INTRODUCTION

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Malaria infection is characterized by both major activation and suppression of the immune system during different phases of the disease. The immune response to the intra-erythrocytic stages of malarial parasites has been best characterized in the rodent model Plasmodium chabaudi chabaudi. In this model the initial cell-mediated response is thought to be mediated by CD4⁺ Th1-cell-dependent activation of effector cells such as macrophages, which mediate nonspecific killing or inactivation of the parasite. This is followed by a sequential switch to Th2 cytokine production, stimulation of antibody-dependent immune mechanisms involved in the final control, and clearance of the parasite (19, 13, 28, 30). Splenomegaly and polyclonal B-cell activation are common phenomena associated with malaria infection in both humans and experimental murine models. The spleen is believed to participate in both the clearing of parasites from the circulation as well as providing a strong hematopoietic response during acute infection (3, 12, 37). We have recently demonstrated the appearance of interleukin-4 (IL-4)-producing FcRI⁺ non-B, non-T cells in the spleens and peripheral blood of P. chabaudi-infected mice during and shortly after peak parasitemia (13) indicative of an altered lymphocyte trafficking during murine Plasmodium infections as described by others (3, 18). The sequential appearance, regulation and development of the different cell populations in the spleen during malaria infections are still poorly understood.

Programmed cell death, apoptosis, is an important mechanism regulating the development, maturation, and activation of lymphocytes. In addition, apoptosis may also prevent and terminate lymphocyte responses (reviewed in references 17 and 33). Lymphocytes may die of "neglect," e.g., lack of proper stimulation, but also from active processes such as signaling through "death receptors" or through cytokines such as IL-2 or tumor necrosis factor alpha (TNF-) (33). During responses to infectious pathogens such as certain viral infections, e.g., lymphocytic choriomeningitis virus infection in mice, the immune response is often associated with high levels of apoptosis in the spleen, both in the selection of antigen-specific cells in the early stages of infection, as well as the silencing of the immune response at the end of the infection (reviewed in reference 36). Polyclonal activators such as lipopolysaccharide or staphylococcal enterotoxin B have been shown to induce strong apoptotic responses when injected into experimental animals, whereby apoptosis may act as a host protective mechanism functioning to limit the excessive inflammatory response (7, 15). In parasitic infections, apoptosis has been shown to be induced in fresh splenocytes and in in vitro-cultured spleen cells from mice infected with Schistosoma mansoni (9, 11), Trypanosoma cruzi (21), and Toxoplasma gondii (16), where it has been implicated as a mechanism whereby parasites escape the immune response. In vitro cultures of human peripheral blood mononuclear cells from patients with acute Plasmodium falciparum malaria (31, 32) have also been demonstrated to display increased numbers of apoptotic cells as compared to healthy controls.

We have investigated changes in T-cell, B-cell, and macrophage populations in the spleen and the peripheral blood during acute blood-stage P. chabaudi AS malaria in mice and determined the number of apoptotic cells in the spleen during the same time period. The phenotypes of the apoptotic cells were determined by three-color flow cytometry. Our results show drastic changes in the cellular composition of the spleen and blood during acute P. chabaudi infections. The cellular composition in spleen and peripheral blood differed significantly from each other. Apoptosis as measured by flow cytometry was shown to be greatly increased during peak parasitemia. Apoptotic cells were found among T cells, B cells, and macrophages, as was increased Fas and Fas-ligand expression, indicating that P. chabaudi-induced apoptosis is, at least in part, a Fas-mediated event. Taken together, our data provide new information on the dramatic cellular changes taking place in the spleen during malaria infection and indicate that apoptosis may be an important mechanism whereby the composition of the spleen in vivo is regulated.

	MATERIALS AND METHODS

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Animals and experimental infections. Female, 6- to 10-week-old BALB/c mice were purchased from B&K Universal (Sollentuna, Sweden). The animals were kept in the animal facility at Stockholm University and supplied with food and water ad libitum. Blood-stage infection with P. chabaudi chabaudi AS (kindly provided by D. Walliker, Edinburgh, United Kingdom) was maintained by weekly passages in naive mice. Experimental blood-stage infections were initiated by intraperitoneal inoculation of 10⁶ infected red blood cells. Parasitemia was monitored daily by Giemsa (BDH, Poole, United Kingdom)-stained thin blood smears made from tailsnips.

Cell preparations. Single cell suspensions were prepared from spleens or peripheral blood leukocytes (PBL) pooled from two to five mice at 3, 6, 9, 12, 15, 18, 23, 30, and 59 days after P. chabaudi infection. At each time point age-matched uninfected mice were included as controls. Splenic red blood cells were lysed by an ammonium chloride (0.15 M)-potassium carbonate (1 mM) buffer, and splenocytes were washed three times with RPMI 1640 supplemented with 2% fetal calf serum (FCS) (Life Technologies, Paisley, Scotland). PBL were prepared by washing peripheral blood cells three times with RPMI-FCS, followed by lysis of erythrocytes. After three additional washings the PBL were resuspended in RPMI-FCS. Both the total number of cells per spleen and the total number of leukocytes per milliliter of blood were calculated for each animal. All cells were kept on ice throughout the whole process.

Flow cytometric analysis. Spleen cells and PBL were incubated with anti-FcRII/III antibody 2.4G2 (Pharmingen, San Diego, Calif.) for 5 min at 4°C to block nonspecific binding and then stained with fluorescein isothiocyanate (FITC)- or phycoerythrin-labeled anti-CD3, anti-CD4, anti-CD8, anti-B220, and anti-Fas (all from Pharmingen); anti-macrophage (monoclonal antibody [MAb] F4-80), T-cell receptor (TCR), or TCR (Caltag, Burlingame, Calif.); or anti-Fas-ligand (Bender MedSystems, Vienna, Austria). A total of 5,000 leukocytes per sample were analyzed on a Coulter Epics XL-MCL.

Statistical analysis. Student's t test was used for assessing statistical significance. A probability of <0.05 was considered significant when comparing the results of P. chabaudi-infected and uninfected mice.

	RESULTS

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Increase in spleen cell and PBL number during P. chabaudi infection. P. chabaudi parasitemia reaches a maximum of approximately 40% in peripheral blood approximately 7 to 8 days after the start of infection. The parasitemia then rapidly decreases and becomes undetectable 12 to 14 days postinfection (Fig. 1A). The number of cells in the spleen increases rapidly during the early phase of infection, reaching a maximum of 280 million cells at day 9. The cellularity increases in two waves with a second peak at day 18 before approaching normal numbers at day 30 (Fig. 1B). The leukocyte counts in peripheral blood increase in one peak, which reaches maximum at day 12, between the two peaks in spleen cellularity (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Malaria parasitemia in peripheral blood (A) and spleen cell numbers () and PBL numbers () (B) during the course of P. chabaudi infection. Parasitemias are from 10 to 20 mice per time point and spleen cells, and PBL counts are from pooled data from six individual experiments with two to six mice per time point ± the standard error of the mean (SEM).

Alterations in the frequencies and absolute numbers of CD4⁺ and CD8⁺ T cells in the spleen and blood during acute blood-stage P. chabaudi infection. The percentage and numbers of CD4⁺ and CD8⁺ T cells in spleen and peripheral blood were analyzed by flow cytometry. As can be seen in Fig. 2, significant decreases in both the percentage of CD4⁺ and CD8⁺ cells were seen in the spleen from day 9 and throughout day 18. On day 12 and 15, the time period when the mice are recovering from the acute infection, almost 50% fewer T cells as are usually found in normal mice were seen (P < 0.05) (Fig. 2A and B). Taking the rapid increase in spleen size into account and calculating the absolute numbers of T cells, a slightly different pattern was observed (Fig. 2C and D). For both CD4⁺ and CD8⁺ T cells a three- to fivefold increase was seen on days 6 and 9 which dropped on days 12 and 15 and then increased again on day 18 (Fig. 2C and D). After day 18 both the percentages and numbers of CD4⁺ and CD8⁺ T cells approached normal levels (Fig. 2A to D).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Percentages () and absolute numbers () of CD4+ and CD8+ T cells in spleen (A to D) and peripheral blood (E to H) during P. chabaudi infection. Unfractionated splenic cells and PBL were examined by flow cytometry. Pooled data from six individual experiments with two to six mice per time point are shown. Bars represent mean values ± the SEM. Solid lines represent mean values of uninfected control mice, with the hatched lines representing ± the SEM. *, P < 0.05 between infected and uninfected mice.

Changes in B-cell and macrophage populations in spleen and peripheral blood during blood-stage P. chabaudi malaria. To investigate if B cells and macrophages also exhibited changes, the cells were stained with antibodies against B220 (B cell) and F4-80 (macrophage) antigens and analyzed by flow cytometry. The results, shown in Fig. 3, revealed that the frequencies of B cells in both the spleen and peripheral blood did not change during the course of the malaria infection (Fig. 3A and E). However, when calculating the absolute numbers of B cells there was a rapid five- to sevenfold increase in the spleen on days 6 and 9 (P < 0.05) which then decreased during later time points (Fig. 3C). This increase in the spleen on days 6 and 9 (Fig. 3C) was followed by a sequential appearance of B cells in the peripheral blood on days 12 and 15 (P < 0.05) (Fig. 3G). Thereafter, the peripheral blood composition gradually returned back to normal values (Fig. 3E and G).

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Percentages () and absolute numbers () of B220+ B cells and F4-80+ macrophages in the spleen (A to D) and peripheral blood (E to H) during P. chabaudi infection. Unfractionated splenic cells and PBL were examined by flow cytometry. Pooled data from six individual experiments with two to six mice per time point are shown. Bars represent mean values ± the SEM. Solid lines represent the mean values of uninfected control mice, with the hatched lines representing ± the SEM. *, P < 0.05 between infected and uninfected mice.

Increased numbers and frequencies of apoptotic cells in the spleen during P. chabaudi infection. To investigate if the changes in the cellular composition seen in the spleens during P. chabaudi AS infections were associated with increased levels of apoptotic cells, spleen cells were stained with Annexin V and analyzed by flow cytometry. A low but consistent percentage of Annexin V-positive spleen cells (< 5%) was found in spleens from normal mice (Fig. 4A). In spleens from P. chabaudi-infected mice an increase in numbers of apoptotic cells started on day 9 (15% apoptotic cells, P < 0.05) (Fig. 4A). Both the percentage as well as the absolute number of apoptotic Annexin V-positive cells increased from days 9 until 18 (Fig. 4) with up to 34% of the spleen cells being Annexin V positive on day 12 (P < 0.05) (Fig. 4A). Thereafter, the frequency and number of apoptotic cells slowly returned to normal (Fig. 4).

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Percentage (A) and absolute numbers (B) of apoptotic cells in spleen during P. chabaudi infection. Unfractionated splenic cells were stained with Annexin and analyzed by flow cytometry. PI-positive cells were excluded from analysis. Pooled data from three individual experiments with two to six mice per time point are shown. Bars represent the mean values ± the SEM. Solid lines represent the mean values of uninfected control mice, with the hatched lines representing ± the SEM. *, P < 0.05 between infected and uninfected mice.

Investigation of the phenotypes of Annexin V-positive cells in the spleen. To phenotypically characterize the apoptotic cells in the spleen the cells were stained with FITC-conjugated antibodies against CD4, CD8, B220, or macrophages, together with Annexin V staining. When we analyzed the various phenotypes among the absolute numbers of apoptotic cells, the results show that the vast majority of the apoptotic cells in the spleen during days 9 to 12 were B cells and macrophages. Up to 50 million cells per spleen were apoptotic B cells at day 12 postinfection (75% of the splenic B cells) (Fig. 5). The absolute numbers of apoptotic CD4⁺ and CD8⁺ T cells also increased slightly. The absolute number of apoptotic cells of all phenotypes rapidly decreased and were approximately back to normal values by day 30 postinfection (Fig. 5).

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Proportions (A) and absolute numbers (B) of apoptotic cell phenotypes in spleen during P. chabaudi infection. Unfractionated splenic cells were stained with Annexin and antibodies against CD4, CD8, B220, and macrophages and were analyzed by flow cytometry. PI-positive cells were excluded from analysis. Pooled data from three individual experiments with two to six mice per time point are shown.

Increase of splenic Fas and Fas-ligand expression in P. chabaudi-infected mice. To investigate if the increased apoptosis seen in the spleens of P. chabaudi-infected mice was a Fas-mediated event, Fas and Fas-ligand expression were analyzed by flow cytometry. As seen in Table 1, the data show that there is a prominent increase in Fas and Fas-ligand expression during a P. chabaudi infection compared to noninfected mice. A massive increase in Fas expression was seen with over 60% of spleen cells being positive at day 6 compared to 7.2% in normal spleens (P < 0.05). The splenic Fas-ligand expression peaked at day 9 at 20.7%, coinciding with the peak of apoptotic Annexin-positive cells (Fig. 4), while the Fas-ligand expression in spleens from normal mice was only 3.5% (P < 0.05). Double-staining experiments revealed that Fas and Fas-ligand positive cells were found among T cells, B cells, and macrophages (data not shown).

	DISCUSSION

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Several lines of evidence suggest that the spleen is an organ of major importance during malaria infection in terms of both its hematopoietic and immunological functions (3, 12, 37). P. chabaudi chabaudi AS infection in BALB/c mice is normally nonlethal and is characterized by a switch in T-helper response from a Th1 response (characterized by gamma interferon and IL-2 production) during the first week of infection, which then is followed by a Th2 response (characterized by IL-4 and antibody production) (13, 19, 28, 30). The switch in this T-helper cell response occurs at the time around or closely after peak parasitemia and correlates with the symptoms of anemia. During the first 1 to 2 weeks of the infection, the splenic microcirculation is altered and a blood-spleen barrier is formed, probably to seal off the hematopoietic sites of the spleen and protect them from circulating parasites (3, 35). As soon as the parasitemia in the peripheral blood decreases, the spleen opens up to its normal capacity and releases newly produced erythrocytes, and the hematocrit is returned to normal.

The complex interactions between cells and the signals regulating expansion and changes in the composition of various cell types during this period are poorly understood. The aim of the present investigation was to elucidate cellular changes in the spleen and PBL during P. chabaudi infections and to relate the degree of apoptosis to these changes.

The flow cytometry analysis revealed that the frequencies of TCR⁺, CD4⁺, and CD8⁺ cells decreased in both spleen and peripheral blood during the days around and shortly after peak parasitemia. During this time point there is an enlargement of the spleen with a great influx of non-B, non-T cells (13). The absolute numbers of TCR⁺, CD4⁺, and CD8⁺ T cells were elevated during peak parasitemia and then dropped to lower levels shortly after which the numbers increased again until the P. chabaudi infection was cleared. At the time points when the numbers were decreased in the spleen there was a parallel increase in the blood. This may reflect a lymphocyte migration to the peripheral blood as reported by others (18). The disappearance of activated T cells from the peripheral blood during acute human P. falciparum infection (14) and murine P. chabaudi infection (20) has been reported. The destination of these activated T cells remains unknown but has been proposed to be the liver (18, 24).

The number of B cells in spleen and peripheral blood increased rapidly during the course of the P. chabaudi infection. As for the T cells there was an earlier appearance of B cells in the spleen than in the peripheral blood. Whether the B cells appearing in the blood represent cells leaving the spleen or if they are naive B cells coming from the bone marrow or if it is an effect of the blood-spleen barrier (35) remains to be established.

There was no increase in the number of monocytes/macrophages in the peripheral blood during P. chabaudi infection. However, there was a slow steady expansion of macrophages in the spleen during the acute phase of the infection probably, reflecting the increased need for removal of infected erythrocytes in the spleen through phagocytosis or the production of proinflammatory cytokines (25, 27).

Apoptosis has been implicated as a regulatory mechanism in the development and homeostasis of the immune response (33). Apoptosis may eliminate self-reactive cells or limit potentially harmful immune reactions. Our data show that there was an increase in both the frequency and the number of apoptotic cells in the spleen during the peak parasitemia in acute blood-stage P. chabaudi infection. During this time period a switch from Th1 to Th2 response takes place and a marked immunosuppression is evident (1, 29). Thus, it is tempting to speculate that part of this suppression may be due to the apoptosis of certain cell types occurring in the spleen at this time.

Apoptotic cells were found among T cells, macrophages, and B cells in the spleens of the P. chabaudi-infected mice. Apoptosis has been described earlier in other parasitic infections and may be involved in downregulating inflammatory Th1 responses (9, 11, 16, 21). In the present study the majority of apoptotic cells in the spleens of P. chabaudi-infected mice were B cells. The mechanism by which B cells become apoptotic is unclear but may involve antigen-induced cell death similar to that seen in T cells (23) or be a result of increased deletion of B cells with the wrong specificity in the germinal centers (8, 17). Both Fas and Fas-ligand expression were increased during the course of infection. Fas expression was prominent already at day 6 postinfection when Fas-ligand expression was still normal. In contrast, at day 9 postinfection, when the number of apoptotic cells reached its peak, Fas-ligand expression also peaked. Thus, P. chabaudi-induced apoptosis is likely to be, at least in part, a Fas-mediated event. Preliminary experiments in our laboratory with Fas-defect lpr mice showed that these mice indeed exhibit lower levels of apoptosis in the spleen (data not shown). However, low levels of apoptosis were still evident, indicating that other mechanisms of apoptosis are also involved during P. chabaudi infection. Further studies of these mechanisms are under way in our laboratory and should provide additional insight into the function of P. chabaudi-induced apoptosis. Other mechanisms involved in the apoptosis may include TNF-, nitric oxide, or reactive oxygen (2, 6, 22), all present, at high concentrations, in the spleens of P. chabaudi-infected mice during acute infection.

The data demonstrated here show an interesting comparison to results obtained from staphylococcal enterotoxin B or lipopolysaccharide-injected mice. These mice display a strong polyclonal activation of spleen cells, one similar to that seen early in a P. chabaudi infection, which is then replaced by massive apoptosis of spleen cells and induction of anergy to both specific and nonrelated antigens (7, 15). Malaria parasites are known to release "malaria toxins," believed to be mainly glycosylphosphatidylinositol-anchored antigens which have been describe as having "lipopolysaccharide-like" activity and which may stimulate naive macrophages to secrete TNF (5, 26). It is therefore likely that these molecules may play an important role in the induction of the splenic apoptosis reported here. However, further work is necessary to establish the induction and role of apoptosis in suppression and anergy during murine malaria infection.

Apoptosis of PBL in the P. chabaudi-infected mice was not determined in this study, but previous reports have shown that peripheral blood mononuclear cells from humans suffering from acute P. falciparum malaria display elevated levels of spontaneous apoptosis (31, 32). This apoptosis could be further enhanced by the addition of parasite extracts to the cultures, indicating that parasite-derived antigens can induce apoptosis in human mononuclear cells. Interestingly, different parasite isolates induced variable levels of apoptosis, and the apoptosis was inversely correlated with proliferation. Thus, distinct parasite isolates induced either apoptosis or proliferation of the lymphocytes (32). These data, together with the data presented in our current study, imply that malaria parasites can, in a way similar to that of schistosomes, toxoplasmas, and trypanosomes, induce apoptosis in host mononuclear cells. This apoptosis may be beneficial for parasite survival, through downregulation of anti-parasite inflammatory responses, or it may act as a host-protective regulatory mechanism to limit the very strong inflammatory Th1 response during the acute infection and/or to keep the density and number of cell populations in balance.

In conclusion, our data show that rapid changes are taking place in the spleen and peripheral blood during blood-stage P. chabaudi AS malaria. The kinetics of the appearance of various cells differ in the spleen and peripheral blood. The rapid changes in cellular populations in the spleen during blood-stage P. chabaudi malaria is influenced by apoptotic events which appear to be, at least in part, Fas mediated. Thus, our data may provide additional mechanistic information about the immunosuppression seen during both human and murine acute malaria infection.