18 h. The number of surviving cells was determined by the MTT colorimetric method (24). Recombinant murine TNF-{alpha} (Genzyme) was used as standard. The assay detection limit was 0.12 U/ml.

Intracellular staining of cytokines

Splenocytes were incubated at 37°C for 90 min with PMA (50 ng/ml), ionomycin (500 ng/ml), and brefeldin A (10 ng/ml) (Sigma-Aldrich) in RPMI 1640 medium containing 10% FCS under in a 5% CO2 atmosphere. Cell samples were stained either with anti-mouse CD8 or anti-mouse CD4 allophycocyanin-conjugated mAbs. Additional cell samples were also incubated with biotinylated anti-F4/80 mAb, washed, and further incubated with streptavidin-FITC. Cells were washed in saline buffer containing 3% FCS and fixed with 4% paraformaldehyde in saline buffer at room temperature for 1 h. The cells were then permeabilized using the perm/wash buffer (BD Pharmingen) before incubation for 1 h with FITC-conjugated anti-IFN-{gamma} (clone XMG1.2; BD Pharmingen) or PE-conjugated anti-TNF-{alpha} (clone MP6-XT22; BD Pharmingen) mAbs diluted in saline buffer containing 3% FCS. Cells (50,000–100,000) were analyzed for each sample. The data were collected using a FACS CANTO flow cytometer and analyzed using the DIVA software (BD Biosciences).

IFN-{gamma} neutralization

On days 0–5 after PbA infection, mice received a single i.p. dose of 2 mg (day 0) and then 1 mg (other days) of the purified anti-IFN-{gamma} mAb XMG-1. Neutralization efficacy of this Ab preparation had been established previously (25).

Blood transfusion

Heparinized blood was obtained from noninfected C57BL/6J mice by retroorbital puncture and further diluted in PBS. Contaminating white cells were eliminated using a Lympholyte gradient (Cedarlane Laboratories). The pellet was collected and washed twice in sterile PBS. Erythrocytes (109 in 100 µl PBS) were injected i.v. in the tail vein starting on day 15 after PbA injection for 3 consecutive days.

Statistical analysis

Difference in survival was assessed with the log-rank Kaplan-Meier test. Differences between two groups were analyzed for statistical significance using the Mann-Whitney U test and for multiple groups using one-way ANOVA and the Dunn posttest. The data were analyzed using GraphPad Prism software (version 3.0) and taking p < 0.05 as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Inhibition of CM in IFN-{alpha}-treated mice

Sixty to 100% of C57BL/6J mice infected with PbA developed CM signs on days 6–12 after parasite inoculation (Table I) and died within 24–48 h after the onset of the signs. Mice were inoculated with PbA and treated with 4 x 105 UI IFN-{alpha}/day (see Materials and Methods) or with diluent. Depending on the experiment, 0–28% of the mice treated daily (from days 0 to 25) with IFN-{alpha} developed CM compared with 66–100% in control mice (Table I and Fig. 1A). In a separate experiment (Fig. 1B), IFN-{alpha} treatment between days 3 and 12 significantly delayed the appearance of neurologic signs in IFN-{alpha}-treated mice. The incidence of CM in mice treated with a single IFN-{alpha} injection at day 0 did not differ significantly from control mice (data not shown). No protection from CM was observed when IFN-{alpha} treatment was initiated on day 6, at the onset of neurologic signs. Indeed, seven of nine (78%) mice in the control group and six of seven (86%) mice in IFN-{alpha}-treated group developed CM.


View this table:
[in this window]
[in a new window]

  		Table I. Effect of IFN-{alpha} on the development of cerebral malaria in PbA-infected micea

 

Figure 1
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 1. Effect of IFN-{alpha} treatment on survival and parasitemia. Survival (A and B) and parasitemia (percentage of parasitized cells per erythrocyte number) (C and D) of mice infected with 106 PbA iRBC and treated with 4 x 105 mouse units of IFN-{alpha} from days 0 to 25 (A and B) or from days 3 to 12 (B and D). Control mice were infected with PbA and received only diluent. Neurologic signs of CM appeared on days 6–12 (shaded area), with death occurring 24–48 h after onset. A, On day 14, as calculated by Fisher’s exact text, CM incidence was significantly (p < 0.05) different between mice treated with IFN-{alpha} and control mice. B, A significant difference between IFN-{alpha}-treated and control mice was observed when calculated by the log-rank test (p < 0.05). C and D, Parasitemia (mean ± SD of six to eight mice per group) was significantly inhibited (p < 0.05, Mann-Whitney U test) by both IFN-{alpha} treatments from day 6 postinfection.

 
Inhibition of parasitemia IFN-{alpha}-treated mice

IFN-{alpha} treatment of PBA-infected mice for 0–25 days or for 3–12 days resulted not only in a marked delay in CM (Fig. 1, A and B) but also in inhibition of parasitemia (Figs. 1, C and D, and 7B) and of parasite load (see Fig. 7C). Nevertheless, mice died subsequently (Figs. 1, A and B, and 7, D and E) with severe anemia (see below).


Figure 7
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 7. Effect of blood transfusion in IFN-{alpha}-treated, PbA-infected mice on survival time (A), parasitemia (B), parasite load (C), erythrocyte count (D), Hb concentrations (E), reticulocytemia (F), and percentage of infected reticulocytes (iR) per total iRBC (G). C57BL/6J mice were inoculated with PbA (106 iRBC) and injected i.p. from days 0 to 25 with IFN-{alpha}. Neurologic signs of CM appear on days 6–12 (shaded area), with death occurring in 24–48 h thereafter. Symbols represent the mean ± SD of 6–8 mice/group. Blood transfusions (109 erythrocytes) were given i.v. on days 15, 16, and 17 after parasite inoculation (arrows). Symbols represent the mean ± SE of 10, 7, or 6 mice, respectively, for PbA, IFN-{alpha}-treated, and IFN-{alpha}-treated and blood-transfused group. After day 8, only 2 of 7 mice remained in the PbA-infected untreated group. Survival time was statistically different (p < 0.05 (log-rank test)) between IFN-{alpha}-treated and IFN-{alpha}-treated and transfused mice. *, p < 0.05 (one-way ANOVA followed by the Tukey multiple comparison test) between IFN-{alpha}-treated and IFN-{alpha}-treated and transfused mice.

 
Inhibition of parasite sequestration in the brains of IFN-{alpha}-treated mice

PbA parasites sequester in various organs (6, 26). We have recently shown using a sensitive quantitative PCR that a small number of PbA-iRBC are sequestered in the brain of mice (B. Lucas, manuscript in preparation), a number below the detection threshold of most currently used imagery techniques (26). IFN-{alpha} treatment decreased the parasite load in the brain (Fig. 2A), and this correlated with a decrease in the numbers of iRBC in the blood (Fig. 2B).


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 2. IFN-{alpha} treatment decreases the number of parasites in the brain and in the blood. A, Quantification of parasite load in the brain. Mice (n = 5) were infected with PbA at day 0 and treated with IFN-{alpha} from days 0 to 7. Control mice were infected and received the diluent. The amount of parasite ribosomal DNA in the brain was measured by quantitative PCR (as described in Materials and Methods) 7 days postinfection. Results are expressed as the mean of brain parasite load ± SD of five mice. B, Blood parasite load (number of parasite per milliliter of blood) was determined in the same groups of mice. The differences between the two groups are statistically significant (p < 0.05, Mann-Whitney U test).

 
IFN-{alpha} treatment prevents up-regulation of ICAM-1 expression in brain endothelial cells in PbA-infected mice

ICAM-1 is up-regulated during PBA infection and has been shown to be essential for the development of CM (27, 28, 29, 30). Thus, we assessed the expression of this adhesion molecule in cerebral vessels. In mice with CM (Fig. 3E), immunostaining for ICAM-1 on endothelium of vessels and microvessels was more intense (42 ± 1.8 ICAM+ vessels per five microscope fields) than that in mice treated with IFN-{alpha} without CM (Fig. 3F; 7.5 ± 0.6 ICAM+ vessels per five microscope fields). In uninfected control and IFN-{alpha}-treated mice, only the major vessels, but not the microvessels, were positive for ICAM-1 expression (data not shown).


Figure 3
View larger version (114K):
[in this window]
[in a new window]

  		FIGURE 3. Brain sections from a PbA-infected mouse dying of CM showing hemorrhages (A) (red arrows) and mononuclear cells (C) accumulated along the endothelial lining (yellow arrows). Brain sections from a PbA-infected mouse treated with IFN-{alpha} without CM showing hemorrhages (B) (red arrows) and note the absence of mononuclear cells in vessels (D) (H&E, original amplification, x500). E, Expression of ICAM-1 in brain microvessels of a mouse infected with PbA and injected with diluent (white arrows); F, absence of staining in a PbA-infected mouse treated with IFN-{alpha}. Staining was observed only in the large vessels of the mouse (see text) (original magnification, x500). Control and IFN-{alpha}-treated infected mice were injected i.p. daily, respectively, with diluent or with IFN-{alpha} (4 x 105 U) and sacrificed at the same time.

 
Absence of leukocyte sequestration in the brains of PbA-infected mice treated with IFN-{alpha}

Qualitative histopathologic analysis of the brains from nontreated mice with CM revealed hemorrhages and microvessels containing iRBC and leukocytes (Fig. 3, A and B). Some of these mononuclear cells were in close contact with the endothelium (Fig. 3C). In contrast, in microvessels of infected mice treated with IFN-{alpha} from days 0 to 25 (without CM), no sequestration or accumulation of leukocytes and iRBC was observed (Fig. 3D). Since the distribution of the leukocytes in the capillaries was observed to be uneven (data not shown), we quantified the total number of cells present in the whole brain of uninfected and infected control and IFN-{alpha}-treated mice. In two separate experiments, all infected control-treated mice (diluent/PbA; Fig. 4) developed neurologic signs between days 7 and 10 and were sacrificed to analyze leukocyte accumulation in their brains. BSL were significantly more numerous in infected mice with CM (diluent/PbA group; Fig. 4) than in uninfected mice or infected mice treated with IFN-{alpha}. BSL from the different mouse groups were phenotyped. We observed a significant increase in the number of F4/80+ macrophages, Gr1+F4/80– neutrophils, and CD3+CD4+ and CD3+CD8+ T lymphocytes in infected mice with CM as compared with uninfected control mice. In contrast, PbA-infected mice treated with IFN-{alpha} did not show an increase in the number of these cell populations in the brain, and in particular those of CD8+ T cells, the subset responsible for CM in mice (Fig. 4). CD4+ and CD8+ T cell numbers in the brain of uninfected mice treated with IFN-{alpha} were similar to those in normal mice or mice treated with the diluent. It was of interest that IFN-{alpha} treatment significantly increased the number of macrophages and neutrophils in the brains of uninfected mice.


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 4. IFN-{alpha} treatment inhibits sequestration of leukocytes in the brains of PbA-infected mice with CM. Five mice were infected with 106 PbA iRBC and treated or with 4 x 105 mouse units of IFN-{alpha} from days 0 to 10. Brains were removed between days 7 and 9 when control PbA-infected mice displayed neurologic signs. Brains from the same number of mice were taken from the other groups. Enumeration of BSL was performed on perfused brains from five mice per group taken between day 7 and day 9. Cell numbers were determined as described in Materials and Methods. Samples of brain leukocyte suspensions were stained with mAbs specific for neutrophils, macrophages, and CD4+ and CD8+ T cells analyzed by flow cytometry. Absolute numbers of a given subset were calculated by multiplying the percentage of positive cells for this subset by the total number of BSL. Values are expressed as mean ± SD. The number of cells for every population tested (leukocytes, macrophages, neutrophils, CD3+, CD4+, and CD8+ T cells) was significantly (*, p < 0.05) higher in the group of mice infected with PbA and receiving the diluent alone than those in all other groups. IFN-{alpha} treatment of infected mice significantly (**, p < 0.05) prevented the accumulation of each leukocyte population investigated as compared with mice infected with PbA-infected, diluent-treated mice.

 
IFN-{alpha} administration has been shown to modify leukocyte circulation and induce peripheral lymphoadenopathy in mice (31). Therefore, we determined whether changes in the pattern of leukocyte migration in infected mice treated with IFN-{alpha} may have accounted for the relative decrease in BSL. Accordingly, spleen and lymph nodes were removed when control mice developed CM (between days 7 and 10 postinfection). As shown in Fig. 5A, PbA infection induced a splenomegaly characterized by increased cellularity, predominantly neutrophils and T cells, particularly CD8+ T cells. IFN-{alpha} treatment resulted in an inhibition of this increase in infected mice. However, while IFN-{alpha} treatment alone did not modify T cell number in the spleen of uninfected mice, IFN-{alpha} treatment in uninfected mice did result in an increase of neutrophil numbers in the spleen.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. IFN-{alpha} treatment prevents modification of peripheral lymphoid organ cellularity induced by PbA infection. Five mice were infected with 106 PbA iRBC and treated or with 4 x 105 mouse units of IFN-{alpha} from days 0 to 9. Spleens and lymph nodes were removed between days 7 and 9 when PbA-infected mice displayed neurologic signs. Spleens and lymph nodes from the same number of mice were taken from the other groups. Enumeration of spleen or lymph node cell subsets was performed on five mice per group. Cell numbers were determined as described in Fig. 2. Values are expressed as mean ± SD. A, IFN-{alpha} treatment inhibits the increase in spleen cellularity induced by PbA infection. The number of total splenocytes, of neutrophils, of CD3+, or of CD8+ T cells was significantly higher (*, p < 0.03) in the group of mice infected with PbA and receiving the diluent than those in the group of uninfected mice or in mice receiving the diluent alone. IFN-{alpha} treatment prevented significantly (**, p < 0.01) the accumulation of total splenocytes, CD3+, CD4+, or of CD8+ T cells but increased significantly (!=, p < 0.002) neutrophil numbers in infected mice as compared with mice infected with PbA and receiving the diluent alone. ND, not done. B, Levels of lymph node cell subsets in IFN-{alpha}-treated mice and infected with PbA. The number of total lymph nodes, of neutrophils, of CD3+, of CD4+, or of CD8+ T cells was reduced significantly (*, p < 0.05) in the group of mice infected with PbA and receiving the diluent alone than those in the group of naive mice and mice receiving the diluent alone. IFN-{alpha} treatment prevented significantly (**, p < 0.05) the decrease in numbers of total lymph node cells or numbers of CD3+, CD4+, or CD8+ T cells, but increased significantly (#, p < 0.05) neutrophil numbers in infected mice as compared with mice infected with PbA and receiving the diluent alone. The increase in neutrophil number induced by the PbA infection, IFN-{alpha}, and the combined IFN-{alpha} treatment, and PbA infection was significant (#, p < 0.005).

 
In contrast to the increased cellularity observed in the spleen of PbA-infected mice, decreased cellularity was observed in the lymph nodes of these mice due to a decrease in both CD4+ and CD8+ T cell numbers (Fig. 5B). IFN-{alpha} treatment inhibited this decrease of cellularity in infected mice. IFN-{alpha} treatment maintained lymph node cell numbers to levels similar to those in uninfected control mice. This treatment also induced a substantial increase in neutrophils.

Production of TNF and IFN-{gamma} in IFN-{alpha}-treated and control PbA-infected mice

TNF (TNF-{alpha} or lymphotoxin) and IFN-{gamma} have been shown to be essential for the development of CM (4, 29, 32, 33). Both cytokine levels were elevated in the blood of PbA-infected mice at the time of CM (Table II). IFN-{alpha} treatment led to a milder rise of TNF-{alpha} level but led to an increased level of IFN-{gamma}. Early production (1 day postinfection) of IFN-{gamma} has been reported to potentially prevent the development of CM (34); however, in an additional experiment, IFN-{alpha} treatment at day 0 did not induce the production of high levels of IFN-{gamma} the next day in PbA-infected mice.


View this table:
[in this window]
[in a new window]

  		Table II. Effect of IFN-{alpha} on the production of TNF and IFN-{gamma} during CM in PbA-infected micea

 
Intracellular cytokine staining on specific splenocyte populations was next performed to identify the cell population(s) able to produce more IFN-{gamma} or less TNF-{alpha} in response to IFN-{alpha} treatment. We chose to analyze the splenocytes because in vitro these cells from IFN-{alpha}-treated, PbA-infected mice were shown to produce higher levels of IFN-{gamma} than those from control PbA-infected mice (Fig. 6A). As shown in Fig. 6B, a higher percentage of splenic CD8+ but not CD4+ cells produce IFN-{gamma} after PMA/ionomycin stimulation. In the absence of this nonspecific stimulation, similar low percentages of IFN-{gamma}-producing CD4+ and CD8+ cells (~3%) were detected in all groups. TNF-{alpha} production was also tested in macrophages (F4/80+ cells) and CD4+ T cells. The percentage of TNF-{alpha}-producing F4/80+ macrophages or TNF-{alpha}-producing CD4+ T cells stimulated or not with PMA/ionomycin were identical in all mouse groups tested (naive, IFN-{alpha}-treated, and control PbA-infected mice).


Figure 6
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 6. Role of IFN-{gamma} in IFN-{alpha}-treated mice and PbA-infected mice. Four to five mice were infected with 106 PbA iRBC and treated or with 4 x 105 mouse units of IFN-{alpha} from days 0 to 5. In two groups, mice were treated with anti-IFN-{gamma} Abs from days 0 to 5. Animal were sacrificed at day 6, and spleens were removed from animals in the different groups. A, In vitro spontaneous IFN-{gamma} production by splenocytes from IFN-{alpha}-treated and control PbA-infected animals. B, Intracellular IFN-{gamma} staining in CD4 and CD8 T cells from naive, IFN-{alpha}-treated, and control PbA-infected mice. Splenocytes were subjected to FACS analysis after intracellular staining for IFN-{gamma}. Cytokine production was analyzed on gated CD8+ and CD4+ cells. C, Parasitemia of IFN-{alpha}-treated, PbA-infected mice treated with an anti-IFN-{gamma} mAb (from days 0 to 5). Controls groups were IFN-{alpha}-treated, PbA-infected mice, control PbA-infected mice, and control PbA-infected mice treated with anti-IFN-{gamma} mAb.

 
Effect of neutralization of IFN-{gamma} in IFN-{alpha}-treated, PbA-infected mice

To demonstrate whether the high levels of IFN-{gamma} were responsible for the inhibition of blood stage parasites, as shown in previous studies (35, 36, 37, 38), PbA-infected, IFN-{alpha}-treated mice were injected for 5 days with an anti-IFN-{gamma} mAb, and this treatment abrogated significantly the inhibitory effect of IFN-{alpha} (Fig. 6C).

Effect of blood transfusion on survival time, parasitemia, and anemia in PbA-infected, IFN-{alpha}-treated mice

Most or all mice treated daily with IFN-{alpha} survived CM (Table I and Fig. 7A), but they died many days subsequent to the onset of severe anemia around day 15, where a 75% decrease in RBC count is observed (Fig. 7D). Thus, we were interested in determining whether several blood transfusions would increase the survival time of IFN-{alpha}-treated mice having survived CM. Three groups of mice were infected with PbA, and on the 15th day (80% of PbA-infected diluent treated mice were dead), one group of six IFN-{alpha}-treated mice received daily transfusion of 109 purified RBC over 3 days while the control group of seven IFN-{alpha}-treated infected mice was not transfused. As can be noted from Fig. 7A, blood transfusion of IFN-{alpha}-treated mice resulted in an increased survival time, as compared with that of IFN-{alpha}-treated mice that were not transfused (p < 0.05) and in significant inhibition of parasitemia (Fig. 7B). However, blood transfusion did not diminish parasite load in IFN-{alpha}-treated mice (Fig. 7C), suggesting that the decrease of parasitemia is due to a dilution effect (i.e., increase of erythrocytes resulting from blood transfusion). It is important to note that transfused and untransfused mice continued to be treated with IFN-{alpha} during this period, and when IFN-{alpha} was discontinued on day 26, there was an increase in parasitemia in blood transfused mice.

We also measured the reticulocyte response to PbA infection. In PbA-infected, diluent-treated anemic mice, there was a brisk reticulocyte response beginning at day 15. IFN-{alpha} treatment inhibited this reticulocyte response. In blood-transfused, IFN-{alpha}-treated infected mice, the reticulocyte response was not only inhibited but markedly delayed until day 25, by which time IFN-{alpha} treatment was discontinued (Fig. 7E).

We determined the ratio of infected reticulocytes/total iRBC in the three groups of PbA-infected mice. It is apparent from Fig. 7G that our strain of PbA can infect both reticulocytes and RBC equally. Whereas the percentage of infected reticulocytes/iRBC increased to 100% in the diluent-treated mice, the ratio remained at ~20% in IFN-{alpha}-treated mice, indicating that 80% of mature RBC in these mice were infected.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have previously reported that treatment of mice, infected with the rodent malaria parasite P. yoelii, with a highly purified recombinant human IFN-{alpha} cross-reactive on mouse cells inhibits parasite development in the blood and thus prevents hepatomegaly and splenomegaly, two pathologies induced by any malaria infection (19). In this article, we have extended this study to determine the effect of IFN-{alpha} treatment on P. berghei infection, an extensively used model of CM.

We have shown here in five different experiments that daily treatment for 25 days with the same IFN-{alpha} preparation used in P. yoelii experiments protected mice from CM (87% of treated mice vs 6% of untreated mice). Protection from CM was most effective when the cytokine was administrated daily for 25 days, starting from the day of inoculation. However, even IFN-{alpha} treatment started 3 days after inoculation still afforded some degree of protection against CM.

The pathogenesis of CM is complex and results primarily from parasite multiplication in the blood and possibly from the sequestration of a small number of iRBC in the brain of infected mice (B. Lucas, unpublished results). IFN-{alpha} treatment resulted in a marked decrease in the number of PbA parasites in the blood, and there were far fewer parasites in the brain. However, it seems that it is not simply reduced parasitemia that accounts for the prevention of CM in IFN-{alpha}-treated mice, as we observed that CM still developed in some mice despite a very low parasitemia (Fig. 1). This suggests that the protective effect of IFN-{alpha} is mediated by additional mechanisms.

We have thus investigated the effect of IFN-{alpha} treatment on different mechanistic pathways that have been shown to be involved in CM. First, we have shown that IFN-{alpha} treatment has an effect on production of IFN-{gamma} and TNF, cytokines known to be essential for the development of CM (29, 32, 33). Circulating TNF levels, accounting for the cumulative levels of TNF-{alpha} and lymphotoxin cytokines as detected in our bioassay (23), were markedly inhibited by IFN-{alpha} treatment in infected animals, as compared with infected, untreated animals. IFN-{alpha} treatment also prevented the up-regulation of ICAM-1 on brain capillaries after PbA infection (Fig. 3, E and F). Increased expression of this adhesion molecule in the brain microvasculature has been implicated in the development of CM (27, 28) and has been shown to be induced by TNF-{alpha}, lymphotoxin and IFN-{gamma} (18, 29, 30). Although the milder rise of circulating TNF level could account for the prevention of up-regulation of ICAM-1 expression in IFN-{alpha}-treated infected mice, this was in conflict with the highly increased levels of IFN-{gamma}. However, Eguchi et al. (39) have shown that IFN-{alpha} is able to down-regulate ICAM-1 expression even on IFN-{gamma}-stimulated endothelial cells.

Although late production (after day 3) of IFN-{gamma} is essential for CM development, a recent report by Mitchell et al. (34) strongly suggested that early production of this cytokine can also prevent the development of CM. In mice coinfected with a different P. berghei strain, Pb K173, which do not develop CM, the high level of circulating IFN-{gamma} detected at day 1 postinfection, but not after, has confirmed a protective role against PbA-induced CM (34). Since in PbA-infected, IFN-{alpha}-treated mice or in control PbA-infected mice 1 day postinfection circulating IFN-{gamma} was not detected, it is thus unlikely that the effect of protective of IFN-{alpha} is mediated by an early production of IFN-{gamma}.

Another striking effect of IFN-{alpha} treatment was the inhibition of leukocyte sequestration in the brain, particularly CD8+ T cells. CD8+ T cells are the key effector cells in CM pathogenesis (7, 40, 41, 42), and we have shown that an accumulation of CD8+ T cells at the time of clinical signs is strongly associated with death due to CM (7). Different hypotheses can be proposed to explain this phenomenon. First, as mentioned above, as increased ICAM-1 expression was prevented by IFN-{alpha} treatment, this could have limited interactions between intravascular leukocytes and T cells and brain endothelial cells. However, this is unlikely, as it was shown recently that in mice deficient for ICAM-1, leukocyte accumulation in the brain during CM is still observed (28, 43). Second, although a single injection of IFN-{alpha} was shown previously to modify leukocyte circulation leading to leukocyte sequestration in lymphoid organs (31), repeated daily injections for 7 days did not induce leukocyte accumulation in spleen or lymph nodes in infected mice. On the contrary, IFN-{alpha} treatment prevented an increase in splenocyte numbers and led to a decrease in cell numbers in lymph nodes (Fig. 5). Thus, the absence of leukocyte accumulation in the brain does not seem to result from the modification of leukocyte circulation or migration to the brain. Thus, the most logical explanation for the absence of leukocyte migration is that it is the result of a decrease in systemic cellular responses, which might be due in part to parasite inhibition.

How do we explain the marked inhibitory effect of IFN-{alpha} treatment on parasitemia? We have previously shown that in a particular P. yoelii parasite strain, which invades only reticulocytes, inhibition of parasite levels by IFN-{alpha} treatment is due to an inhibition of production of reticulocytes in response to the infection (19). PbA parasites’ ability to invade reticulocytes and normocytes (Fig. 7G) (44) was not modified by IFN-{alpha} treatment, suggesting the involvement of an immune mechanisms in parasite inhibition. The most likely candidate was IFN-{gamma} since high levels of IFN-{gamma} are induced in infected mice by IFN-{alpha}. Ab-mediated neutralization (Fig. 6C) and splenocyte intracellular staining (Fig. 6B) demonstrated that IFN-{gamma} secreted by CD8+ T cells was mediating the inhibitory effect of IFN-{alpha}. IFN-{gamma} is important for the activation of, and phagocytosis by, macrophages and neutrophils, and these mechanisms have been associated with the control of the acute phase of parasitemia in rodent malaria (35, 36, 37, 38). The fact that, despite strong inhibition, parasites were not cleared and that when IFN-{alpha} treatment was discontinued parasite multiplication resumed suggests that protective adaptive immune responses did not develop. Even in those IFN-{alpha}-treated mice that received blood transfusions to counteract the development of a lethal anemia, protection was observed only when IFN-{alpha} treatment was maintained. However, it is possible that a longer transfusion protocol might have helped control parasite development.

We observed that, despite the inhibition of PbA parasitemia, the onset and degree of anemia was similar for both control and IFN-{alpha}-treated mice. In a previous report using P. yoelii-infected C57BL/6J mice, we showed that there was an inhibition in blood cell precursors (CFU-E) in IFN-{alpha}-treated mice, which resulted in the reduced reticulocytosis and thus contributed to the profound anemia in these mice. As already postulated, inhibition of erythropoiesis by IFN-{alpha} may have been due to a direct effect (45, 46, 47, 48) or to an indirect effect through the induction of other molecules, which may inhibit erythropoiesis such as NO or IFN-{gamma} (49, 50, 51).

To date, there have been a few reports (52, 53, 54) showing that IFN-{alpha} is produced during infection with P. falciparum. In a study with infected African children, IFN-{alpha} levels were found to be significantly higher in children with mild malaria than in those with severe malaria (52). Recently, Aucan et al. (55) have found an association with a polymorphism in the IFN-{alpha} receptor promoter and protection against CM but not severe anemia in Gambia. This suggests that high levels of IFN-{alpha} may well be protective against CM.

Finally, viral infections are very common in most areas where malaria is endemic, and these infections may induce interferonemia (56). Thus, repeated viral infections might influence the outcome of CM in Plasmodium-infected humans. These and previous studies support the theory that IFN-{alpha}, which is being widely used in the treatment of viral, neoplastic, and autoimmune disease, may also prove useful in the adjunct treatment of falciparum malaria.


   Acknowledgments
 
We thank Veronique Amani for technical help at the early stage of the project. We also thank Sarah Potter for reviewing the manuscript and for helpful comments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported in part by grants from INSERM, Fundação para Ciencias e Technologia, Fondation de La Recherche Médicale (10000017), Association Française du Sang (96007), and Fondation Electricité et Santé (to L.R.). A.M.V. held a fellowship from Fundação para a Ciência e a Tecnologia (BD9255/96). E.B. held a fellowship from Ministere de L’Education Nationale, de la Recherche et de la Technologie. A.C.G. was supported by a grant from the Carlsberg Foundation. Back

2 Current address: Center for Vaccinology and Neonatal Immunology, University of Geneva, Centre Medical Universitaire, Geneva, Switzerland. Back

3 Address correspondence and reprint requests to Dr. Laurent Rénia, Département d’Immunologie, Institut Cochin, Hôpital Cochin, Bâtiment Gustave Roussy, 27 rue du Fb St. Jacques, 75014 Paris, France. E-mail address: renia{at}cochin.inserm.fr Back

4 Abbreviations used in this paper: CM, cerebral malaria; BSL, brain-sequestered leukocyte; Hb, hemoglobin; iRBC, infected RBC; PbA, Plasmodium berghei ANKA. Back

Received for publication September 27, 2006. Accepted for publication March 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Miller, L. H., D. I. Baruch, K. Marsh, O. K. Doumbo. 2002. The pathogenic basis of malaria. Nature 415: 673-679. [Medline]
  2. Coltel, N., V. Combes, N. H. Hunt, G. E. Grau. 2004. Cerebral malaria—a neurovascular pathology with many riddles still to be solved. Curr. Neurovasc. Res. 1: 91-110. [Medline]
  3. Schofield, L., G. E. Grau. 2005. Immunological processes in malaria pathogenesis. Nat. Rev. Immunol. 5: 722-735. [Medline]
  4. Hunt, N. H., G. E. Grau. 2003. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 24: 491-499. [Medline]
  5. Engwerda, C. R., E. Belnoue, A. C. Gruner, L. Renia. 2005. Experimental models of cerebral malaria. Curr. Top. Microbiol. Immunol. 297: 103-143. [Medline]
  6. Hearn, J., N. Rayment, D. N. Landon, D. R. Katz, J. B. De Souza. 2000. Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature. Infect. Immun. 68: 5364-5376. [Abstract/Free Full Text]
  7. Belnoue, E., M. Kayibanda, A. M. Vigario, J. C. Deschemin, N. van Rooijen, M. Viguier, G. Snounou, L. Renia. 2002. On the pathogenic role of brain-sequestered {alpha}betaCD8+ T cells in experimental cerebral malaria. J. Immunol. 169: 6369-6375. [Abstract/Free Full Text]
  8. Menendez, C., A. F. Fleming, P. L. Alonso. 2000. Malaria-related anaemia. Parasitol. Today 16: 469-476. [Medline]
  9. Rosenberg, E. B., G. T. Strickland, S. L. Yang, G. E. Whalen. 1994. IgM antibodies to red cells and autoimmune anemia in patients with malaria. Am. J. Trop. Med. Hyg. 22: 146-152.
  10. Looareesuwan, S., T. M. Davis, S. Pukrittayakamee, W. Supanaranond, V. Desakorn, K. Silamut, S. Krishna, S. Boonamrung, N. J. White. 1991. Erythrocyte survival in severe falciparum malaria. Acta Trop. 48: 263-270. [Medline]
  11. Weatherall, D. J., S. H. Abdalla. 1982. The anemia of Plasmodium falciparum malaria. Brit. Med. Bull. 38: 147-151. [Free Full Text]
  12. Clark, I. A., G. Chaudhri. 1988. Tumor necrosis factor may contribute to the anemia of malaria by causing dyserythropoiesis and erythrophagocytosis. Br. J. Haematol. 70: 99-103. [Medline]
  13. Chang, K. H., M. M. Stevenson. 2004. Malarial anemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria. Int. J. Parasitol. 34: 1501-1516. [Medline]
  14. McGuire, W., J. C. Knight, A. V. S. Hill, C. E. M. Allsopp, B. M. Greenwood, D. P. Kwiatkowski. 1999. Severe malarial anemia and cerebral malaria are associated with different tumor necrosis factor promoter alleles. J. Infect. Dis. 179: 287-290. [Medline]
  15. Othoro, C., A. A. Lal, B. L. Nahlen, D. Koech, A. S. S. Orago, V. Udhayakumar. 1999. A low interleukin-10 tumor necrosis factor {alpha} ratio is associated with malaria anemia in children residing in a holoendemic malaria region in Western Kenya. J. Infect. Dis. 179: 279-282. [Medline]
  16. Kurtzhals, J. A. L., V. Adabayeri, B. Q. Goka, D. B. Akanmori, J. O. Oliver-Commey, F. K. Nkrumah, C. Behr, L. Hviid. 1988. Low plasma concentrations of interleukin 10 in severe malarial anemia compared with cerebral and uncomplicated malaria. Lancet 351: 1768-1772.
  17. Tzung, S. P., T. C. Mahl, P. Lance, V. Andersen, S. A. Cohen. 1992. Interferon {alpha} prevents endotoxin-induced mortality in mice. Eur. J. Immunol. 22: 3097-3101. [Medline]
  18. Amani, V., M. I. Boubou, S. Pied, M. Marussig, D. Walliker, D. Mazier, L. Renia. 1998. Cloned lines of Plasmodium berghei ANKA differ in their abilities to induce experimental cerebral malaria. Infect. Immun. 66: 4093-4099. [Abstract/Free Full Text]
  19. Vigario, A. M., E. Belnoue, A. M. Cumano, M. Marussig, F. Miltgen, I. Landau, D. Mazier, I. Gresser, L. Renia. Inhibition of Plasmodium yoelii blood-stage malaria by interferon {alpha} through the inhibition of the production of its target cell, the reticulocyte. Blood 97: 3966-3971.
  20. Taverne, J., N. Sheikh, J. B. De Souza, J. H. L. Playfair, L. Probert, G. Kollias. 1994. Anemia and resistance to malaria in transgenic mice expressing human tumor necrosis factor. Immunology 82: 397-403. [Medline]
  21. Meister, A., G. Uze, K. E. Mogensen, I. Gresser, M. G. Tovey, M. Grutter, F. Meyer. 1986. Biological activities and receptor binding of two human recombinant interferons and their hybrids. J. Gen. Virol. 67: 1633-1643. [Abstract/Free Full Text]
  22. Hulier, E., P. Petour, G. Snounou, M. P. Nivez, F. Miltgen, D. Mazier, L. Renia. 1996. A method for the quantitative assessment of malaria parasite development in organs of the mammalian host. Mol. Biochem. Parasitol. 77: 127-135. [Medline]
  23. Espevik, T., J. Nissen-Meyer. 1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods 95: 99-105. [Medline]
  24. Mosmann, T. R.. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63. [Medline]
  25. Belnoue, E., F. T. M. Costa, T. Frankenberg, A. M. Vigario, T. Voza, N. Leroy, M. M. Rodrigues, I. Landau, G. Snounou, L. Renia. 2004. Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment. J. Immunol. 172: 2487-2495. [Abstract/Free Full Text]
  26. Franke-Fayard, B., C. J. Janse, M. Cunha-Rodrigues, J. Ramesar, P. Buscher, I. Que, C. Lowik, P. J. Voshol, M. A. den Boer, S. G. van Duinen, et al 2005. Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration. Proc. Natl. Acad. Sci. USA 102: 11468-11473. [Abstract/Free Full Text]
  27. Favre, N., C. Da Laperrousaz, B. Ryffel, N. A. Weiss, B. Imhof, W. Rudin, R. Lucas, P. F. Piguet. 1999. Role of ICAM-1 (CD54) in the development of murine cerebral malaria. Microbes Infect. 1: 961-968. [Medline]
  28. Li, J., W. L. Chang, G. Sun, H. L. Chen, R. D. Specian, S. M. Berney, D. Kimpel, D. N. Granger, H. C. van der Heyde. 2003. Intercellular adhesion molecule 1 is important for the development of severe experimental malaria but is not required for leukocyte adhesion in the brain. J. Investig. Med. 51: 128-140. [Medline]
  29. Rudin, W., H. P. Eugster, G. Bordmann, J. Bonato, M. T. Muller, M. Yamage, B. Ryffel. 1997. Resistance to cerebral malaria in tumor necrosis factor {alpha}/beta-deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response. Am. J. Pathol. 150: 257-266. [Abstract]
  30. Bauer, P. R., H. C. van der Heyde, G. Sun, R. D. Specian, D. N. Granger. 2002. Regulation of endothelial cell adhesion molecule expression in an experimental model of cerebral malaria. Microcirculation 9: 463-470. [Medline]
  31. Gresser, I., D. Guy-Grand, C. Maury, M. T. Manoury. 1981. Interferon induces peripheral lymphoadenopathy in mice. J. Immunol. 127: 1569-1575. [Abstract]
  32. Amani, V., A. M. Vigario, E. Belnoue, M. Marussig, L. Fonseca, D. Mazier, L. Renia. 2000. Involvement of IFN-{gamma} receptor-mediated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection. Eur. J. Immunol. 30: 1646-1655. [Medline]
  33. Engwerda, C. R., T. L. Mynott, S. Sawhney, J. B. De Souza, Q. D. Bickle, P. M. Kaye. 2002. Locally up-regulated lymphotoxin {alpha}, not systemic tumor necrosis factor {alpha}, is the principle mediator of murine cerebral malaria. J. Exp. Med. 195: 1371-1377. [Abstract/Free Full Text]
  34. Mitchell, A., A. M. Hansen, L. Hee, H. J. Ball, S. M. Potter, J. C. Walker, N. H. Hunt. 2005. Early cytokine production is associated with protection from murine cerebral malaria. Infect. Immun. 73: 5645-5653. [Abstract/Free Full Text]
  35. Shear, H. L., R. Srinivasan, T. Nolan, C. Ng. 1989. Role of IFN-{gamma} in lethal and nonlethal malaria in susceptible and resistant murine hosts. J. Immunol. 143: 2038-2044. [Abstract]
  36. Meding, S. J., S. C. Cheng, B. Simon-Haarhaus, J. Langhorne. 1990. Role of {gamma} interferon during infection with Plasmodium chabaudi chabaudi. Infect. Immun. 58: 3671-3678. [Abstract/Free Full Text]
  37. van der Heyde, H. C., B. Pepper, J. Batchelder, F. K. Cigel, W. P. Weidanz. 1997. The time course of selected malarial infections in cytokine-deficient mice. Exp. Parasitol. 85: 206-213. [Medline]
  38. Yoneto, T., T. Yoshimoto, C. R. Wang, Y. Takahama, M. Tsuji, S. Waki, H. Nariuchi. 1999. {gamma} Interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical. J. Immunol. 67: 2349-2356.
  39. Eguchi, K., A. Kawakami, M. Nakashima, H. Ida, S. Sakito, N. Matsuoka, K. Terada, M. Sakai, Y. Kawabe, T. Fukuda, et al 1992. Interferon {alpha} and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells. Clin. Exp. Immunol. 88: 448-454. [Medline]
  40. Nitcheu, J., O. Bonduelle, C. Combadiere, M. Tefit, D. Seilhean, D. Mazier, B. Combadiere. 2003. Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis. J. Immunol. 170: 2221-2228. [Abstract/Free Full Text]
  41. Boubou, M. I., A. Collette, D. Voegtle, D. Mazier, P. A. Cazenave, S. Pied. 1999. T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR Vbeta8 during experimental cerebral malaria. Int. Immunol. 11: 1553-1562. [Abstract/Free Full Text]
  42. Yanez, D. M., D. D. Manning, A. J. Cooley, W. P. Weidanz, H. C. van der Heyde. 1996. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J. Immunol. 157: 1620-1624. [Abstract]
  43. Sun, G., W. L. Chang, J. Li, S. M. Berney, D. Kimpel, H. C. van der Heyde. 2003. Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria. Infect. Immun. 71: 6553-6561. [Abstract/Free Full Text]
  44. Deharo, E., F. Cocquelin, A. G. Chabaud, I. Landau. 1996. The erythrocytic schizogony of two synchronized strains of Plasmodium berghei, NK65 and ANKA, in normocytes and reticulocytes. Parasitol. Res. 82: 178-182. [Medline]
  45. Broxmeyer, H. E., L. Lu, E. Platzer, C. Feit, L. Juliano, B. Y. Rubin. 1983. Comparative analysis of the influences of human {gamma}, {alpha} and beta interferons on human multipotential (CFU-GM) progenitor cells. J. Immunol. 131: 1300-1305. [Abstract]
  46. Means, R. T., S. B. Krantz. 1996. Inhibition of human erythroid colony-forming units by interferons {alpha} and beta: differing mechanisms despite shared receptor. Exp. Hematol. 24: 204-208. [Medline]
  47. Raefsky, E. L., L. C. Platanias, N. C. Zoumbos, N. S. Young. 1985. Studies of interferon as a regulator of hematopoietic cell proliferation. J. Immunol. 135: 2507-2512. [Abstract]
  48. Tarumi, T., K. Sawada, N. Sato, S. Kobayashi, H. Takano, T. Yasukouchi, T. Takahashi, S. Sekiguchi, T. Koile. 1995. Interferon {alpha}-induced apoptosis in human erythroid progenitors. Exp. Hematol. 23: 1310-1318. [Medline]
  49. Angulo, I., R. Rodriguez, B. Garcia, M. Medina, J. Navarro, J. L. Subiza. 1995. Involvement of nitric oxide in marrow-derived natural suppressor activity: its dependence on IFN-{gamma}. J. Immunol. 155: 15-26. [Abstract]
  50. Shami, P. J., J. B. Weinberg. 1996. Differential effects of nitric oxide on erythroid and myeloid colony growth from CD341 human bone marrow cells. Blood 87: 977-982. [Abstract/Free Full Text]
  51. Zoumbos, N. C., J. Y. Djeu, N. S. Young. 1984. Interferon is the suppressor of hematopoiesis generated by stimulated lymphocytes in vitro. J. Immunol. 133: 769-774. [Abstract]
  52. Luty, A. J., D. J. Perkins, B. Lell, R. Schmidt-Ott, L. G. Lehman, D. Luckner, B. Greve, P. Matousek, K. Herbich, D. Schmid, et al 2000. Low Interleukin-12 activity in severe Plasmodium falciparum malaria. Infect. Immun. 68: 3909-3915. [Abstract/Free Full Text]
  53. Ojo-Amaize, E. A., L. S. Salimonu, A. I. Williams, O. A. Akinwolere, R. Shabo, G. V. Alm, H. Wigzell. 1981. Positive correlation between degree of parasitemia, interferon titers, and natural killer cell activity in Plasmodium falciparum-infected children. J. Immunol. 127: 2296-2300. [Abstract]
  54. Ronnblom, L., E. A. Ojo-Amaize, L. Franzen, H. Wigzell, G. V. Alm. 1985. Plasmodium falciparum parasites induce interferon production in human peripheral blood "null" cells in vitro. Parasite Immunol. 5: 165-172.
  55. Aucan, C., A. J. Walley, B. J. Hennig, J. Fitness, A. J. Frodsham, L. Zhang, D. P. Kwiatkowski, A. V. S. Hill. 2003. Interferon {alpha} receptor-1 (IFNAR1) variants are associated with protection against cerebral malaria in The Gambia. Genes Immun. 4: 275-282. [Medline]
  56. Kurane, I., B. L. Innis, S. Nimmannitya, A. Nisalak, A. Meager, F. A. Ennis, Jr. 1993. High levels of interferon {alpha} in the sera of children with Dengue virus infection. Am. J. Trop. Med. Hyg. 48: 222-229. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
M.-F. Penet, M. Abou-Hamdan, N. Coltel, E. Cornille, G. E. Grau, M. de Reggi, and B. Gharib
Protection against cerebral malaria by the low-molecular-weight thiol pantethine
PNAS, January 29, 2008; 105(4): 1321 - 1326.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
F. E. Lovegrove, S. A. Gharib, S. N. Patel, C. A. Hawkes, K. C. Kain, and W. C. Liles
Expression Microarray Analysis Implicates Apoptosis and Interferon-Responsive Mechanisms in Susceptibility to Experimental Cerebral Malaria
Am. J. Pathol., December 1, 2007; 171(6): 1894 - 1903.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vigário, A. M.
Right arrow Articles by Rénia, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vigário, A. M.
Right arrow Articles by Rénia, L.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS