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Protective Roles of Mast Cells and Mast Cell-Derived TNF in Murine Malaria

Takahisa Furuta^1,*, Takane Kikuchi^2,*, Yoichiro Iwakura and Naohiro Watanabe

^* Department of Microbiology and Immunology, Division of Infectious Genetics, and Division of Cell Biology, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and Department of Tropical Medicine, Jikei University School of Medicine, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TNF plays important roles in the protection and onset of malaria. Although mast cells are known as a source of TNF, little is known about the relationship between mast cells and pathogenesis of malaria. In this study, mast cell-deficient WBB6F₁-W/W^v (W/W^v) and the control littermate WBB6F₁^+/+ (+/+) mice were infected with 1 x 10⁵ of Plasmodium berghei ANKA. +/+ mice had lower parasitemia with higher TNF levels, as compared with W/W^v mice. Diminished resistance in W/W^v mice was considered to be due to mast cells and TNF. This fact was confirmed by experiments in W/W^v mice reconstituted with bone marrow-derived mast cells (BMMCs) of +/+ mice or of TNF^–/– mice. W/W^v mice with BMMCs of +/+ mice exhibit lower parasitemia and mortality accompanying significantly higher TNF levels than those of W/W^v mice. Parasitemia in W/W^v mice with BMMCs of TNF^–/– mice was higher than that in +/+ mice. Activation of mast cells by anti-IgE or compound 48/80 resulted in release of TNF and decrease of parasitemia. In addition, splenic hypertrophy and increased number of mast cells in the spleen were observed after infection in +/+ mice and W/W^v mice reconstituted with BMMCs of +/+ mice as compared with W/W^v mice. These findings propose a novel mechanism that mast cells and mast cell-derived TNF play protective roles in malaria.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Malaria is the most serious parasitic disease of humans in the world. Approximately 2.7 million people die from the disease every year, and most of them are children under 5 years old (1). Malaria infections can cause fever, severe anemia, coma, and renal failure in children and adults (2, 3), and poor birth outcomes in pregnant women (4). The pathogenesis of malaria is complex, and the immune system may mediate both protection from malaria and development of disease. Many studies (3, 5, 6, 7, 8, 9, 10, 11) indicate that elevations in immune mediators such as IL-1, IL-6, IL-8, IL-10, TNF, and NO have been associated with disease severity. Especially, high concentrations of TNF have been associated with disease severity in human Plasmodium falciparum infections and several animal malarias (8). Mast cells can be a major source of TNF (12, 13), suggesting that they may play important roles in protection and disease in malaria. Despite these observations, little is known about the relationship between mast cells and malaria pathogenesis.

Mast cells are a pivotal effector cell in allergic disease by their capacity to respond rapidly to stimuli and release a wide range of preformed and newly generated proinflammatory mediators (14). They are best known for IgE-mediated immediate-type hypersensitivity reactions. Mast cells are abundant in tissues exposed to the external environment including the skin, intestinal tract, and trachea, and also normally present in heart, lymph nodes, spleen, and CNS (15). Because these sites are also common portals of infection, mast cells are likely to be one of the first inflammatory cells to make contact with invading pathogens. Mast cells can be activated by a multitude of stimuli such as Abs, cytokines, chemokines, and neuropeptides, and also exert their biological effects by releasing preformed and de novo-synthesized mediators such as histamine, proteases, leukotrienes, PGs, and various cytokines, including TNF (16, 17, 18, 19, 20). Although blood monocytes, tissue macrophages, and Kupffer cells of liver are the best known sources of TNF, mast cells are the only cell type capable of storing presynthesized TNF (18, 2). Because of this unique capability, mast cells provide the only readily available source of TNF within peripheral tissues during the early course of infection. In deed, mast cells secrete TNF within minutes of bacterial challenge and then clear invading bacteria (12, 13, 21, 22). Recently, mast cells have been reported to bind various bacteria via TLRs 2 and 4 (23) and then secrete TNF. The mast cell therefore represents not only a sustainable source but also a potential "early" source of TNF, which may be especially important when rapid mobilization of local responses is critical, as in innate immune responses to bacteria and in acquired immune responses to pathogens. Despite the findings that mast cells are vital in mediating clearance of pathogens at sites of infection through the release of TNF and recruitment of immune inflammatory cells (24), little is known about the relationship between mast cells and malaria. In this study, we demonstrated for the first time the importance of mast cells for host immunity in malaria infection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice and infection of Plasmodium berghei ANKA

Genetically mast cell-deficient WBB6F₁-W/W^v (W/W^v) and the control WBB6F₁^+/+ (+/+) mice, and C57BL/6 were obtained from Japan SLC. TNF^–/– mice were bred and maintained under specific pathogen-free conditions in an environmentally controlled clean room at the Center for Experimental Medicine, Institute of Medical Science (University of Tokyo, Tokyo, Japan) (25). Five- to 6-wk-old W/W^v, +/+, and C57BL/6 mice were inoculated i.v. with 1 x 10⁵ P. berghei-infected erythrocytes to examine the parasite growth. Male Wistar rats aged 8 wk old were also obtained from Japan SLC. The animal experiments were approved by the Committee for Animal Experiment of the Institute of Medical Science, University of Tokyo.

In vitro infectivity of P. berghei ANKA to RBC obtained from W/W^v and +/+ mice

To examine the infectivity of P. berghei ANKA to RBCs obtained from W/W^v and +/+ mice, in vitro culture of P. berghei ANKA was performed according to the method previously reported by Janse and Waters (26), and P. berghei ANKA merozoites were prepared from P. berghei ANKA-infected rats. Briefly, Wistar rats were infected with P. berghei ANKA to serve as a source of blood-stage parasites for the culture and purification. Rats were infected i.p. with 1 x 10⁷ P. berghei ANKA-infected erythrocytes, and then phenylhydorazine-HCl (40 mg/kg body weight; Sigma-Aldrich) was injected i.p. to induce reticulocytosis at day 2 after the infection. Blood with parasitemia of 3% was collected by cardiac puncture under anesthesia at day 5 after the infection. The infected blood was washed by RPMI 1640 (Invitrogen Life Technologies) with pH 7.3, 20% heat-inactivated FBS (Invitrogen Life Technologies) and then resuspended in 150 ml of culture medium. Culture flasks were incubated with gas mixture (10% O₂, 5% CO₂, 85% N₂) at 37°C with shaking. After overnight incubation, thin blood smears stained with Giemsa were made of erythrocyte culture to check mature schizont formation. After observed predominant mature schizonts, the schizonts were separated from uninfected erythrocytes by 55% Nycodenz (Sigma-Aldrich)/PBS density gradient. After centrifugation for 25 min at 200 x g, schizonts were collected on interface layer, providing preparations consisting of >95% pure schizonts. To collect pure viable merozoites, the schizonts in medium were ruptured by vigorously spinning a magnetic bar, and culture medium containing merozoites were passed through a polycarbonate sieve of 2-µm pore size. Different dose of P. berghei ANKA merozoites obtained as described was added in RBC culture at 5% hematocrit (Ht),³ and then the infectivity of merozoites to RBCs of W/W^v or +/+ mice was examined in vitro. After the incubation with gas mixture at 37°C for 20 h, blood smears stained with Giemsa were made from each culture well to examine the parasitemia.

Bone marrow-derived mast cell (BMMCs) differentiation and reconstitution

Bone marrow cells were harvested from both femurs of wild-type^+/+ or TNF^–/– mice and cultured in complete RPMI 1640 supplement with 10% FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate, 50 mM 2-ME, and 4 ng/ml IL-3 (27). This addition consistently increased the viability of the cultured cells. BMMCs were used at >99% purity, as determined by flow cytometric analysis (FcRI⁺c-kit^high) and normal granular staining with metachromatic dyes such as toluidine blue. At the time of reconstitution, BMMCs (5 x 10⁶/mouse) were injected i.v. into groups of five to seven W/W^v mice. Mice were housed for 8 wk before infection with P. berghei ANKA.

Evaluation of anemic condition in W/W^v mice after BMMC transfer

To examine the possibility that reconstitution of W/W^v mice with BMMCs affects macrocytic anemia in W/W^v mice, we examined Ht and mean corpuscular volume (MCV) in W/W^v mice with or without transfer of BMMCs to evaluate the condition of macrocytic anemia. W/W^v mice were transferred i.v. with 5 x 10⁶ BMMCs from +/+ mice, and then mice were sacrificed 8 wk later to collect blood. Blood with anticoagulant (1.5 mg/ml dipotassium EDTA; Sigma-Aldrich) was collected from mice by cardiac puncture under anesthesia. To evaluate states of macrocytic anemia in W/W^v mice after BMMCs transfer, total RBC count and Ht in W/W^v mice with BMMCs were determined using Coulter Micro Diff II machine (Beckman Coulter) to compare those of age-matched normal W/W^v and +/+ mice. MCV (femtoliter) was calculated as follows: Ht (percentage)/RBCs (mm³) x 10.

Cytokine assay by ELISA

To assay cytokine level, serum was collected from infected and uninfected mice as indicated. TNF level was assessed using TNF- ELISA set according to the manufacturer’s instructions (R&D Systems).

TNF bioassay

TNF activity in the serum was measured by bioassay using a modified L-929 cytotoxicity that is based on reduction of tetrazolium dye (28, 29). Murine TNF-sensitive L-929 cells were cultured in 96-well plates as a concentration of 2.5 x 10⁴/well in RPMI 1640 with L-glutamine, penicillin, and streptomycin containing 10% FBS. After overnight incubation at 37°C, the samples such as 2-fold dilutions of murine recombinant TNF- (R&D Systems) or sera obtained from P. berghei ANKA-infected mice were made in RPMI 1640 containing 3% FBS and final concentration of 0.5 µg/ml actinomycin-D (Sigma-Aldrich), and the dilutions were added in 0.1-ml volumes in triplicate to the cultured cells. Cell control wells received only medium, whereas the lysis control received 0.05% Nonidet P-40 (Sigma-Aldrich) lysis buffer. After 20 h, supernatants were removed from each well, and 0.05 ml of 2 mg/ml MTT dye (Sigma-Aldrich) in saline solution was added, and then the culture plates were incubated for an additional 4 h. Supernatants were removed without disturbing the formed dark blue crystals. DMSO (Wako Pure Chemical) was used in 0.2 ml/well to solubilize these crystals, and the OD₅₅₀ was measured. Cell wells exhibiting an A₅₅₀ closest to 50% of the lysis control are considered to represent 50% lysis of the L-929 cells. TNF units per milliliter of a sample were calculated by multiplying the reciprocal of the highest dilution resulting in 50% lysis.

Treatment of mouse anti-TNF Ab

Anti-mouse TNF polyclonal rabbit Ab (25 µg/mouse; Pierce Biotechnology) or an isotype control Ab (rabbit IgG 25 µg/mouse; Pierce Biotechnology) was injected i.p. into C57BL/6 mice at the same time when 1 x 10⁵ P. berghei ANKA was inoculated. Injection of the Abs was repeated on the third day after the infection until end of the infection.

Effect of mast cell activation by anti-IgE () mAb or compound 48/80

P. berghei ANKA-infected C57BL/6 mice were injected i.p. with 2 mg of anti-mAb 6HD5 (rat IgG2a) or normal rat IgG2a at day 8 or 11 after the infection (30). The parasitemia and TNF levels in the blood were measured 24 h later. Compound 48/80 (Sigma-Aldrich) was injected i.v. into P. berghei ANKA-infected C57BL/6 mice at a dose of 1.2 mg/kg body weight of mice with normal saline as a vehicle (31). The percentage of parasitemia in the blood was measured 24 h later.

Effects of compound 40/80 or LPS on mast cells and macrophages

To examine the effect of compound 48/80 or LPS on the parasites and macrophages in vivo, we examined parasite growth in P. berghei ANKA-infected W/W^v mice after treatment of compound 48/80 or LPS. W/W^v and +/+ mice were infected i.v. with 1 x 10⁵ P. berghei ANKA, and then W/W^v mice were divided into three groups after the infection. The first group of mice were treated i.v. with compound 48/80 (1.2 mg/kg/mouse), the second group were treated i.p. with LPS (1 µg/mouse; Sigma-Aldrich), and other groups of W/W^v and +/+ mice were left without any treatment. Compound 48/80 or LPS was injected into C57BL/6 mice at the same time when 1 x 10⁵ P. berghei ANKA was inoculated and then repeated the third day after the infection until end of the infection. Parasitemia in blood smears obtained from those mice was monitored daily after the infection.

Histopathology

The main organs of mice were fixed in 10% neutral-buffered formalin and then embedded in paraffin. Tissue sections were stained with H&E for evaluation of pathologic changes or with toluidine blue for detection of mast cells.

TNF production in peritoneal mast cells and macrophages in vitro

To confirm the source of cell type secreting TNF in vivo, we measured TNF levels in mast cells and macrophages obtained from the peritoneal cavity of mice. Peritoneal mast cells and macrophages were obtained from P. berghei ANKA-infected C57BL/6 mice at day 10 after the infection. Mast cells were collected from the peritoneal cavity of the mice by washing with Tyrode’s buffer, and then peritoneal mast cells were isolated using 22.5% Nycodenz density gradients, according to the method previously reported (32). The isolated cells were washed with Tyrode’s buffer three times. The purity of recovered peritoneal mast cells was >98% according to May-Grünwald-Giemsa staining. Peritoneal macrophages were obtained from the peritoneal cavity of mice by washing with ice-cold HBSS (Invitrogen Life Technologies). Cells were cultured for 2 h and washed with HBSS to remove nonadherent cells. Adherent cells were used as peritoneal macrophages. A total of 1 x 10⁵ of mast cells or macrophages was seeded in 96-well plates and cultured in RPMI 1640 supplemented with 10% FBS and then incubated with soluble P. berghei ANKA Ag (20 µg/well) for 24 h. Soluble P. berghei ANKA Ag was prepared from P. berghei ANKA-infected RBCs by sonication at 5A for 30 s, after twice freezing and thawing. After incubation, culture supernatants were collected, and then TNF- production was measured by ELISA (R&D Systems).

Parasitemia

For assessment of parasite growth in P. berghei ANKA-infected mice, thin blood smears were made using aliquots of RBCs obtained by tail vein puncture and stained with Giemsa. More than 1000 erythrocytes were counted by microscopy to determine the percentage of parasitized cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Growth of P. berghei ANKA on mast cell-deficient W/W^v mice

To directly evaluate the in vivo role of mast cells in murine malaria, mast cell-deficient W/W^v and their control normal +/+ mice were infected with P. berghei ANKA. W/W^v mice developed significantly higher parasitemia than control +/+ mice, as indicated by higher daily parasitemia (Fig. 1A). This finding suggests the presence of mast cell-mediated resistance against P. berghei infection. Because mast cells can be a major source of TNF, we tested TNF levels in the circulation following infection in both W/W^v and +/+ mice. TNF levels in sera of +/+ mice with the lower parasitemia were significantly higher than those in W/W^v and uninfected +/+ mice (Fig. 1B), suggesting important roles of mast cells in resistance to P. berghei NKA infection. The number of RBCs in W/W^v mice was about two-thirds that of +/+ mice because of severe macrocytic anemia. To examine the possibility that the severe anemia affected parasite growth in W/W^v mice, we transferred RBC obtained from normal +/+ mice to W/W^v mice to adjust the number of RBCs to the same levels as in +/+ mice. We then compared the growth of the parasite in W/W^v and +/+ mice. A significant difference in parasite growth was still observed between W/W^v mice with the adjusted number of RBCs and +/+ mice (Fig. 1A). Thus, the anemia did not appear to affect the parasite growth in W/W^v mice.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Parasitemia of P. berghei ANKA in mast cell-deficient W/WV and control +/+ mice. A, Mast cell-deficient W/WV and +/+ mice (n = 6 each) were inoculated i.v. with 1 x 105 P. berghei ANKA. To examine the possibility that the severe anemia affected parasite growth in W/Wv mice, we transferred RBCs obtained from normal +/+ mice to W/Wv mice to adjust the number of RBCs to be the same level as in +/+ mice at 1 day before infection. We then compared the parasitemia in them (mean ± SD; *, p < 0.03). This experiment was repeated three times with similar results. B, The levels of serum TNF in mast cell-deficient W/WV and +/+ mice (n = 5 each) were measured by ELISA at day 10 after 1 x 105 P. berghei ANKA infection (mean ± SD; *, p < 0.01). This experiment was repeated three times with similar results.

W/W^v mice have a macrocytic anemia due to a defect in erythropoiesis. Because RBCs in W/W^v mice are abnormal and probably are at different maturation states than those in +/+ mice, this defect alone might result in the differences in parasitemia between W/W^v and +/+ mice. To examine this possibility, we performed in vitro culture to examine the infectivity of parasites to RBCs obtained from W/W^v and +/+ mice. Different dose of P. berghei ANKA merozoites as indicated in Fig. 2 was added in RBC culture at 5% Ht, and then the infectivity of merozoites to RBCs was examined in vitro. After incubation for 20 h, blood smears stained with Giemsa were made from each culture well to compare the parasitemia between RBCs from W/W^v and +/+ mice. As shown in Fig. 2, the results showed that merozoites of P. berghei ANKA could invade both erythrocytes, and no significant difference was found on parasitemia between RBCs from W/W^v and +/+ mice, suggesting that P. berghei ANKA merozoites invade both RBCs equally.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. In vitro infectivity of P. berghei ANKA to RBCs obtained from W/Wv and +/+ mice. In vitro culture of P. berghei ANKA merozoites was performed to examine the infectivity of parasites to RBCs obtained from W/Wv and +/+ mice. Different dose of P. berghei ANKA merozoites as indicated in Fig. 2 was added in RBC culture at 5% Ht to examine their infectivity in vitro. After the incubation for 20 h, blood smears stained with Giemsa were made from each culture well to examine the parasitemia between RBCs from W/Wv and +/+ mice (mean ± SD of triplicate determinations). This experiment was repeated three times with similar results.

W/W^v are genetically mast cell-deficient mice, and this deficiency can be repaired by adoptive transfer of bone marrow cells from the control normal +/+ mice. If mast cell deficiency accounts for the differences in parasite growth between W/W^v and +/+ mice, reconstitution of the mast cell population in these animals should restore disease severity to the level of control animals. To directly evaluate the in vivo role of mast cells in murine malaria, we performed mast cell reconstitution in W/W^v recipients by i.v. injection of BMMCs derived from +/+ mice. A total of 5 x 10⁶ BMMCs was transferred into W/W^v mice via tail vein injection, and after 8 wk, mice were infected with P. berghei ANKA, and then parasitemia was examined. The mast cell-reconstituted W/W^v mice showed low parasitemia (Fig. 3A) and mortality (data not shown) associated with significantly increased concentration of TNF as compared with W/W^v. The TNF in sera was also detected by a bioassay method for TNF activity as well as ELISA, and TNF level in W/Wv mice was significantly low compared with +/+ mice and BMMCs-reconstituted W/W^v mice at day 10 after P. berghei ANKA infection (Fig. 3, B and C). These data suggest that mast cells have protective roles in murine malaria. There is a possibility that reconstitution of W/W^v mice with BMMCs affects their anemic condition to modify the susceptibility to P. berghei ANKA infection. To evaluate states of macrocytic anemia in W/W^v mice after transfer of 5 x 10⁶ BMMCs from +/+ mice, we examined Ht and MCV in W/W^v mice with or without BMMCs transfer. Total RBC count and Ht in W/W^v mice with BMMC transfer were determined using Coulter Micro Diff II machine (Beckman Coulter) to compare those of age-matched normal W/W^v and +/+ mice. MCV was calculated as follows: Ht (percentage)/RBCs (mm³) x 10. As the results show, total RBC count was 6.2 1 x 10⁶ ± 42.1/mm³ in W/W^v, 6.3 x 10⁶ ± 48.4/mm³ in W/W^v with BMMC transfer, and 1.05 x 10⁷ ± 25/mm³ in +/+ mice; Ht (percentage) was 38 ± 1.5 in W/W^v, 39 ± 1.7 in W/W^v with BMMC transfer, and 49 ± 1.2 in +/+ mice; and MCV was 62.8 ± 1.5 in W/W^v, 61 ± 1.2 in W/W^v with BMMC transfer, and 45.1 ± 0.32 in +/+ mice (n = 5 each), suggesting that no significant differences in Ht and MCV were noted between W/W^v and W/W^v with BMMC transfer.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Parasitemia of P. berghei ANKA in mast cell-deficient W/WV mice with or without reconstitution of cultured BMMCs. A, Parasitemia of P. berghei ANKA in mast cell-deficient W/WV mice with or without reconstitution of cultured BMMCs of +/+ mice was examined after 1 x 105 P. berghei ANKA infection (mean ± SD; n = 5 each). *, p < 0.05; **, p < 0.01. This experiment was performed three times with similar results. The concentration of serum TNF in +/+ mice and W/WV mice with or without BMMC reconstitution of +/+ mice was measured by ELISA (B) and TNF bioassay method (C) at day 10 after 1 x 105 P. berghei ANKA infection. TNF bioassay was performed by a modified L-929 cytotoxicity that is based on reduction of tetrazolium dye (mean + SD of triplicate determinations; *, p < 0.05 (B); *, p < 0.01 (C)). This experiment was performed three times with similar results. D, To define the specific function of mast cell-derived TNF in protective roles against P. berghei ANKA infection, we transferred cultured-BMMCs derived from TNF–/– mice to W/Wv mice and then infected P. berghei ANKA in these mice as described before (n = 5 each; mean ± SD; *, p < 0.01). This experiment was repeated three times with similar results.

W/W^v or W/W^v mice reconstituted with cultured BMMCs of +/+ or TNF^–/– mice were sacrificed at day 12 after P. berghei ANKA infection to examine the localization of mast cells in the spleen. Spleen wet weight in W/W^v mice with BMMC reconstitution of +/+ mice was increased as compared with W/W^v mice reconstituted with BMMCs of TNF^–/– mice or without BMMCs (Fig. 4A). The histopathology of the spleen revealed that there was an increased number of mast cells in spleens of +/+ or W/W^v mice reconstituted with BMMCs of +/+ or TNF^–/– mice, as compared with W/W^v mice after P. berghei ANKA infection (Fig. 4, B–E). Many mast cells were distributed in splenic red pulp. Although we detected mature mast cells in the spleen from +/+ mice or W/W^v mice reconstituted with BMMCs of +/+ or TNF^–/– mice after P. berghei ANKA infection, increased spleen weight was not found in W/W^v mice reconstituted with BMMCs of TNF^–/– mice. Mast cells were absent in spleens of P. berghei ANKA-infected W/W^v (Fig. 4E) or a few in spleens of uninfected +/+ mice (Fig. 4F). These results indicate that cultured BMMCs of the +/+ mice could survive and differentiate into functional mast cells in murine malaria after the injection into W/W^v mice.

View larger version (46K): [in this window] [in a new window]   FIGURE 4. Spleen hypertrophy in W/Wv mice with BMMCs reconstitution of +/+ mice. A, Spleen wet weight of +/+ mice and W/Wv mice with or without BMMCs reconstitution of TNF–/– or +/+ mice was examined at day 10 after the infection (n = 5 each; mean ± SD; *, p < 0.03. This experiment was repeated three times with similar results. The histopathology of the spleen revealed that there was an increased number of mast cells in spleens of W/Wv mice with the reconstitution of BMMCs from +/+ or TNF–/– mice, as compared with W/Wv mice after P. berghei ANKA infection (n = 5 each). Although we detected mature mast cells in the spleen from +/+ mice (B; bar, 10 µm) and W/Wv mice reconstituted with BMMCs of +/+ (C; bar, 10 µm) or TNF–/– mice (D; bar, 10 µm) after P. berghei ANKA infection, increased spleen wet weight was not observed in W/Wv mice reconstituted with BMMCs of TNF–/– mice (D; bar, 10 µm). Mast cells were absent in P. berghei ANKA-infected W/Wv (E; bar, 30 µm) or a few in normal +/+ mice (F; bar, 10 µm).

P. berghei ANKA-infected mice were treated with anti-TNF Ab to examine whether TNF-mediated resistance occurs in murine malaria. Anti-TNF Ab was injected i.p. into mice at the same time when parasites were inoculated, and then the treatment was repeated every third day until the end of the experiment. Parasitemia in mice treated with anti-TNF was significantly higher than that in control mice associated with high mortality (Fig. 5). These data suggested that TNF-mediated resistance occurred in murine malaria.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Administration of anti-TNF Ab to P. berghei ANKA-infected mice. Anti-mouse TNF polyclonal rabbit Ab (25 µg/mouse) or rabbit IgG (isotype control Ab, 25 µg/mouse) was injected i.p. to C57BL/6 mice at the same time when 1 x 105 P. berghei ANKA was inoculated. Injection of Abs was repeated every third day after the infection until end of the infection. Parasitemia (A) and mortality (B) were examined daily after the infection (n = 5 each; mean ± SD; *, p < 0.01; **, p < 0.005). This experiment was performed twice with similar results.

We attempted to determine whether anti-IgE-mediated mast cell activation would stimulate resistance to P. berghei ANKA infection. P. berghei ANKA-infected mice were injected with an anti-mouse IgE mAb to induce TNF from mast cells by bridging of FcRI. C57BL/6 mice were inoculated i.v. with 1 x 10⁵ P. berghei ANKA and then divided into two groups at day 10 after the infection. One group of mice was treated i.p. with rat anti-mouse IgE Ab (50 µg/mouse), and the other group of mice was treated with rat IgG (50 µg/mouse) as a control. Parasitemia and serum TNF level in the mice was examined on day 11 after the infection. Anti-mouse IgE injection caused significantly low parasitemia with high TNF levels compared with the control mice (Table I), suggesting that TNF from mast cells activated by anti-mouse IgE Ab is likely to play a key modulatory role in P. berghei ANKA infection. To provide further support for the function of mast cells in resistance to P. berghei ANKA infection, we injected a mast cell chemical activator compound 48/80 into P. berghei ANKA-infected mice, and then parasitemia was examined. C57BL/6 mice were inoculated i.v. with 1 x 10⁵ P. berghei ANKA and then divided into two groups at day 8 (I) or 11(II) after the infection. One group of mice was treated i.v. with compound 48/80 (1.2 mg/kg), and the other group of mice was treated with vehicle as a control. We observed decreased parasitemia at 9 or 12 days through mast cell activation by compound 48/80, accompanied by significantly increased concentrations of TNF in sera (Table II).

View this table: [in this window] [in a new window]   Table I. Decreased parasitemia in P. berghei ANKA-infected mice after mast cell activation by anti-IgE () mAba

To examine the effect of compound 48/80 or LPS on the parasites and macrophages in vivo, we examined parasite growth in P. berghei ANKA-infected W/W^v mice after treatment of compound 48/80 or LPS. W/W^v and +/+ mice were inoculated with 1 x 10⁵ P. berghei ANKA, and then W/W^v mice were divided into three groups after the infection. The first group of mice was treated i.v. with compound 48/80 (1.2 mg/Kg/mouse), the second group was treated i.p. with LPS (1 µg/mouse), and other groups of W/W^v and +/+ mice were left without any treatment. As shown Fig. 6, parasitemia in W/W^v mice with or without those treatments was comparable and significantly increased compared with +/+ mice. From these findings, it was thought that compound 48/80 or LPS did not appear to affect parasites and macrophages in P. berghei ANKA-infected W/W^v mice.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Growth of P. berghei ANKA in W/Wv mice treated with compound 48/80 or LPS. To examine the effect of compound 48/80 and LPS on parasite growth, we examined parasitemia in P. berghei ANKA-infected W/Wv mice after treatment of compound 48/80 (1.2 mg/kg/mouse, i.v.) or LPS (1 µg/mouse, i.p.). Compound 48/80 or LPS was injected to W/Wv mice at the same time when 1 x 105 P. berghei ANKA was inoculated and then repeated every third day until end of the infection after the infection. Parasitemia was examined daily after the infection (n = 5 each) (mean + SD). This experiment was repeated three times with similar results; *, p < 0.01.

It has been reported that TNF is secreted from macrophages as well as mast cells during the infection (33). To identify the cell secreting TNF in vivo, we measured TNF level in mast cells and macrophages obtained from the peritoneal cavity of P. berghei ANKA-infected C57BL/6 mice (day 10 after the infection). A total of 1 x 10⁵ of mast cells or macrophages obtained from C57BL/6 mice was cultured in RPMI 1640 supplemented with 10% FBS and then incubated with or without soluble P. berghei ANKA Ag (20 µg/well). After 24 h of incubation, culture supernatants were collected, and then TNF production was measured by ELISA. As shown in Fig. 7, the level of TNF in mast cells and macrophages was comparable and significantly higher than those in the control. This result suggests that macrophages secrete TNF as well as mast cells in vitro.

View larger version (11K): [in this window] [in a new window]   FIGURE 7. TNF production in peritoneal mast cells and macrophages in vitro. Mast cells and macrophages were obtained from the peritoneal cavity of P. berghei ANAK-infected C57BL/6 mice at day 10 after the infection. A total of 1 x 105 cells of peritoneal mast cells or macrophages was cultured with RPMI 1640 supplemented with 10% FBS and incubated with or without soluble P. berghei ANKA Ag (20 µg/well) for 24 h. Culture supernatants were collected, and then TNF production was measured by ELISA. This experiment was repeated three times with similar results (mean ± SD of triplicate determinations; *, p < 0.01).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Regarding the possible mechanisms involved in the TNF-mediated protection, studies with P. falciparum in vitro (10) and Plasmodium yoelii and Plasmodium chabaudi AS in mice (34) indicated that activated macrophages and neutrophils are probably involved in TNF-mediated parasite killing. In addition, Taverne et al. (34) suggested that the effect of TNF on erythropoiesis might play a role in the suppression of parasitemia (35, 36). In the present studies, those parasite-killing mechanisms might be involved in the protection of murine malaria.

Many kinds of pathways for mast cell activation may contribute to protection to malaria parasites by inducing TNF release. The examples established in this study are cross-linking of FcRI by anti-IgE and chemical activation of mast cell by compound 48/80. These stimulations to mast cells initiated TNF release and suppressed parasite growth. Another candidate for mast cell activation in malaria infection is complement. Studies of human malaria suggest that complement activation, particularly by the classical pathway, plays a role in host defense against infection (37, 38, 39, 40). Anaphylatoxin derived from the classical pathway can release mediators from mast cells, including TNF. Immune complex formation commonly occurs in malaria (41, 42, 43, 44, 45, 46), and thus complement fixing IgM and IgG Abs could stimulate mast cells and release TNF and other mediators. IgG immune complexes are also capable of releasing cytokines from mast cells through FcR without complement activation (47). Mast cell activation through immune complexes and IgE Ab is an expression of acquired immunity.

Mast cells can be activated also by molecules involving innate immunity. Although the mechanisms of direct activation of mast cells by various microorganisms have not been studied precisely, it has been reported recently that activation of mast cells by various bacteria was mediated by TLR2 and -4 (23). It is probable that malaria parasites have molecules with binding activity to TLRs. Complement activation through the alternative pathway by malarial components is also a possible mechanism for mast cell activation. It is noteworthy that presynthesized TNF in mast cells is a candidate for the immediate initiation of the host response to infection. Because the secretion of TNF by other cell types is greatly delayed by the time required to complete de novo synthesis of this cytokine (17), involvement of mast cell activation through innate immunity or direct stimulation is likely to function in the present model. Indeed, higher parasitemia was found in W/W^v mice in an early stage of infection when acquired immune responses might not operate (Fig. 1A).

IgE-mediated protection through FcRI on mast cells occurred in murine malaria associated with an increase in serum TNF level (Table I). So far, little direct evidence regarding the possible protective or pathogenic role of IgE has been reported in malaria. In humans, 85% of individuals living in areas of high P. falciparum endemicity have significantly elevated levels of total IgE and specific IgE Ab against P. falciparum. Plasmodial infection may have directly contributed to IgE elevation (48, 49). It has been reported that anti-P. falciparum IgE Ab plays a protective role in pregnant women (4). In contrast, cerebral malaria patients had significantly higher anti-P. falciparum IgE Ab levels than those with uncomplicated disease, providing a possible relation between IgE and TNF in the pathogenesis (48, 49). Despite these observations, little is known about the relationship between mast cells and malaria pathogenesis.

Spleen hypertrophy in P. berghei ANKA-infected W/W^v mice was enhanced by transfer of BMMCs of +/+ mice. Lymphoid hyperplasia and lymphocyte proliferation were found in the marginal zones of lymphoid follicles accompanied by increased numbers of mast cells as compared with control W/W^v mice. These results indicate that cultured BMMCs of the +/+ mouse could survive and differentiate into functional mast cells after the injection into W/W^v mice. Regarding the mechanisms of spleen hypertrophy in murine malaria, we suppose that once mast cell activation occurs, the release of numerous mast cell mediators could act at several levels to influence disease induction and/or progression. For example, degranulation and secretion of preformed mediators that are stored in the cytoplasmic granules, such as vasoactive amines, neutral proteases, proteoglycans, the de novo synthesis of proinflammatory lipid mediators, and the synthesis and secretion of cytokines and chemokines will have occurred. McLachlan et al. (22) reported that TNF production from mast cells can substantially enhance T cell recruitment to local lymph nodes, accompanying lymph node enlargement during experimental infection with Escherichia coli. Thus, mast cells can regulate tissue changes that confer either benefit or harm, depending on the specific circumstances (50). Mast cells have been found to be activated in various T cell-mediated inflammatory processes and to reside in close physical proximity to T cells (51). Recently, it has been reported that murine mast cells are stimulated to degranulate in association with direct contact with activated murine T cells (52, 53). In the present studies, because spleen hypertrophy was observed in mast cell-transferred mice accompanied by increase of TNF production, but not in mast cell-deficient mice after P. berghei ANKA infection, we supposed that mast cells and mast cell-derived TNF are important for pathogenesis of spleen hypertrophy as well as for protection of malaria. Thus, because mast cells and mast cell-derived TNF play important roles for the protection and pathogenesis in murine malaria, these findings may provide a basis for a new strategy in antimalaria therapy of human.

	Acknowledgments

 
We thank Dr. Dean Befus for helpful discussions and critical reading of this manuscript.

	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 Address correspondence and reprint requests to Dr. Takahisa Furuta, University of Tokyo, Division of Infections Genetics, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: furuta{at}ims.u-tokyo.ac.jp

² Current address: Department of Veterinary Biosciences, College of Veterinary Medicine, Ohio State University, 1925 Coffey Road, Columbus, OH 43210-1093.

³ Abbreviations used in this paper: Ht, hematocrit; BMMC, bone marrow-derived mast cell; MCV, mean corpuscular volume.

Received for publication June 22, 2005. Accepted for publication June 15, 2006.

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