Cell Distribution Striking but Temporary Changes in Splenic

Plasmodium chabaudi chabaudi Infection in Mice Induces Strong B Cell Responses and Striking But Temporary Changes in Splenic Cell Distribution This information is current as of September 25, 2021. Ariel H. Achtman, Mahmood Khan, Ian C. M. MacLennan and Jean Langhorne J Immunol 2003; 171:317-324; ; doi: 10.4049/jimmunol.171.1.317 http://www.jimmunol.org/content/171/1/317 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Plasmodium chabaudi chabaudi Infection in Mice Induces Strong B Cell Responses and Striking But Temporary Changes in Splenic Cell Distribution1

Ariel H. Achtman,* Mahmood Khan,† Ian C. M. MacLennan,† and Jean Langhorne2*

B cells and Abs play a key role in controlling the erythrocytic stage of malaria. However, little is known about the way the humoral response develops during infection. We show that Plasmodium chabaudi chabaudi causes major, but temporary changes in the distribution of leukocytes in the spleen. Despite these changes, an ordered response to infection develops, which includes vigorous extrafollicular growth of plasmablasts and germinal center formation. Early in the response, the lymphocytes in the T zone and follicles become widely spaced, and the edges of these compartments blur. This effect is maximal around the peak of parasitemia. Germinal centers are apparent by day 8, peak at day 20, and persist through day 60. Extrafollicular foci of plasmablasts are visible Downloaded from from day 4 and initiate a very strong plasma cell response. Initially, the plasma cells have a conventional red pulp distribution, but by day 10 they are unconventionally sited in the periarteriolar region of the white pulp. In this region they form clusters occupying part of the area normally ﬁlled by T cells. B cells are absent from the marginal zone for at least 30 days after the peak of infection, although ﬂow cytometry shows their continued presence in the spleen throughout infection. Relatively normal splenic architecture is regained by day 60 of infection. These results show that the changes in splenic cell distribution are linked to the

presence of parasites and do not seem to interfere with the development of the humoral response. The Journal of Immunology, http://www.jimmunol.org/ 2003, 171: 317–324.

cells and Abs are necessary for eliminating the erythro- still be found up to 1 year after infection with P. chabaudi cytic stages of malaria in most experimental models. chabaudi, but their number is significantly reduced compared with B Mice lacking B cells are unable to clear infections with the response 3 mo after infection (14). Together, these data indi- Plasmodium yoelii and Plasmodium chabaudi chabaudi, although cate that the generation or maintenance of memory B cells and in the latter, the early acute phase is controlled to some extent in long-lived plasma cells could be impaired in malaria infection. the absence of Ab (1–3). A role for the humoral immune response One way of examining B cell memory generation is to assess the in protective immunity in human malaria is inferred from the ex- balance between the follicular and extrafollicular B cell response. by guest on September 25, 2021 perimental models and from immuno-epidemiological studies in T-independent Ab responses, which typically induce extrafollicu- which high Ab titers and in some cases restricted Ig isotypes to lar Ab responses, are not associated with functional germinal cen- particular Ags or regions of Ags correlate with immunity. Transfer ters or memory formation (15). Under exceptional conditions, T of immune serum into naive mice or humans can diminish or pre- cell-independent germinal centers can form but do not complete vent infection in the recipient (4, 5). their development (16). By contrast, T-dependent Ags, which pro- Although Ab appears to be important in the blood stages of duce both extrafollicular responses and germinal centers, are as- human malaria, immunity only develops after repeated infections sociated with affinity maturation and sustained Ab responses (17, and is lost without continued exposure to infection. Longitudinal 18). The differentiation between the follicular and extrafollicular studies in malaria-exposed human populations show that Ab levels pathways is performed by studying the four major B cell popula- to several important malarial proteins drop rapidly at the end of the tions of the spleen: naive recirculating B cells, which are mainly malaria transmission season (6–10). This drop in Ab levels is also located in the follicular mantle of the white pulp; germinal center seen after people have left the transmission area (11). These data B cells as the source of memory B cells and long-lived plasma contrast with Ab responses of noninfected mice to nonrenewable cells; plasma cells and their plasmablast precursors; and marginal protein Ags that can be sustained for years (12, 13). In mice, zone B cells, which include both naive and memory cells and give splenic B cells able to respond to infected erythrocytes in vitro can rise to rapid extrafollicular thymus-dependent and thymus-independent Ab responses (19–21). In the present study, C57BL/6 mice were infected with P. *Division of Parasitology, National Institute for Medical Research, London, United chabaudi chabaudi (AS), and the resulting changes in the splenic Kingdom; and †Medical Research Council Center for Immune Regulation, University architecture as well as the four major B cell populations of the of Birmingham, Edgbaston, Birmingham, United Kingdom spleen were studied by immunohistology and flow cytometry. We Received for publication December 24, 2002. Accepted for publication April 29, 2003. conclude that there are major structural changes in the spleen, but these reflect an ordered response to the parasite rather than a sal- 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 vage response to indiscriminate damage. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Medical Research Council, U.K., and in part by the Materials and Methods European Union, International Cooperation with Developing Countries CT980363. Animals 2 Address correspondence and reprint requests to Dr. Jean Langhorne, Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 Female C57BL/6 mice bred in the specific pathogen-free unit at the Na- 1AA London, U.K. E-mail address: [email protected] tional Institute for Medical Research (London, U.K.) were used at 6–12 wk

of age. They were conventionally housed on sterile bedding, food, and hydrated in graded alcohols, cleared with Histoclear (National Diagnostics, water. Hull, U.K.), and mounted in DPX (BDH). Parasites and infection Flow cytometry P. chabaudi chabaudi clone AS was routinely injected from frozen stocks. Single-cell suspensions of spleens were prepared, the erythrocytes lysed 5 Further infections were initiated by i.p. injection of 10 parasitized RBCs with 0.16 M NH4Cl and the cells resuspended in FACS buffer (1% weight obtained from infected mice before the peak of parasitemia, and the to volume ratio BSA, 5 mM EDTA, 0.01% sodium azide in PBS, or FACS infection monitored by Giemsa-stained thin blood films as previously buffer without sodium azide for syndecan-1 staining). Cells (5 ϫ 105) were described (22). incubated with appropriately diluted Abs in the presence of anti-Fc receptor Ab to prevent Ab binding via the Fc receptor. Samples were acquired ␥ 3 Immunization with chicken -globulin (CGG) on a FACSCalibur and analyzed with CellQuest software (BD Biosciences, CGG (1 mg/ml; The Jackson Laboratory, Bar Harbor, ME) was mixed 1:1 San Jose´, CA). with 9% potassium-aluminum-sulfate and precipitated with 1 M sodium hydroxide. Mice were immunized i.p. with 25 ␮g alum-precipitated Ag in Cell death ␮ 100- l saline solution. 7-Aminoactinomycin D (7-AAD, Sigma-Aldrich) was used to detect apoptotic cells, with the necrotic cell marker propidium iodide used in parallel Abs and other reagents for immunohistology and flow cytometry ϩ to ascertain that the majority of the 7-AAD cells were actually apoptotic. The following Abs and reagents were used in immunohistology: rat anti- Splenocytes were stained as described in the previous section. In the final mouse CD3 (KT3) and rat anti-mouse IgM H chain (LO-MM-9) (Serotec, step, either 7-AAD (1 ␮g/2.5 ϫ 105 cells) or propidium iodide (1 ␮g/2.5 ϫ Oxford, U.K.); rat anti-mouse syndecan-1 (CD138, 09341D; BD Phar- 105 cells; Sigma-Aldrich) was added for 20 or 5 min, respectively. Cells Mingen, Oxford, U.K.); biotinylated peanut agglutinin (PNA; Vector Lab- were then analyzed on the FACSCalibur without further washes. Downloaded from oratories, Burlingame, CA); sheep anti-mouse IgD and HRP-labeled don- key anti-sheep/goat Ig (The Binding Site, Birmingham, U.K.); biotinylated ELISA rabbit anti-rat Ig and alkaline phosphatase (AP)-labeled StreptABC com- Total IgG and malaria-specific IgG were measured using affinity-purified plex (DAKO, Cambridgeshire, U.K.). goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, The Abs for flow cytometry were obtained from BD PharMingen: FITC- AL) and crude malaria extract (22) as coating reagents. IgG was revealed labeled anti-mouse CD21/CD35 (7G6), anti-mouse IgD (11-26c.2a), and with AP-labeled goat anti-mouse IgG Abs (Southern Biotechnology Asso- anti-mouse GL7 (GL7); PE-labeled anti-mouse CD23 (B3B4), anti-mouse ciates) and p-nitrophenyl phosphate as described (1, 22). Normal plasma CD19 (1D3), and anti-mouse syndecan-1 (CD138; 281-2); biotinylated anti- http://www.jimmunol.org/ was used as a negative control, and plasma from P. chabaudi chabaudi mouse CD19 (1D3). Streptavidin-Tricolor was from Caltag Laboratories immune mice was used as a standard (22). Data are expressed as milli- (Burlingame, CA). Rat anti-mouse B220 (RA3 3A1) (23) and rat anti- grams per milliliter and arbitrary units (AU), based on a immune plasma mouse Fc receptor (2.4G2) Abs (24) were purified from hybridoma value of 100, respectively, for total IgG and malaria-specific Abs. supernatants. Tissue preparation for immunohistology Statistical analysis Data were analyzed by a two-tailed Student t test using GraphPad Instat Spleens were snap frozen by repeated dipping in liquid nitrogen and stored Ͻ at Ϫ70°C in gripseal plastic bags. The enlarged spleens from later stages software (San Diego, CA). Values of p 0.05 were considered to be of infection were halved before freezing. The spleens were embedded in significantly different.

cold OCT compound (Sakura, Zoeterwede, The Netherlands), and serial by guest on September 25, 2021 5-␮m cryosections were mounted on four spot-glass slides (Hendley Essex, Results Loughton, U.K.). Slides were allowed to air dry at room temperature for at Characteristics of the P. chabaudi chabaudi infection least 1 h. They were fixed in 90% acetone at 4°C for 20 min, air dried, and stored in gripseal bags at Ϫ20°C until use. P. chabaudi chabaudi (AS) infection with 105 parasitized RBCs becomes patent in C57BL/6 mice on day 3 of infection, following Immunohistology which parasitemia rises exponentially, peaking on day 9 or day 10 Immunohistology was performed as previously described (25). In brief, of infection with up to 30% of erythrocytes infected. Parasitemia primary Abs were added to thawed sections for1hatroom temperature, then drops rapidly, followed by a much lower recrudescence of followed by washing and HRP- or biotin-labeled secondary Abs for 45 ϳ min. After another wash, AP-labeled StreptABC complex was added. HRP 1% at day 20–25 and becomes undetectable around day 30–35 activity was detected with diaminobenzidine tetrahydrochloride solution (Fig. 1a). The infection is accompanied by splenomegaly with containing hydrogen peroxide to inhibit endogenous peroxidases. AP ac- spleen weights increasing more than 10-fold (e.g., Ref. 26) and tivity was detected using the substrate Naphthol AS-MX phosphate (0.4 nucleated splenocyte numbers increasing 8-fold (Fig. 1b). There is mg/ml) and the chromogen Fast Blue BB salt (1 mg/ml, Sigma-Aldrich, a 3-fold increase in total plasma IgG peaking at 14 days of infec- Poole, U.K.) in 50 mM TBS (pH 9.2), containing 0.8 mg/ml levamisole Ϯ Ϯ (Sigma-Aldrich) to inhibit endogenous phosphatases, and 3.8% v/v N,N- tion (6.14 0.53 mg/ml compared with 2.32 0.10 mg/ml in dimethylformamide (Sigma-Aldrich). The slides were then mounted in Im- normal plasma, p Ͻ 0.01, n ϭ 5) and levels remain elevated at 28 muno-Mount (Thermo Shandon, Pittsburgh, PA). days (5.558 Ϯ 0.22 mg/ml compared with normal plasma, p Ͻ ϭ Size measurements in histological sections 0.01, n 5). Malaria-specific IgG was detectable only after 20 days of infection (day 20, 3 Ϯ 0.7 AU; day 28, 30 Ϯ 10 AU). The size of the germinal centers (PNAϩ areas in the follicular mantle) was determined by systematic perusal of the entire spleen section through the General changes in white pulp after infection grid of the microscope ocular while counting the number of grid intersec- tions that fell within the germinal center. This measurement was converted The four major B cell populations of the spleen were examined into millimeters squared by comparison with the grid of a Neubauer count- during a course of infection with P. chabaudi chabaudi. Immuno- ing chamber at the same magnification. Where spleens had to be halved for histology was performed to determine: 1) the distribution of T cells cryosectioning, germinal center numbers were extrapolated for whole spleens. and naive recirculating B cells (also referred to as follicular B cells) by staining for CD3 and IgD (Fig. 1c); 2) the extrafollicular H&E staining of splenic sections Ab response by staining cells committed to plasma cell differen- Spleens were fixed for 24 h in 10% neutral buffered formalin, embedded in tiation with syndecan-1 (CD138) (Fig. 1d); 3) the development and fibrowax (BDH, Poole, U.K.), sectioned, and then stained with H&E, de- persistence of germinal centers by staining for IgD and CD3, where germinal centers appear as unstained areas in the follicles 3 Abbreviations used in this paper: CGG, chicken ␥-globulin; AP, alkaline phospha- (Fig. 1c), or positive staining of germinal centers for PNA binding tase; 7-AAD, 7-aminoactinomycin D; AU, arbitrary units; PNA, peanut agglutinin. (Fig. 2a); and 4) the involvement of marginal zone B cells by IgM The Journal of Immunology 319 Downloaded from

FIGURE 1. The basic splenic white pulp architecture and plasma cell response during malaria infection. a, Course of parasitemia in C57BL/6 mice http://www.jimmunol.org/ after i.p. injection of 105 P. chabaudi chabaudi (AS)-parasitized RBCs. b, Numbers of nucleated splenocytes per spleen during the infection. c, The basic division of the white pulp into the T zone and the follicular mantle is deﬁned by Abs against T cells (CD3 in blue) and naive B cells (IgD in brown). d, Ab-containing plasmablasts and plasma cells are revealed by Abs against syndecan-1 (blue) by guest on September 25, 2021 compared with naive B cells (brown). GC, germinal centers; FM, follicular mantle; T, T zone; ex foc, extrafollicular focus. Dark spots in the later images are caused by the malarial pigment hemo- zoin. The size bar corresponds to 100 ␮m. Spleens from days 0, 8, 10, 20, and 60 of infection were examined. The sections shown are representative of three to six mice at each time point. 320 SPLENIC HUMORAL RESPONSES IN MOUSE MALARIA

small B cells at the outer surface of the germinal centers (day 10, Fig. 1c). Between days 10 and 40, the cuff of T cells in the periarteriolar region reforms, but strikingly, this region is temporarily shared by large accumulations of Ab-containing cells, which form a subcompartment displacing T cells to only part of the periarteriolar sheath (Fig. 2, d and e). Although the white pulp organiza- tion is changed to an extraordinary extent at the peak of infection, this effect reverses with the waning of the parasitemia and by day 60 of infection, the white pulp has regained fairly normal appearance (day 60, Fig. 1c).

Red pulp changes Before infection, the red pulp contains many widely spaced IgDϩ and CD3ϩ cells (day 0, Fig. 1c). On days 8 and 10 there has been a massive increase in the number of Ab-containing cells in the red pulp (days 8 and 10, Fig. 1d). Some of these contain IgM, but many have switched to IgG (data not shown). Between days 10 and 20 of infection, the red pulp appears devoid of IgDϩ and CD3ϩ Downloaded from lymphocytes (day 10 and 20, Fig. 1c). Flow cytometric analyses show that the numbers of naive recirculating B cells (Fig. 3b) and of T cells (27) are not decreased at the peak of infection, suggesting that the lymphocytes are just more dispersed in the enlarged spleens. To assess the type of cells occupying the expanded red pulp, H&E staining was performed. There is a massive but transient increase in the level of hematopoiesis in the red pulp in agree- http://www.jimmunol.org/ ment with previous observations in P. yoelli infections (28). Large islands of erythroid precursors and megakaryocytes were observed at day 10 (Fig. 2f). Macrophage densities were not increased ac- cording to F4/80 or nonspeciﬁc esterase staining (data not shown).

B cell development in malarial spleens The location of the four major splenic B cell populations (naive recirculating B cells, germinal center B cells, Ab-containing cells, by guest on September 25, 2021 marginal zone B cells) during infection was determined by immunohistology. Small numbers of PNA-positive germinal centers are present in the spleens of uninfected control mice (Table I). Slightly increased numbers of small germinal centers are seen on days 7–9, FIGURE 2. Location of B cell populations in malarial spleens. Sections and by day 10 the germinal center response is firmly established from spleens infected with 105 P. chabaudi chabaudi (AS)-parasitized (Table I, Figs. 1c and 2a), representing the most constant element RBCs are shown. a, PNA (blue) and anti-IgD (brown) show well-devel- in the changing white pulp structure. The germinal centers con- oped germinal centers on day 10 of infection. b, Areas of apoptotic cells tinue to increase in size and number up to day 20. Normal germinal with condensed nuclei are circled in a germinal center stained with H&E. centers are present up to day 40 and still visible on day 60 in a c, Marginal zone B cells are positive for IgM and negative for IgD. Arrows more weakly stained form. Germinal centers are slightly larger and point to the marginal zone, which no longer contains visible marginal zone B cells on day 10 of infection. Serial sections stained with Abs against IgD more frequent on day 10 of a P. chabaudi chabaudi infection than (brown) and either CD3 (d) or syndecan-1 (e) show that T cells and Ab- after immunization with a conventional Ag, alum-precipitated containing cells occupy nonoverlapping areas of the T zone from day 12 of CGG, and at day 20 of infection, the number and average size of infection, as shown by the arrows. f, H&E staining shows that the infected the germinal centers are significantly higher those seen 10 days spleen at the peak of infection contains many more hematopoietic precur- after CGG immunization (Table I, p Ͻ 0.05). sors than an uninfected spleen. These include erythroid precursors (islands Small foci of Ab-containing cells (differentiating plasmablasts of small nuclei) and megakaryocytes (Meg), which compress the splenic and Ab-secreting plasma cells) detected by anti-syndecan-1 Abs sinuses and cords of Billroth visible on day 0. The size bar corresponds to are found on day 4 of infection, all in the typical red pulp location ␮ ␮ 100 mina and cÐf and to 10 minb. Spleens from days 0, 8, 10, 20, and (data not shown). The numbers of Ab-containing cells increase 60 of infection were examined. The sections shown are representative of rapidly, reaching peak density at day 9 or day 10 of infection (days three to six mice at each time point. 8 and 10, Fig. 1d). In the rapid expansion period between days 7 and 10, a large number of IgDϩ Ab-containing cells can be seen staining in the marginal zones (Fig. 2c). Parallel studies were con- among the syndecan-1ϩ cells in the red pulp (data not shown). ducted using flow cytometry. At day 10 of infection, the majority of the syndecan-1ϩ cells are Immunohistology revealed that marked changes occur in splenic located in the T cell zone of the white pulp (day 10, Fig. 1d) rather lymphocyte distribution during the peak of infection (days 8–10). than the red pulp. Between day 12 and at least day 20 of infection By day 7–8 of infection, the T zone and the follicular mantle begin (day 20, Fig. 2, d and e, and Fig. 1d), as the total number of to lose their clear outlines (day 8 in Fig. 1c). By 10 days of in- syndecan-1ϩ cells begins to decrease and more T cells are seen in fection, only a few T cells are detected in the T zone, and the the T zone, T cells and Ab-containing cells occupy nonoverlapping indistinct follicular mantle consists mainly of IgDϩ recirculating areas of the T zone. By day 30, the Ab-containing cells are again The Journal of Immunology 321

FIGURE 3. Flow cytometric analysis of B cell populations in malarial spleens. Flow cytometric proﬁles are shown for representative time points of infection along with graphs of the absolute numbers of each population in the spleen. Live cells in the Downloaded from nucleated splenocyte fraction were used as the basis for further population deﬁnitions. a, Total B cells (CD19ϩ), b, recirculating naive B cells (CD21int CD23ϩ), c, marginal zone B cells (CD21ϪCD23ϩ), d, germinal center B cells (GL7ϩCD19ϩ), e, Ab- containing cells (syndecan-1ϩCD19ϩ). Bars repre- sent the arithmetic mean from three to six spleens, http://www.jimmunol.org/ with up to 15 spleens for time point 0, as an uninfected control was included in each experiment. Er- ror bars show SE. MZ, marginal zone B cells; FO, follicular B cells; GC, germinal centers; PC, plasma cells; ND, not done. by guest on September 25, 2021

found in the red pulp (data not shown) with numerous foci still visible at day 60 (day 60, Fig. 1d). Table I. Germinal center growth after P. chabaudi chabaudi infectiona Marginal zone B cells (identified in immunohistology as ϩ Ϫ IgM IgD ) are normally located at the edge of the white pulp, as Day Germinal Center (n) Average size (mm2) can be seen in the spleens of uninfected mice (day 0, Fig. 2c). After infection, the marginal zone B cells decrease in number, and after 0 1.5 Ϯ 0.5 0.002 Ϯ 0.0003 Ϯ Ϯ day 10 are no longer detectable in the marginal zones (day 10, Fig. 8 21.2 6.5 0.004 0.0005 9 47.0 Ϯ 1.0 0.009 Ϯ 0.003 2c). The cells may have migrated to other parts of the spleen, but 10 70.0 Ϯ 20.0 0.016 Ϯ 0.002 ϩ immunohistology cannot distinguish between IgM plasma cells 11 77.7 Ϯ 29.7 0.010 Ϯ 0.002 and IgMϩ B cells of marginal zone origin without the additional 20b 152 Ϯ 23.1 0.040 Ϯ 0.001 ϩ Ϯ Ϯ locational indicator. By day 40, the marginal zones contain IgM 30 110.0 23.2 0.028 0.011 60 35.2 Ϯ 2.6 0.013 Ϯ 0.0007 B cells again, but even at day 60 they form a less continuous CGGc 51.7 Ϯ 3.67 0.013 Ϯ 0.002 structure than before the disruption (data not shown). a The arithmetic means and SE of germinal center measurements in cryosections Quantification of splenic B cell populations are shown for two to three spleens at each time point. Germinal center numbers and areas were determined as described in Materials and Methods. Due to the large increase in spleen size during infection, which b The number and size of germinal centers on day 20 are significantly different from those induced 10 days after CGG immunization ( p Ͻ 0.005). involves unequal expansion of the red and white pulp, an increase c Spleens were taken 10 days after immunization with alum-precipitated CGG. in a cell population may be masked by the overall increase in 322 SPLENIC HUMORAL RESPONSES IN MOUSE MALARIA

Table II. Levels of apoptosis in splenic B cells at the peak of Plasmodium chabaudi chabaudi infectiona

CD19ϩ B Cells Germinal Center B Cells Marginal Zone B Cells

Total no. of cells Total no. of cells Total no. of cells Day Percentageb (ϫ107)c Percentage (ϫ105) Percentage (ϫ105)

0 10.2 Ϯ 1.5 0.4 Ϯ 0.09 24.4 Ϯ 6.4 0.2 Ϯ 0.08 10.7 Ϯ 1.8 8.0 Ϯ 3.5 8 7.7 Ϯ 0.06 1.4 Ϯ 0.36 3.1 Ϯ 0.9 3.3 Ϯ 0.86 4.0 Ϯ 0.3 18 Ϯ 1.3 9 8.7 Ϯ 5.0 2.0 Ϯ 0.35 4.4 Ϯ 2.5 4.7 Ϯ 0.61 5.4 Ϯ 3.1 4.5 Ϯ 0.5 10 14.1 Ϯ 0.9 1.7 Ϯ 0.29 5.6 Ϯ 1.5 3.5 Ϯ 0.24 6.6 Ϯ 0.8 2.6 Ϯ 0.4

a Each data set represents the arithmetic mean and SE from a group of three mice. b The percentage of apoptotic cells in each B cell population, as determined by 7-AAD. c Total numbers of apoptotic cells of this type in the spleen. spleen size. We therefore analyzed the four major splenic B cell ( p Ͻ 0.05, 0.01, and 0.05, respectively). This contrasts with the populations quantitatively by flow cytometry. The Ab combina- apparent complete loss of marginal zone B cells seen in immuno- tions used to define the B cell populations are shown for two rep- histology, suggesting that the marginal zone B cells have migrated resentative time points of infection and compared with uninfected into other parts of the spleen. control mice in Fig. 3. Abs against CD19 (Fig. 3a) were used to Downloaded from determine total B cell numbers. The naive follicular B cells are Apoptosis defined as CD21intCD23ϩ (Fig. 3b, labeled FO), while marginal Previous studies have shown that malarial infection causes large zone B cells are CD21ϩCD23Ϫ (Fig. 3c, labeled MZ) (20). Ger- numbers of host cells to undergo apoptosis (26, 27). To determine minal center B cells were identified by positive staining with Abs whether apoptosis preferentially affects any particular population against GL7 and CD19 (Fig. 3d, labeled GC), as there was a con- of B cells, CD19ϩ B cells, germinal center B cells, and marginal siderable level of nongerminal center binding of PNA in the in- zone B cells were labeled with 7-AAD. Splenocytes from days 8, http://www.jimmunol.org/ fected spleens. Ab-containing cells (plasma cells and plasmablasts) 9, and 10 of infection were studied, as the parasite density and are defined as syndecan-1ϩCD19int (Fig. 3e, labeled PC). splenic disruption are highest during this period. The graphs in Fig. 3 show how the relative population sizes, as Although the total number of apoptotic CD19ϩ, germinal cen- determined by flow cytometry, translate into absolute numbers for ter, and marginal zone B cells increases ϳ5-fold, 24-fold, and the different B cell populations. The total number of CD19ϩ B 2-fold, respectively, by day 8 or day 9 of infection (Table II), there cells is greatest at day 8 of infection when there are roughly twice were no significant differences in the percentages of apoptotic cells as many B cells as in uninfected mice (Fig. 3a, p ϭ 0.0012). After in any of the B cell populations when compared with control un- that, numbers drop slightly and fluctuate around a value that is still infected mice ( p Ͼ 0.05 for all B cell populations). This suggests significantly higher than that in uninfected mice ( p Ͻ 0.01 for days that the increase in total numbers of apoptotic B cells is entirely by guest on September 25, 2021 10, 20, 30, and 40 postinfection). The naive recirculating B cell explained by the size increase in these populations during infection population (CD21intCD23ϩ) grows in the early and late stages of (see Fig. 3). Examination of the H&E-stained sections from days infection, but even at the peak of infection, numbers are not sig- 8 and 10 of infection supports this notion, as clusters of cells with nificantly lower than in naive mice (Fig. 3b). Thus, the marked apoptotic nuclei (condensed chromatin) were only observed in the transient reduction of these cells in the follicular mantles observed germinal centers, where apoptosis is an integral part of affinity- by immunohistology between days 8 and 12 is not reflected in the based B cell selection (Fig. 2b). total population size. The total numbers of germinal center B cells and Ab-containing Discussion cells agree well with the immunohistological observations. The Infection of C57BL/6 mice with P. chabaudi chabaudi leads to numbers of germinal center B cells (GL7ϩCD19ϩ) rise between severe but reversible disruption of the splenic white pulp architec- days 3 and 5 postinfection, with significant increases above unin- ture, with transient loss of follicular mantle and T zone integrity, fected controls by day 6 (Fig. 3d, p Ͻ 0.01). Numbers are maximal atypical location of plasma cells, and migration of marginal zone on day 20 and remain elevated relative to uninfected controls up to B cells. These observations extend previous studies on P. chabaudi day 40 of infection ( p Ͻ 0.01 comparing day 0 with days 10–40) and P. yoelii infections where less specific histological analysis and even at this late stage, levels are five times higher than at day indicated splenic disruption and partial lymphocyte depletion (29– 10 after CGG immunization ( p Ͻ 0.01 comparing day 40 postin- 31). In human Plasmodium falciparum malaria, disturbed T cell fection with CGG-immunized mice). areas and changes in splenic architecture have also been observed Ab-containing (syndecan-1ϩ) cell numbers increase even more (B. Urban, unpublished observations). sharply (Fig. 3e) with the peak levels at day 10 of infection ϳ300- The disturbance of the B and T cell areas does not appear to fold higher than uninfected levels ( p Ͻ 0.01 comparing day 0 with affect either generation of germinal centers or the production of days 8–12 postinfection) and over 100-fold higher than on day 10 Ab-containing cells (plasma cells and plasmablasts) in the spleen. after CGG immunization ( p Ͻ 0.0001). After that, levels drop, but This finding suggests that organized primary B cell follicles and T are still over four times higher than uninfected levels 60 days after cell areas may not be necessary for the generation of germinal infection ( p Ͻ 0.01). centers and supports previous findings that B cells can colocalize The marginal zone B cell population exhibits a strong peak at with the follicular dendritic cell network and develop fully func- days 5 and 6 of infection, with significantly higher numbers than tional germinal centers in mice incapable of forming primary those observed in uninfected mice ( p Ͻ 0.01) followed by a sharp splenic B cell follicles (32). decline at day 7 and a gradual decrease up to day 16 (Fig. 3c). The kinetics of germinal center appearance after infection are After that, the population regains and maintains a level signifi- similar to those observed after immunization with classical hapten cantly higher than the uninfected level for days 16, 30, and 40 Ags (e.g., (4-hydroxy-3-nitrophenyl)acetyl nitropropene), where The Journal of Immunology 323

PNAϩ germinal centers are visible within 8 days of immunization thought to play a role in plasma cell migration (45), but it is un- (33). The lifespan of germinal centers depends on the duration of known whether T cells supply further migration signals via che- Ag exposure, and hapten-specific germinal centers are present in mokines or direct cellular interaction. the spleen for ϳ3 wk after immunization (e.g., Ref. 19). However, This study shows that B cells disappear from the marginal zones germinal centers in the spleens of P. chabaudi-infected mice can at the peak of infection. Flow cytometry reveals that the cells are persist for at least 60 days, which is reminiscent of the persistence still present in the spleen and do not increase their level of apo- of germinal centers at least 4 mo after murine mammary virus ptosis, suggesting that they migrate to other parts of the spleen. infection (25). In visceral leishmaniasis on the other hand, which LPS causes transient loss of B cells from marginal zones (46) and resembles malaria in causing splenomegaly and long parasite per- immune complexes can induce transient migration of B cells from sistence, germinal centers diminish after 4 wk of infection in par- marginal zones to the follicles (47). In addition, LPS injection allel with destruction of follicular dendritic cells (34). This shows stimulates IgDϩ blast formation reminiscent of that observed in that closer examination of Ag-processing cells and APC during our model (48), but malaria-infected spleens do not exhibit the malaria infection must still be performed to understand better the rapid repopulation of the marginal zone seen after injection of functioning of the B cells. nonreplicating Ags (47). The long-lasting changes in the marginal These results indicate that if B cell memory is impaired it is not zone B cells suggest that these cells may play a role during the through a deficiency in germinal center formation or maintenance. early acute malarial infection. Therefore, it would be interesting to Other possible causes for the reported short-lived Ab responses or see whether this phase of infection is altered in a mouse lacking poor memory responses (6–10), such as ablation of specific B cells marginal zone B cells, such as the Pyk-2-deficient mouse (21). by soluble Ag (35, 36) or generation of short-lived rather than A possible function of the marginal zone B cells may lie in their Downloaded from long-lived plasma cells are currently being investigated. It has capability to develop into Ab-secreting plasma cells more quickly been shown previously that large amounts of soluble Ag can in- than naive follicular B cells, particularly in response to particulate duce apoptosis of specific B cells in germinal centers and thus T-independent Ags (49, 50). This possibility is supported by our affect affinity maturation and B cell memory (35, 36). Because observation that marginal zone B cell numbers roughly double on malaria infections generate a lot of circulating Ag (37), this would days 5 and 6 of infection, with a sizable fraction (18% as compared

be an attractive mechanism of interference with memory develop- with 3% in naive animals, data not shown) of the marginal zone B http://www.jimmunol.org/ ment in malaria. However, we found no evidence that enhanced cell population observed on day 6 of plasmablast size. apoptosis or reduction in germinal center size takes place in ger- In summary, our findings reveal no obvious deficiencies in the minal centers during the acute infection. follicular or extrafollicular B cell pathways. On the contrary, the B We have also shown that plasmablast and plasma cell numbers cell response is much stronger than that seen in classical immuni- expand greatly up to the peak of the infection and then rapidly zation experiments despite the gross alterations of the splenic ar- disappear, which fits with the spleen’s limited capacity to support chitecture at the peak of infection. Although only P. chabaudi continued plasma cell survival (38). Most plasma cells generated (AS) was studied, the observation of splenic disruption and abnor- during an immune response are short lived, with only a small pro- mal plasma cell distribution in infections with other species of portion of long-lived cells remaining in the spleen or migrating to Plasmodium support the assumption that other clones of P. by guest on September 25, 2021 the bone marrow (39–41). Increased plasma cell numbers were chabaudi and other rodent malaria species will elicit a similar B still found in the red pulp 60 days after the mice were infected. It cell response. is unclear whether these persistent plasma cells are long-lived cells The overall strength of the B cell response could be misleading, derived from the extrafollicular response or are recent emigrants because malaria is known to generate a large polyclonal B cell from germinal centers. All of these sources contribute to persistent response (51, 52). The kinetics of this response correspond well red pulp plasma cells in responses of noninfected mice to hapten with the peak prevalence of Ab-containing cells in the infected protein (38). It would be valuable to determine the balance be- spleens. However, the level of specific malarial IgG does not rise tween the acute short-lived plasma cells in the spleen and the long- above the levels in normal serum until 3 wk of infection. This lived plasma cells in the bone marrow because this could provide result suggests that a large fraction of the early B cell response important information on the longevity of the Ab response in may not be specific for malarial Ags. Therefore, it is essential to malaria. study how the B cell response specific for a malarial Ag develops Normally, plasma cells move from the edges of the T zone in the spleen and the bone marrow. through the bridging channels into the extrafollicular foci of the red pulp (33). 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