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The Journal of Immunology, 2006, 177: 2518-2526.
Copyright © 2006 by The American Association of Immunologists

CD8+ T Lymphocyte-Mediated Loss of Marginal Metallophilic Macrophages following Infection with Plasmodium chabaudi chabaudi AS1

Lynette Beattie*,{dagger},{ddagger}, Christian R. Engwerda*, Michelle Wykes* and Michael F. Good2,*,{dagger}

* Molecular Immunology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia; {dagger} Cooperative Research Centre for Vaccine Technology, Brisbane, Queensland, Australia; and {ddagger} School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The splenic architecture is essential for the quick resolution of a primary infection with Plasmodium. A critical component of this architecture is the marginal zone (MZ), an area of the spleen that separates the reticuloendothelial red pulp of the spleen from the lymphoid white pulp compartment. There are two unique macrophage populations found in the MZ: MZ macrophages (MZM) found on the outer border of the MZ, and marginal metallophilic macrophages (MMM) found on the inner border, adjacent to the white pulp. We investigated the homeostasis of MMM and MZM following infection with Plasmodium chabaudi and demonstrated that a complete loss of both MMM and MZM occurred by the time of peak parasitemia, 8 days after infection. The loss was not induced by up-regulation of the inflammatory cytokines TNF or IFN-{gamma}. In contrast, following only CD8+ T cell depletion (not dendritic cell), MMM but not MZM were retained, implicating CD8+ T cells in the P. chabaudi-induced loss of MMM. Retention of MMM occurred in mice deficient in CD95, CD95-ligand, and perforin, indicating that these signals are involved in the death pathway of MMM. These data have significant implications for the understanding of the immune-mediated pathology of the spleen as a result of infection with Plasmodium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
After several decades of research aimed at developing a malaria vaccine, malaria still remains one of the world’s leading causes of morbidity and mortality. Although several malaria vaccine candidates are currently undergoing clinical trials (1), the mechanisms that lead to immune protection and pathology as a result of infection are still poorly understood.

The spleen functions as a blood-filtering system that is capable of removing particulate matter such as bacteria, as well as effete RBC, from the circulation (2). It also functions as a lymphatic organ and facilitates the generation of immune responses to blood-borne Ags (3). There are three segregated areas of the spleen: the white pulp, the red pulp, and the marginal zone (MZ).3 The white pulp is the lymphoid compartment of the spleen, while the red pulp is the reticuloendothelial compartment, responsible for removal of particulate matter. The MZ forms a distinctive border between the red and white pulp and is the entry point for blood entering the splenic circulation (4).

In addition to trafficking leukocytes, four types of cells are constitutively present in the MZ of murine spleen: MZ B cells, MZ dendritic cells, MZ macrophages (MZM), and marginal metallophilic macrophages (MMM). MMM are located on the inner border of the MZ directly adjacent to the white pulp, whereas MZM are located on the outer border of the splenic MZ, at the border with the red pulp.

MMM are not considered to be highly phagocytic, but are essential for the initiation of an immune response to T-dependent particulate Ags (5, 6) and T-independent type 2 Ags (7). MMM are capable of migrating from the MZ, into a developing germinal center during the course of an immune response, suggesting that MMM are involved in the transportation of unprocessed Ag from the MZ into the B cell follicle (8, 9), and are thus thought to be important in the generation of immune responses.

MZM are highly phagocytic and are located on the outer border of the splenic MZ. They are large cells with elongated processes that facilitate a close association with MZ B cells (4). MZM are characterized, and quite often defined, by their capacity to undergo selective uptake of neutral polysaccharides. MZM selectively take up the carbon particles and are easily identified in splenic tissue sections by light microscopy (10, 11).

A small number of studies have examined the MZ following Plasmodium infection. Mice resistant to infection with Plasmodium chabaudi have more MMM than susceptible A/J mice (12). Dissolution of the MZ was noted following infection in humans (13) and in rodents (14), with a loss of MZ B cells. Other reports suggest that the ring of MMM broadens in BALB/c mice following P. chabaudi adami infection (15). A loss of MZM staining in the spleens of malaria-infected mice has been reported (12, 15); however, it is not yet established whether this is due to loss of the cells or a change of surface phenotype. A recent study reported loss of MZM, but not MMM, following infection with P. chabaudi. The authors concluded that, as a similar loss occurred following administration of TNF, the MZM loss was the result of up-regulation of TNF in the infected mice (16).

We used the P. chabaudi model of malaria infection to investigate changes to the architecture of the splenic MZ following infection of C57BL/6 mice. We found that a loss of both MMM and MZM occurred by the time of peak parasitemia as a result of apoptosis and was induced in MMM by CD8+ T cells, due to perforin and CD95/CD95-ligand (CD95-L) interactions. These data have significant implications for the understanding of the immune-mediated pathology as a result of Plasmodium infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Female mice (6–12 wk old at the commencement of experiments) were used. C57BL/6J and B6.CD45.1 mice were purchased from the Animal Resource Centre. B6.TNF–/– were bred at the Queensland Institute of Medical Research animal facility. B6.IFN-{gamma}–/–, B6.lpr (CD95-deficient) (17), and B6.gld (CD95-L-deficient) (18) mice were bred at the Herston Medical Research Centre. B6.Pfp–/– mice (19) were a gift from Dr. M. Smyth (Cancer Immunology Program, Tresconthick Laboratories, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia) and were bred at the Peter MacCallum Cancer Centre. All experiments were reviewed and approved by the Queensland Institute of Medical Research Animal Ethics Committee.

Parasites

P. chabaudi chabaudi AS was obtained from Richard Carter (Institute of Cell, Animal, and Population Biology, Edinburgh, Scotland) and maintained by passaging parasitized RBC from cryogenically stored stocks into naive mice. Mice were challenged with 105 pRBC i.v. in all experiments. Parasitemias were monitored by peripheral blood smears stained with Diff Quik (Lab Aides).

Time course study of the changes to the splenic MZ following infection with P. chabaudi

Spleens were collected from groups of three mice on days 0 (naive), 4, 8, 12, 20, 30, and 60 postinfection. Immunohistology to detect MMM was performed using 3D6 (20) or MOMA-1 (21) (Serotec) mAbs. Immunohistology to detect MZM was performed using the ER-TR9 mAb (22). Each section was also labeled with B220 (anti-CD45R) to detect follicular B cells, so that the relative localization of the B cell follicle and MMM or MZM could be examined.

Preinjection with india ink for detection of MZM

Three groups of three C57BL/6 mice were preinjected with india ink according to the method described previously (10), and two of the groups of mice were infected with 105 P. chabaudi pRBC. Four or 8 days after infection, the groups of mice were sacrificed and spleen sections were examined for the presence of india ink by light microscopy.

Immunofluorescent labeling of frozen spleen sections

Tissue was collected and placed in Tissue-Tek Optimal Cutting Temperature embedding medium (Sakura Finetek) on dry ice (CO2). The Optimal Cutting Temperature embedding medium was allowed to set and the blocks then transferred to –70°C until sectioning. Sections (6–10 µM) were fixed in 100% cold acetone for 10 min and rehydrated in PBS. Sections were blocked in 5% (v/v) normal rabbit serum (Vector Laboratories) for 1 h at room temperature (RT). Primary Ab, diluted in 5% (v/v) normal rabbit serum in PBS, was added to the sections for 1 h at RT. The sections were incubated with biotinylated rabbit anti-rat IgG (Vector Laboratories) in 5% (v/v) rabbit serum in PBS, for 1 h at RT. The sections were washed and incubated in streptavidin Alexa Fluor 594/488 conjugate (Molecular Probes) for 30 min at RT in the dark. For double-color labeling, the sections were then blocked in 100 µg/ml purified rat Ig (Sigma-Aldrich), incubated with anti-mouse CD45R-Alexa 488 conjugate (BD Pharmingen), and washed after all labeling steps. All incubations were performed for 1 h at RT in the dark. Slides were mounted in ProLong Gold mounting medium for fluorescence (Molecular Probes) and examined on a Zeiss Axioskop 2 (Zeiss), and photographs were captured on a Zeiss high sensitivity monochromatic camera (Zeiss) using the Axioskop 2 software (Zeiss). Color was added electronically to the black and white photos after acquisition.

To quantify the loss of cells, the number of positive cells was counted either as the number of cells in 20 (magnification, x200) fields for each mouse at each time point or as the number of cells/follicle for the total number of follicles per section for each mouse at each time point. The number of cells was expressed as the average number ± the SEM. Differences between groups were compared using a one-way ANOVA with 95% confidence intervals and a Tukey posttest to compare the differences between the individual groups.

Detection of apoptotic cells in frozen spleen sections by the TUNEL assay

To detect apoptotic cells in frozen spleen sections, the TUNEL assay (in situ cell death detection kit) (Roche Diagnostic Systems) was used. Frozen spleen sections were fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich), and the cells were permeabilized in 0.1 M sodium citrate (BDH Chemicals) with 0.1% Triton X-100 for 2 min on ice. If MMM were to be detected, the frozen sections were labeled with 3D6, according to the method outlined above. Once labeled with 3D6, the sections were labeled with the TUNEL assay according to the manufacturer’s instructions. India ink uptake was used to identify MZM in these experiments due to the need to fix the sections in paraformaldehyde. The sections were washed in three changes of PBS and mounted in ProLong Gold mounting medium for fluorescence (Molecular Probes) and examined under a fluorescent microscope (Zeiss Axioskop 2) (Zeiss).

In vivo depletion of CD8+ cells

For depletion of CD8+ T cells only, 500 µg of anti-CD8beta Ab (clone 53-5.8) or rat Ig control was injected i.p. on days –1 and 4. Mice were infected with 105 P. chabaudi AS pRBC on day 0, and parasitemia was monitored for 8 days when spleens were collected and frozen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MMM are lost following infection with P. chabaudi

To examine the effect of infection on the splenic MZ, several cohorts of C57BL/6 mice were infected with P. chabaudi and the parasitemia was monitored by peripheral blood smear for 60 days (Fig. 1a). Four days after infection with P. chabaudi, the pattern of MMM was similar to that observed in naive controls with a clear ring of MMM surrounding the B cell follicle (Fig. 1b). However, 8 days after infection, MMM were undetectable by CD169 labeling, which specifically labels MMM. The clear ring of MMM surrounding the B cell follicle was absent in the day 8 group, indicating either a loss of MMM or a down-regulation of CD169 on the surface of the MMM. MMM remained undetectable until day 30 postinfection, at which time some follicles were observed with surrounding MMM (Fig. 1b). By day 60, the ring of MMM was fully restored and was similar to that seen in naive controls.


Figure 1
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  		FIGURE 1. Loss and repopulation of MMM following infection with P. chabaudi. Mice were infected with P. chabaudi pRBC. a, The course of parasitemia in the infected mice. b, Frozen spleen sections labeled with 3D6 (red; left) to detect MMM and B220 (CD45R; green, middle) to detect follicular B cells (overlay of red and green on right). c, The number of MMM present at each time point. Bars represent the average number of 3D6-positive cells counted in 20 x 200 magnification fields. d, Frozen spleen sections labeled with MOMA-1 (red; left) to detect MMM and B220 to detect follicular B cells (green, middle) (overlay of red and green, right). All images are x100 magnification. Error bars represent the SEM.

 
Cell counts confirmed that a significant loss of cells expressing CD169 occurred by day 8 of infection when compared with the naive controls (Fig. 1c) (p = 0.0002). A significant increase in the number of cells expressing CD169 was observed by day 20 postinfection, when compared with the number seen at day 8 postinfection (p = 0.011). The number of cells that expressed CD169 did not return to the numbers seen in the naive control until day 60 postinfection when the difference between the two groups was not significant (p > 0.05).

To determine whether the reduction in the number of cells expressing CD169 was due to a loss of MMM or a down-regulation of CD169 expression, MMM were labeled with an alternative Ab, MOMA-1, which binds to a molecule regulated differentially to CD169 and expressed on the surface and in the cytoplasm of MMM (21). Although clear rings of MOMA-1-positive cells, surrounding the B cell follicles, were observed in the spleens from the naive and the day 4 postinfection mice, the rings were completely absent in the spleens of the mice that were 8 days postinfection with P. chabaudi (Fig. 1d). The loss in detection of two, differentially regulated, MMM-specific molecules strongly suggested that a loss of MMM, rather than a down-regulation in expression of the molecules, was occurring.

MZM are lost following infection with P. chabaudi

Following the observation that MMM were lost from the spleen 8 days after infection with P. chabaudi, investigations were conducted to determine the fate of MZM following infection, using ER-TR9 labeling. The number of MZM decreased slightly, but not significantly, 4 days after infection with P. chabaudi, when compared with the naive controls (Fig. 2a). A loss of labeling was observed by day 8 postinfection. The clear rings of MZM surrounding the B cell follicle were absent in the group that were 8 days postinfection, indicating that either a loss of MZM or a down-regulation of the molecule that binds ER-TR9 had occurred. The loss of MZM was maintained for nearly 60 days postinfection, when small numbers of ER-TR9-positive cells were present, but the numbers were significantly fewer than the number present in naive controls (Fig. 2b) (p < 0.0001).


Figure 2
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  		FIGURE 2. The loss and repopulation of MZM following infection with P. chabaudi. Mice were preinjected with india ink and then infected with P. chabaudi pRBC. a, Frozen spleen sections labeled with ER-TR9 (red, left) to detect MZM and B220 (CD45R; green, middle) to detect follicular B cells (overlay of red and green, right). All images are x100 magnification. b, The number of MZM present at each time point. Bars represent the average number of ER-TR9-positive cells counted in 20 x 200 magnification fields. Error bars represent the SEM. c, MZM detected by india ink on days 0, 4, and 8 after infection as detected by bright field microscopy. Magnification, x100, left; magnification, x250, right.

 
To confirm that the loss of ER-TR9 labeling was due to a loss of MZM, india ink uptake (a functional test for the presence of MZM) was used. Although rings of cells that had taken up india ink were visible in the spleens of the naive and day 4 postinfection groups, the rings were not visible in the spleens of the mice 8 days postinfection (Fig. 2c), strongly suggesting that P. chabaudi infection caused a loss of MMM, in addition to a loss of MZM by day 8 of infection.

MMM and MZM colocalize with apoptotic bodies 4 days after infection with P. chabaudi

To determine whether apoptosis was the mechanism of MMM and MZM loss, frozen spleen sections were examined by the TUNEL assay.

A number of apoptotic cells were observed in the MZ area in the naive spleens, seen as bright green TUNEL-positive cell nuclei. However, the apoptotic cell nuclei did not colocalize with expression of MMM- or MZM-specific markers (Figs. 3 and 4). At 4 days postinfection, the number of apoptotic cells in the MZ increased and the positive nuclei were closely associated with both MMM and MZM (Figs. 3 and 4).


Figure 3
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  		FIGURE 3. The localization of MMM and apoptotic cells in mice 4 or 8 days postinfection with P. chabaudi. Mice were infected with P. chabaudi pRBC, and the spleens were collected 0, 4, and 8 days later. Sections were labeled with 3D6 to detect MMM (red, left) and the TUNEL assay to detect cells undergoing apoptosis (green, middle) (overlay of red and green to show colocalization on right). Top row, Magnification, x250. Bottom row, Magnification, x400. Arrows highlight areas of colocalization. RP = red pulp. Data are representative of duplicate experiments with a minimum of three mice/group.

 

Figure 4
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  		FIGURE 4. The localization of MZM and apoptotic cells in mice 4 or 8 days postinfection with P. chabaudi. Mice were preinjected with india ink and then infected with P. chabaudi pRBC. The spleens were collected 0, 4, and 8 days later. Sections were examined under bright field to detect MZM by india ink uptake (left) and labeled with the TUNEL assay to detect cells undergoing apoptosis (green, middle) (overlay of the two images to show colocalization; right). Top row, Magnification, x250. Bottom row, Magnification, x400. Arrows highlight areas of colocalization. RP = red pulp. Data are representative of duplicate experiments with a minimum of three mice/group.

 
Although the possibility that MMM or MZM were taking up apoptotic bodies could not be excluded, the extensive loss of MMM and MZM by day 8 of infection and the close association of MMM and MZM with apoptotic-positive cell nuclei suggested that both MMM and MZM were undergoing apoptosis as a result of infection.

The loss of MMM and MZM is not due to TNF or IFN-{gamma}

Studies in other infection models including Leishmania have shown that the proinflammatory cytokine, TNF, causes major changes to the splenic architecture following infection, including a loss of MZM (11). In addition, studies in rodent models of malaria infection have shown that an apoptotic deletion of Plasmodium-specific T cells occurs via a mechanism that is dependent upon the presence of IFN-{gamma} (23). Therefore, we investigated the role of TNF and IFN-{gamma} in the loss of MMM and MZM in cytokine-deficient mice. Spleens were collected from TNF- and IFN-{gamma}-deficient mice along with control C57BL/6 mice on day 7 postinfection, a time at which all mice had similar peak parasitemia levels (data not shown). Naive control TNF–/– mice and naive control IFN-{gamma}–/– mice had similar numbers of MMM and MZM as observed in naive C57BL/6 controls (Fig. 5). A similar loss of MMM and MZM occurred in the spleens of TNF–/– mice, IFN-{gamma}–/– mice, and C57BL/6 wild-type controls 7 days after infection with P. chabaudi. The loss of MMM and MZM following infection with P. chabaudi can therefore occur independently of the production of the inflammatory cytokines TNF or IFN-{gamma}.


Figure 5
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  		FIGURE 5. Frozen spleen sections demonstrating MMM and MZM loss in B6.TNF–/– and B6.IFN-{gamma}–/– mice following infection with P. chabaudi. Mice were infected with P. chabaudi pRBC, and the spleens were collected 7 days later. Frozen spleen sections were labeled with 3D6 and B220 (left) or ER-TR9 and B220 (right). All images are x100 magnification. Data are representative of duplicate experiments with a minimum of three mice/group.

 
CD8+ T cells cause the loss of MMM, but not MZM, following infection with P. chabaudi

Studies in mice infected with lymphocytic choriomeningitis virus found that MMM were lost following infection, but the loss did not occur if CD8+ cells were depleted (24), implicating CD8+ cells in the loss of MMM. To determine whether CD8+ cells mediated the loss of MMM or MZM following infection with P. chabaudi, depletion studies were performed, as described (see Materials and Methods).

Following infection, MMM were clearly present in the anti-CD8beta-treated group, with obvious rings of 3D6-positive cells surrounding the B cell follicle areas (Fig. 6a). MMM were not present in the depletion control group, demonstrating that CD8+ T cells were involved in the loss of MMM following infection with P. chabaudi.


Figure 6
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  		FIGURE 6. Frozen spleen sections demonstrating MMM and MZM loss in the absence of CD8+ CTL, following infection with P. chabaudi. Mice were treated with anti-CD8b Ab or rat Ig control on days –1 and +4 by i.p. injection. On day 0, the mice were infected with P. chabaudi pRBC, and the spleens were collected on day 8 postinfection. a, Spleen sections were labeled with 3D6 and B220 (left) or ER-TR9 and B220 (right). b, Bars represent the average number of 3D6-positive cells counted/follicle for each group. c, Bars represent the average number of ER-TR9-positive cells counted/follicle for each group. Error bars represent the SEM, and the data shown are representative of duplicate experiments with a minimum of five mice/group.

 
Cell counts confirmed that following infection, the anti-CD8beta-treated mice had a similar number of MMM as the naive controls (p > 0.05) (Fig. 6b). Both the naive control animals and the anti-CD8beta-treated animals had significantly more MMM than the rat Ig-treated controls (p < 0.001). CD8+ T cells are therefore critical to the deletion of MMM following infection with P. chabaudi.

In contrast, MZM were still lost following anti-CD8beta depletion and infection with P. chabaudi (Fig. 6a). The number of MZM present in the anti-CD8beta-treated mice was not significantly different from the rat Ig-treated controls (p > 0.05) (Fig. 6c). There were significantly fewer MZM present in the CD8beta-depleted and infected group than the naive controls (p = <0.001). CD8+ T cells can therefore be excluded as contributing to the loss of MZM following infection with P. chabaudi.

CD95/CD95-L interactions partially mediate the loss of MMM, but not MZM, following infection with P. chabaudi

To investigate the role of CD95/CD95-L in the CD8+ T cell-mediated loss of MMM following infection with P. chabaudi, CD95-deficient (B6.lpr) and CD95-L-deficient (B6.gld) mice were used. Spleens were collected from all mice 7 days postinfection with P. chabaudi, the time at which all mice had similar peak parasitemia levels in previous experiments (data not shown).

MMM were partially retained in the spleens of the infected CD95–/– and CD95-L–/– mice (Fig. 7a). Significantly fewer MMM were present in the spleens of the infected C57BL/6 mice than in the naive controls (p < 0.001) (Fig. 7b). However, MMM were not lost to a significant degree in CD95–/– mice (p > 0.05). In contrast, MMM were partially lost in CD95-L–/– mice (p < 0.05). The data suggest that a proportion of the CD8+-mediated death signal given to MMM following infection with P. chabaudi is mediated by CD95/CD95-L interactions.


Figure 7
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  		FIGURE 7. Frozen spleen sections demonstrating MMM and MZM loss in CD95 (B6.lpr)- and CD95-L (B6.gld)-deficient mice following infection with P. chabaudi. Mice were infected with P. chabaudi pRBC, and the spleens were harvested 8 days later. a, Frozen spleen sections labeled with 3D6 and B220 (left) or ER-TR9 and B220 (right). b, Bars represent the average number of 3D6-positive cells/follicle (left) or the average number of ER-TR9-positive cells/follicle (right). Error bars represent the SEM. Data shown are representative of duplicate experiments with three to five mice per group.

 
A similar loss of MZM was observed in the C57BL/6 control mice and the CD95–/– and CD95-L–/– mice following infection (Fig. 7a). The loss of MZM was significant when compared with the controls for both strains of mice (Fig. 7b). Signaling via CD95 is therefore not required for the P. chabaudi-induced loss of MZM.

MMM are not lost from the spleen following infection with P. chabaudi in perforin-deficient mice

To investigate the role of perforin in the CD8+ T cell-mediated loss of MMM following infection with P. chabaudi, perforin-deficient mice (B6.Pfp–/–) were used. Spleens were collected from perforin-deficient mice 9 days postinfection with P. chabaudi, the time at which the mice had similar peak parasitemia levels to C57BL/6 mice, which peaked 2 days earlier, on day 7 (data not shown).

MMM were lost following infection of C57BL/6 mice with P. chabaudi (Fig. 8a), as previously shown. MMM were retained in the spleens of the infected B6.Pfp–/– mice. Similar numbers of MMM were present in the spleens of the infected B6.Pfp–/– mice and the naive B6.Pfp–/– controls (p > 0.05). In contrast, significantly fewer MMM were present in the spleens of the infected C57BL/6 mice than in the naive controls (p < 0.001) (Fig. 8b). It was therefore concluded that the death signal given to MMM by CD8+ T cells following infection with P. chabaudi is mediated by perforin, in addition to CD95.


Figure 8
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  		FIGURE 8. Frozen spleen sections demonstrating MMM and MZM loss in perforin-deficient mice following infection with P. chabaudi. Mice were infected with P. chabaudi pRBC, and the spleens were harvested 8 days later. a, Frozen spleen sections were labeled with 3D6 and B220 (left) or ER-TR9 and B220 (right). b, Bars represent the average number of 3D6-positive cells/follicle (left) or the average number of ER-TR9-positive cells/follicle (right). Error bars represent the SEM. Data shown are representative of three mice per group.

 
The loss of MZM in B6.Pfp–/– mice also was investigated. A similar loss of MZM was observed in the C57BL/6 control mice and in the B6.Pfp–/– mice following infection (Fig. 8a). The loss of MZM was significant when compared with the controls for both strains of mice (Fig. 8b). Perforin signaling therefore is not required for the P. chabaudi-induced loss of MZM.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Understanding the complex cellular interactions that lead to tissue damage as a result of immune-mediated pathology is crucial to the development of effective vaccines, and can lead to the development of novel immunotherapies. Inflammatory immune responses result in immune-mediated pathology in the spleen during many infectious states, including malaria (12, 13, 14, 25, 26), leishmaniasis (11), and lymphocytic choriomeningitis virus infection (24). The current study aimed to investigate immune-mediated pathology in the spleen following infection with P. chabaudi.

P. chabaudi infection in C57BL/6 mice caused a loss of both MMM and MZM by the time of peak parasitemia. This loss of the cells was confirmed by secondary cell-specific markers and functional studies. Studies with the TUNEL assay showed that the loss of MMM and MZM may have been the result of apoptosis. The loss occurred in the absence of the inflammatory cytokines TNF and IFN-{gamma}; however, depletion of CD8+ T cells prevented the loss of MMM, but not MZM. Hence, CD8+ T cells contribute to the loss of MMM following infection with P. chabaudi. Studies in CD95/CD95-L-deficient and perforin-deficient mice showed that perforin and CD95/CD95-L pathways mediated the loss of MMM, but not MZM. These studies have thus described a novel interaction between MMM and CD8+ CTL following infection with Plasmodium and have significant implications for the understanding of the changes to the spleen following infection.

Further studies to confirm the loss of MMM and MZM and to further investigate the mechanism of the loss ideally would have incorporated flow cytometry studies, and were attempted. However, the need to use collagenase digestion to isolate both MMM and MZM resulted in phenotypic changes and death of the cells in vitro, and is the reason that large amounts of research involving these cells have been performed either on fixed splenic tissue sections or by in vivo depletion studies (4, 11, 15, 27). We therefore concluded that the loss of two MMM-specific markers in addition to functional studies demonstrated that MMM and MZM are lost from the spleen following infection with P. chabaudi.

The function of MMM is still poorly understood. They are essential for the initiation of immune responses to T-dependent particulate Ags (5, 6) and T-independent type 2 Ags (7). MMM are capable of migrating from the MZ into a developing germinal center during the course of an immune response (8, 9), and may be of considerable importance in the generation of immune responses to previously unseen Ags. The exact role of MMM in the control of infection with Plasmodium and the effect that MMM have on the development of parasite-specific immunity have not been thoroughly investigated and require further studies. Initial studies suggest that germinal center development is compromised following infection with P. chabaudi when compared with the response to a model Ag (L. Beattie, M. Wykes, Y. Zhou, and M. F. Good, unpublished observations), an effect that may be due to the absence of MMM, but further investigations are required. The results of the current study indicate that despite the loss of MMM, C57BL/6 mice cleared parasitemia and controlled infection, indicating a minor role for this cell population in control of a primary malaria infection. However, the prominent position of MMM within the splenic architecture indicates that the cells most likely have an important role in cell movement. Trafficking of nonlymphocyte populations may be affected in the absence of MMM, or the loss of MMM may have an effect on the ability of the host to control secondary infections with Plasmodium. Further studies are required to determine the functional outcome of MMM loss following Plasmodium infection.

Although CD8+ T cells are not considered to play a major role in the control of blood stage infection with Plasmodium (28), they clearly mediate pathology in the spleen during the blood stage. Although the specificity of these T cells needs to be further investigated, the close colocalization of pRBC with MMM following i.v. injection (data not shown) would suggest that MMM may be able to take up parasite Ags, possibly resulting in presentation of these Ags in association with class I on the cell surface. Further studies would need to be undertaken to confirm this. Also, it is important to determine whether there is an immune advantage to the CD8+ T cell activation that occurs, or whether intervention strategies designed to limit CD8+ T cell activation would also limit pathology of the spleen and therefore be an important mechanism of limiting the pathology associated with malaria disease. There is a well-characterized correlation between Plasmodium infection and immune suppression in humans (29) with an increase in susceptibility to bacterial infections (30, 31). Because clearance of bacterial infections has been proposed to be the role of the splenic MZ (32) and splenectomy also results in an increased susceptibility to bacterial infections, it is proposed that, due to the loss of macrophages in the splenic MZ, Plasmodium infection induces a state similar to that of asplenia in the host, leading to an increased susceptibility to bacterial infections. Hence, parasite-induced pathology of the spleen may cause an impaired ability of the host to clear bacterial infections, a factor that needs to be taken into consideration in malaria disease treatment strategies.

There are two signals that CD8+ T cells use to induce apoptosis of target cells: the perforin/granzyme pathway and the CD95/CD95-L pathway. We found that MMM were retained following infection with P. chabaudi in both CD95-deficient and perforin-deficient mice, suggesting that both pathways may be required for CD8+ T cell-mediated killing of MMM to occur. Studies to determine the mechanism of pancreatic beta cell destruction in NOD mice showed a similar phenotype as that observed in the current study, with a reduced susceptibility to disease in both CD95- (33) and perforin-deficient (34) NOD mice, suggesting that both pathways were required for initiation of disease. This, however, has not always been shown to be true (35).

We also observed a loss of MZM following infection with P. chabaudi. This loss was identified through a loss of ER-TR9 labeling, and confirmed by india ink uptake, a functional test for the presence of MZM (10). A loss of MZM-specific labeling following infection with P. chabaudi was suggested previously (12, 15, 16). It is possible that the loss of MZM following infection with P. chabaudi was the result of migration of MZM away from the spleen to a different tissue site. Karlsson et al. (36) suggested that following Staphylococcus aureus infection, MZM migrated from the MZ to the red pulp. However, if MZM were undergoing migration within the spleen in the current study, then the presence of india ink-positive cells in regions of the spleen other than the MZ would have been detected. A more likely explanation is that MZM died by apoptosis in situ and the apoptotic bodies were taken up by other phagocytes in the area, including red pulp macrophages, leading to the dispersal of india ink particles throughout the spleen, and strongly india ink-positive cells would not have been detected.

The findings in this study have implications for the understanding of how immune-mediated pathology of the spleen occurs following infection with Plasmodium and provide some clues as to the functional outcome of that pathology. Further studies are required to elucidate the mechanism and functional outcome of MZM loss, and to determine the functional outcome of killing of MMM. In addition, methods to prevent immune-mediated pathology of the spleen should be investigated, as they may have the capacity to limit the immune suppression that is associated with malaria disease.


   Acknowledgments
 
We acknowledge Yonghong Zhou and Rachel Kuns for technical assistance, and William Heath and Trevor Forster for helpful discussions.


   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 was supported by the National Health and Medical Research Council of Australia. L.B. received financial support from Queensland University of Technology and the Cooperative Research Centre for Vaccine Technology. Back

2 Address correspondence and reprint requests to Dr. Michael F. Good, Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, P. O. Royal Brisbane Hospital, Brisbane 4029, Australia. E-mail address: Michael.Good{at}qimr.edu.au Back

3 Abbreviations used in this paper: MZ, marginal zone; CD95-L, CD95-ligand; MMM, marginal metallophilic macrophage; MZM, MZ macrophage; RT, room temperature. Back

Received for publication February 17, 2006. Accepted for publication May 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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