Fatal Plasmodium falciparum Malaria Causes Specific Patterns of Splenic Architectural Disorganization

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1 INFECTION AND IMMUNITY, Apr. 2005, p Vol. 73, No /05/$ doi: /iai Copyright 2005, American Society for Microbiology. All Rights Reserved. Fatal Plasmodium falciparum Malaria Causes Specific Patterns of Splenic Architectural Disorganization Britta C. Urban, 1 * Tran T. Hien, 2 Nicholas P. Day, 1,3 Nguyen H. Phu, 2 Rachel Roberts, 4 Emsri Pongponratn, 3 Margret Jones, 4 Nguyen T. H. Mai, 2 Delia Bethell, 3 Gareth D. H. Turner, 4 David Ferguson, 4 Nicholas J. White, 1,3 and David J. Roberts 4,5 Nuffield Department of Clinical Medicine 1 and Nuffield Department of Clinical Laboratory Sciences, 4 University of Oxford, and National Blood Service, 5 John Radcliffe Hospital, Oxford, United Kingdom; Centre for Tropical Diseases, Cho Quan Hospital, Ho Chi Minh City, Vietnam 2 ; and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 3 Received 7 July 2004/Returned for modification 23 August 2004/Accepted 14 December 2004 The spleen is critical for host defense against pathogens, including Plasmodium falciparum. It has a dual role, not only removing aged or antigenically altered erythrocytes from the blood but also as the major lymphoid organ for blood-borne or systemic infections. The human malaria parasite P. falciparum replicates within erythrocytes during asexual blood stages and causes repeated infections that can be associated with severe disease. In spite of the crucial role of the spleen in the innate and acquired immune response to malaria, there is little information on the pathology of the spleen in human malaria. We performed a histological and quantitative immunohistochemical study of spleen sections from Vietnamese adults dying from severe falciparum malaria and compared the findings with the findings for spleen sections from control patients and patients dying from systemic bacterial sepsis. Here we report that the white pulp in the spleens of patients dying from malaria showed a marked architectural disorganization. We observed a marked dissolution of the marginal zones with relative loss of B cells. Furthermore, we found strong HLA-DR expression on sinusoidal lining cells but downregulation on cordal macrophages. P. falciparum infection results in alterations in splenic leukocytes, many of which are not seen in sepsis. Infection with Plasmodium falciparum causes a wide variety of clinical syndromes ranging from a mild febrile illness to life-threatening conditions such as severe malarial anemia and cerebral malaria (46). Clinical immunity develops only after repeated exposure to the parasite and largely depends on the humoral immune response to variant and conserved parasite antigens (6). This immunity is complex but imperfect, allowing infection but regulating parasite density, thus preventing severe disease and attenuating symptoms. At least one family of parasite-derived variant antigens, expressed on the surface of infected red blood cells (irbc), also mediates adhesion of mature irbc stages, trophozoites and schizonts, to host receptors expressed on endothelial cells (25). Therefore, usually only young forms of irbc, the so-called ring stages, can be detected in the peripheral circulation, while mature forms are sequestered in capillaries and venules of vital organs. This process of sequestration is the pathological hallmark of falciparum malaria. The expression of variant antigens and the associated sequestration are under the control of the spleen and are eventually lost in splenectomized hosts (4, 15, 22). Thus, the spleen seems to have an important role in both controlling and establishing chronic P. falciparum infection, although the precise mechanisms remain elusive. The spleen has a highly organized architecture designed to * Corresponding author. Mailing address: Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, University of Oxford, Old Road, Oxford, OX3 7LJ, United Kingdom. Phone: Fax: britta.urban@ndm.ox.ac.uk. allow coordination of its phagocytic and cellular immune functions. It consists of lymphoid follicles, the white pulp, and intervening sinusoids, the red pulp. Blood vessels running through the white pulp terminate in the red pulp just outside the white pulp in the perifollicular zone. The majority of leukocytes migrate actively from the perifollicular zone into the marginal zone and then deeper into the white pulp to localize in specialized areas, such as the T-cell zones in the periarteriolar lymphatic sheath and B-cell follicles (37, 38). The spleen removes irbc debris resulting from the rupture of schizonts and irbc opsonized by immunoglobulins and/or complement in the perifollicular zone and in the cords of the red pulp. In addition, the spleen can directly extract Plasmodium parasites from young irbc in a process called pitting (2, 12). In acute malaria there is a lower splenic threshold for the removal of rigid erythrocytes, antibody-coated erythrocytes, and irbc, whereas splenectomized malaria patients have a prolonged clearance period for irbc and parasite products (13, 20, 26, 28). Phagocytosis of irbc and parasite debris by antigen-presenting cells in the marginal zone, such as monocytes, macrophages, and dendritic cells, can initiate adaptive immune responses. Provided antigen-presenting cells receive inflammatory signals, either from the pathogens themselves or from components of the innate immune system responding to the infection, they migrate deeper into the white pulp and activate naïve and memory T cells (3). Comparisons of the phenotypes and localizations of leukocytes within the highly organized splenic compartments can provide insights into the pathophys- 1986

2 VOL. 73, 2005 FATAL MALARIA CAUSES CHANGES IN SPLENIC ARCHITECTURE 1987 TABLE 1. Patient details Case Sex a Age (yr) Diagnosis b Drug c Complication(s) d parasitemia (no./ Maximum l of blood) Admission parasitemia (no./ l of blood) Last parasitemia (no./ l of blood) e Time to death (h) Malaria 1 M 28 CM A An, ARF, DIC, hypoglycemia 585,808 99,475 6, Malaria 2 M 20 CM Q Jn, shock 37,177 37,178 ND f 38 Malaria 3 M 24 CM A GI bleeding, Jn 67,824 67,824 67, Malaria 4 F 40 NCM Q An, Jn, mixed P. falciparum and P. vivax 4,522 4, Malaria 5 M 38 NCM A An, Jn, HyperP 790, , Malaria 6 F 50 NCM Q ARF, Jn, shock 17,082 8,290 6, Malaria 7 M 25 NCM A ARF, Jn Malaria 8 M 25 NCM Q ARF, An, Jn 335, , Malaria 9 F 43 NCM Q An, HyperP 824, , Malaria 10 M 69 CM A An, Jn, shock 112,537 92, Malaria 11 M 22 CM A HyperP, Jn 614, , , Malaria 12 M 63 CM Q ARF, DIC, hypoglycemia 136, , Malaria 13 F 52 CM A An, Jn, shock 7,536 7,536 7, Malaria 14 F 19 NCM A HyperP, shock 164, , , Malaria 15 M 63 CM A Jn, shock 84,403 84, Sepsis 1 F 87 Sepsis PE, pelvic abscess and sepsis 0 ND Sepsis 2 F 31 Sepsis MSOF secondary to sepsis, leukemia 0 ND Sepsis 3 F 69 Sepsis Staphylococcus sepsis secondary to pyelonephritis 0 ND Sepsis 4 M 84 Sepsis Sepsis secondary to bronchopneumonia 0 ND Sepsis 5 M 69 Sepsis Sepsis and stroke following cholecystitis and MI 0 ND Sepsis 6 F 54 Sepsis Septic with subphrenic abcess 0 ND Sepsis 7 M 63 Sepsis Bronchopneumonia/ARDS, AAA repair 0 ND Sepsis 8 M 62 Sepsis APKD, Enterobacter septicemia following MVR 0 ND Sepsis 9 F 73 Sepsis Peritonitis due to colonic perforation 0 ND Sepsis 10 M 76 Sepsis Bronchopneumonia 0 ND Sepsis 11 F 84 Sepsis Ruptured aneurysm 0 ND Sepsis 12 M 66 Sepsis Staphylococcal abscess (heart) 0 ND a M, male; F, female. b CM, cerebral malaria; NCM, noncerebral malaria (other severe complications). c A, artemether; Q, quinine. d ARF, acute renal failure; GI, gastrointestinal; DIC, disseminated intravascular coagulation; Jn, jaundice; Hyperp, hyperparasitemia; An, Anemia; PE, pulmonary embolus; MSOF, multisystem organ failure; MI, myocardial infarct; ARDS, adult respiratory distress syndrome; AAA, abdominal aortic aneurism; APKD, adult polycystic kidney disease; MVR, myocardial valve repair. e The last peripheral blood parasitemia was assessed within 6 h before death. These data were not available for 1 of the 15 malaria cases (Malaria 2). f ND, not determined. Downloaded from iological processes of infectious diseases. However, only a few studies have examined the splenic architecture and distribution of leukocytes in the human spleen (34, 35). For malaria, the majority of pathological studies have been studies of rodent models. One study showed that marginal zone macrophages are absent during malaria infection (39). Furthermore, Plasmodium chabaudi chabaudi irbc are not retained and phagocytosed by macrophages in the marginal zone but filter directly into the red pulp (47). In a recent study Achtmann et al. (1) observed transient changes in the migration of B cells during acute P. chabaudi chabaudi infection. All of these alterations may have consequences for the immune response to malaria. Here we describe the first immunohistochemical study of spleen sections from patients dying from severe falciparum malaria. We provide evidence that there were changes in the architecture of the spleen during fatal P. falciparum malaria infection and marked changes in the distribution of leukocytes within the spleen, which were specific for malaria compared to changes seen in sepsis or control patients. MATERIALS AND METHODS Tissue samples. Spleen samples were taken postmortem from Vietnamese adults who died from P. falciparum malaria. These patients were studied as part of a double-blind control drug trial for the treatment of severe malaria with quinine or artemether in a special research ward at the Centre for Tropical Diseases in Ho Chi Minh City, Vietnam (19). Patients were recruited into the drug trial study on admission to the hospital when they gave informed consent, had asexual P. falciparum blood stages as determined by a peripheral blood smear, and had one or more of the following complications: a Glasgow coma score of less than 11 (cerebral malaria), anemia (hematocrit, 20%), jaundice (serum billirubin concentration, 2.5 mg/dl), renal impairment (urine output of 400 ml in 24 h and serum creatinine concentration of 3 mg/dl), hypoglycemia (blood glucose concentration, 40 mg/dl), hyperparasitemia ( 10% parasitemia), and systolic blood pressure below 80 mm of Hg with cool extremities. Patients received either arthemether (50 mg/ml) or quinine dihydrochloride (250 mg/ml) for a minimum of 72 h. All patients underwent full clinical examination on admission to the hospital, and the level of peripheral blood parasitemia was determined every 4 h for the first 24 h and then every 6 h until three consecutive blood smears were negative or the patient died. For the patients who recovered from P. falciparum infection, the median time to total parasite clearance was 72 h (interquartile range [IQR], 54 to 102 h) for treatment with arthemether and 90 h (IQR, 66 to 108 h) for treatment with quinine. The median times to discharge of patients from the hospital were 288 h (IQR, 216 to 432 h) and 240 h (IQR, 192 to 336 h) for treatment with artmether and quinine, respectively (19). A detailed description of the clinical management of patients has been published previously (19). Some patients died more than 100 h after admission to the hospital (19). These deaths were due to late clinical complications and irreparable organ damage, such as acute renal failure, intractable shock, respiratory arrest with continued pulse, and metabolic acidosis as a direct result of P. falciparum infection. A full autopsy was performed on each of the patients who died if informed consent was obtained from relatives. Autopsies were performed shortly after death (median time, 7 h; IQR, 3 to 12 h) (33). We performed a histological and immunohistochemical study of the spleen for samples from 15 adults who died with severe falciparum malaria (Table 1). Eight of these patients had cerebral malaria, and seven patients suffered from at least one other complication of malarial disease, such as hyperparasitemia (four patients), anemia (three patients), and acute respiratory failure (four patients). When all malaria cases were considered, the average maximum level of parasitemia ranged from 480 to 1,644,104 irbc per l of blood, the median level of parasitemia was 112,537 irbc per l of blood, and the median admission level on August 23, 2018 by guest

3 1988 URBAN ET AL. INFECT. IMMUN. TABLE 2. Histological and immunohistological features of the spleen in different case groups a Parameter Control (n 16) Malaria (n 15) Sepsis (n 12) Area covered by red pulp (%) 62 ( ) 65 (57 81) 69.6 ( ) Spleen wt (g) 130 (90 170) 300 ( ) c 252 ( ) % of reactive B-cell follicles 8.9 ( ) 0 (0 16) d 3.6 (0 8.3) d No. of B cells in marginal zone (0.01 mm 2 ) 30 ( ) 10 (6 11) c 28 (26 32) No. of B cells in red pulp (0.1 mm 2 ) 23 ( ) 6.6 (1 12.3) c 6.6 ( ) c No. of B cells in red pulp corrected by wt b 2,880 (1,696 4,680) 2,283 (200 4,200) 628 (89 1,870) c No. of T cells in red pulp (0.1 mm 2 ) 19.5 ( ) 10 ( ) 21 ( ) No. of T cells in red pulp corrected by wt b 2,100 (893 3,147) 6,700 (3,392 11,152) d 3,820 (1,302 9,516) No. of macrophages in red pulp (0.1 mm 2 ) 75 (61 100) 67 (55 95) 83 (71 97) No. of macrophages in red pulp corrected by wt b 10,240 (5,480 23,004) 24,175 (14,025 34,950) d 13,476 (8,654 24,368) No. of neutrophils in red pulp (0.1 mm 2 ) 101 (84 111) 61 (49 74) d 66 (58 80) d No. of neutrophils in red pulp corrected by wt b 12,750 (8,960 14,140) 25,750 (16,550 35,950) 13,227 (8,654 24,368) a The data are the medians and (in parentheses) the 25th and 75th percentiles. b The observed increase in spleen weight in patients with malaria was associated with increased spleen volume, most likely due to an increased number of cells. Because data for spleen volume in these patients were not available, we used splenic weight as a crude estimate of volume and multiplied the number of cells observed per area counted by spleen weight to obtain an estimate of whether the overall number of cells in a particular cell population remained constant or changed. c P as determined by the Mann-Whitney U test for a comparison of the control data and the malaria or sepsis data. d P 0.01 as determined by the Mann-Whitney U test for a comparison of control data and the malaria or sepsis data. of parasitemia was 92,442 irbc per l of blood (IQR, 38,057 to 217,037 irbc per l of blood). The median level of parasitemia before death was 20 irbc per l of blood (IQR, 10 to 3,401 irbc per l of blood). In order to detect features in the spleens from malaria patients which were specific for infection with P. falciparum rather than signs of systemic infectious disease, we also analyzed spleen sections from 12 United Kingdom patients dying from primary or secondary disseminated bacterial sepsis proven by premortem blood culture (Table 1). The normal controls were tissue samples from 16 trauma patients who had their spleens removed during surgery (control) but had normal splenic histology because such spleen specimens were not available from Vietnamese adults. These patients were older than the malaria patients or the control group of trauma patients; the median ages of the malaria, control, and sepsis patients were 39 years (IQR, 24 to 52 years), 38 years (IQR, 28 to 69 years), and 69 years (IQR, 62 to 82 years), respectively. Collection of postmortem tissues and the protocols used for this study were approved by the Central Scientific and Ethics Committee of the Centre for Tropical Diseases in Vietnam and Oxford. Autopsies were performed only after the patients families gave informed consent. Histology. Following fixation in 10% phosphate-buffered formalin, tissue blocks were dehydrated in graded alcohol by using standard histological techniques, embedded in paraffin, and sectioned with a microtome. Sections were cut for immunohistochemistry onto Snowcoat Xtra slides (Surgipath, Petersborough, United Kingdom). The histological appearance was observed following counterstaining with hematoxylin and eosin. In some cases silver staining was used to reveal reticulin fibers of the connective tissue framework of the spleen. Immunohistochemistry. Unless stated otherwise, reagents were purchased from DAKO (Cambridge, United Kingdom). An immunohistochemistry analysis was performed with a panel of antibodies defining common leukocyte subgroups normally found in the spleen. These antibodies included CD20 (clone L26) defining B cells, CD3 (rabbit polyclonal antiserum or clone F7.2.38) defining T cells, CD68 (clone PGM-1) defining monocytes/macrophages, neutrophil elastase (clone NP54) defining neutrophils, and prion protein (3F4) recognizing myeloid dendritic cells. Staining for major histocompatibility complex (MHC) class II molecules (clone CR3/43) was also performed to examine patterns of immune activation among splenic leukocytes. Tissue sections were dewaxed and rehydrated by using standard procedures. For antigen retrieval, sections were heated in either 20 mm Tris 5 mm EDTA (ph 9) (CD20, MHC class II, monoclonal antibody against CD3), 10 mm citric acid (ph 6) (polyclonal antibody against CD3, CD68), or 2 mm HCl (normal prion protein) for 4 min and then washed in 20 mm Tris-HCl (ph 7.5) 100 mm NaCl. The sections were stained with antibodies as indicated above by using the indirect alkaline phosphatase method (APAAP method) and were developed by using New Fuchsin Red (14). The sections were counterstained with hematoxylin and mounted. Electron microscopy. The tissues were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (ph 7.4) and processed as described previously (33). Thin sections were stained with uranyl acetate and lead citrate prior to examination with a Jeol 1200 EX II transmission electron microscope. Quantitative analysis. For quantitative ultrastructural analysis of spleen sections, the squares of a copper grid were used as the reference area. A minimum of 100 infected and noninfected red blood cells were counted in 10 grid squares in three different areas, and the percentage of irbc and the percentage of knob-negative irbc were determined. For quantitative immunohistochemical analysis, at least three independent areas were counted for each parameter. Blind counting of spleen sections was not possible because of the even macroscopic dark brown appearance of spleen sections from malaria cases. Different regions within the white pulp were distinguished by a combination of morphological features and reactivity with antibodies. Thus, primary B-cell follicles (or tangentially sectioned corona) were densely packed with cells. In the marginal zone surrounding the follicles, the cells were less densely packed and consisted mainly of B cells, as well as various amounts of macrophages, dendritic cells, and T cells (37). Cell numbers were determined by using a graticule, divided into 100 squares, and expressed as the number of cells in 0.1 mm 2 or 0.01 mm 2 (in the case of dendritic cells and B cells in the marginal zone) of the section. B cells, T cells, macrophages, neutrophils, and prion protein (PrP)-positive dendritic cells were counted at a magnification of 400. The ratio of reactive lymphoid follicles to all lymphoid follicles and the areas covered by red pulp and white pulp (1 mm 2 ) in the section examined were assessed by using a magnification of 40. A statistical analysis was performed with SPSS (SPSS Inc., Chicago, Ill.) by using the Mann-Whitney U test, the chi-square test, or Spearman s correlation. RESULTS Gross splenic architecture. Splenomegaly is usual in malaria and indeed has been used historically as an epidemiological index for the intensity of malaria transmission in areas where malaria is endemic. We first compared splenic weight and gross architecture for all groups. The weight of the spleen was significantly increased in malaria cases (P 0.001), whereas the weight of the spleen in sepsis cases was slightly but not significantly increased compared to controls (Table 2). The red pulp was frequently congested with erythrocytes both in cases of malaria and in cases of sepsis. The ratios of the area covered by the red pulp to the area covered by the white pulp were similar for malaria cases and controls and for sepsis cases and controls (Table 2). We therefore assumed that the increase in weight in malaria cases was due to expansion of both compartments, the red pulp and the white pulp, as has been reported previously for rodent malaria (44). Additional silver staining of extracellular fibers revealed that the red pulp and the white pulp

4 VOL. 73, 2005 FATAL MALARIA CAUSES CHANGES IN SPLENIC ARCHITECTURE 1989 remained clearly separated (Fig. 1a), implying that the overall architecture of the spleen was retained despite distortion and changes in the different cellular compartments within the white pulp, as described below. Changes in the distribution of B and T lymphocytes. In the spleen, B cells are located in B-cell follicles in the white pulp and in the marginal zone and are scattered throughout the red pulp. While primary B-cell follicles contain naïve B cells, the marginal zone contains memory B cells, as well as marginal zone B cells. Both B-cell subsets respond rapidly to invading pathogens by secretion of antibodies. Interestingly, we observed a profound depletion of B cells from the marginal zone surrounding B-cell follicles only in malaria cases (Fig. 1b and 2 and Table 1). This depletion did not result from migration of activated B cells into the red pulp to form B-cell foci or into B-cell follicles to induce germinal center reactions. Indeed, the frequency of germinal centers within lymphoid follicles was significantly reduced in malaria cases compared with controls (Table 2), indicating that differentiation of B cells to plasma cells and memory cells was compromised in these patients. A similar trend was observed for sepsis cases. Activated B cells and plasma cells can migrate into the red pulp to form B-cell foci and undergo differentiation and to secrete antibodies, respectively. However, the number of B cells in the red pulp was decreased in both malaria and sepsis cases compared to controls, indicating that the depletion of B cells from the marginal zone in malaria cases was not due to migration into the red pulp. Because in malaria cases splenic weight was increased significantly, we corrected the number of B cells found in the red pulp for variation in the splenic weight in the disease groups, assuming that the splenic volume was proportional to the splenic weight. Although only a rough estimate, this correction could give an indication of whether the expansion of the red pulp and the white pulp in the spleens of malaria cases was due to changes in the number of cells of a particular leukocyte subset. After correction for splenic weight, in malaria cases the reduction in the number of B cells per unit of area in the red pulp that we observed was no longer significant. In contrast, the overall number of B cells in sepsis cases remained significantly reduced after correction for splenic weight (Table 2). The number of CD3-expressing T cells in the red pulp varied widely in both disease groups (Table 2 and Fig. 1c). When we corrected the number of T cells for splenic weight as described above, the overall number of T cells in the red pulp was significantly increased in malaria cases. In addition, we observed in malaria cases but not in sepsis cases that T cells were not segregated into separate areas but were scattered diffusely in the white pulp and in B-cell follicles (Yates-corrected 2 for malaria cases versus controls, 9.31 [P 0.01]; Yates-corrected 2 for sepsis cases versus controls, 0.01 [P 0.93]) (Fig. 1b and c). Changes in the distribution and phenotype of myeloid cells. Macrophages in the cords of the red pulp are important for the clearance of irbc and parasite products. CD68-positive cordal macrophages in the red pulp were loaded with hemozoin, the polymerized form of heme resulting from digestion of hemoglobin by the parasite (Fig. 1d). This phenomenon accounted for the even macroscopic dark brown appearance of the spleens from malaria cases. The number of macrophages in the red pulp tended to be lower in malaria and sepsis cases than in controls, but the differences were not significant (Table 2). When we corrected for splenic weight as described above, the relative number of macrophages in the red pulp was significantly increased in malaria cases (Table 2). Expression of the MHC class II molecule on cordal macrophages was reduced in malaria cases compared to controls or sepsis cases (Fig. 1e). A similar phenomenon was observed for interdigitating dendritic cells in the white pulp, which were defined by their morphology and high levels of expression of MHC class II molecules (Fig. 1f). In contrast to the weak staining of MHC class II molecules on macrophages and interdigitating dendritic cells, we observed high levels of expression on sinusoidal lining cells in the red pulp in almost all spleen sections from malaria cases ( 2 for malaria cases versus controls, 4.01 [P 0.05]; 2 for sepsis cases versus controls, 0.06 [P 0.8]) (Fig. 1e). These results indicate that ongoing inflammatory processes within the spleens of malaria patients were sufficient to induce upregulation of MHC class II molecules on sinusoidal lining cells but not on macrophages and interdigitating dendritic cells. The perifollicular zone and to a certain extent the marginal zone contain numerous antigen-presenting and phagocytic cells, including macrophages, myeloid dendritic cells, and neutrophils. Neutrophils were enriched in the red pulp adjacent to white pulp, presumably the perifollicular zone, as has been described previously (37) (Fig. 1g). The number of neutrophils in this area was reduced in malaria and sepsis cases compared to controls (Table 2). When we corrected the number of neutrophils in the red pulp for splenic weight, the difference was no longer significant in malaria cases. The normal form of PrP is expressed on myeloid dendritic cells in the marginal zone and in the red pulp, but interdigitating dendritic cells in the white pulp express this protein only weakly (9). The overall number of PrP-expressing myeloid dendritic cells was increased in malaria cases compared to control or sepsis cases. PrP-expressing myeloid dendritic cells were scattered throughout the red pulp, and in one-half of the control cases they accumulated immediately adjacent to the white pulp, presumably in the perifollicular zone (Fig. 1 h). In malaria cases, PrP-positive myeloid dendritic cells were significantly enriched in the red pulp and marginal zone (Fig. 3). The number of PrP-positive myeloid dendritic cells in the red pulp was correlated with the number in the marginal zone (Spearman s, 0.815; P 0.001) but was inversely correlated with maximum peripheral blood parasitemia (Spearman s, 0.579; P 0.05). Staining of consecutive section confirmed that PrP-expressing myeloid dendritic cells were distinct from neutrophils or macrophages (data not shown). Mature parasite-infected red blood cells in the spleen. Using transmission electron microscopy, we analyzed the quality and quantity of intact irbc in spleen sections of six patients who died with malaria. We identified infected cells using ultrastructural evidence of parasitic infection within the RBC and expression of electron-dense knob proteins on the erythrocyte surface. The percentages of irbc reflected the percentages seen in the brain and peripheral blood irrespective of whether the patients suffered from cerebral malaria or other severe complications (Table 3). In most patients the percentage of irbc in the spleen was lower than the percentage in the brain but

5 1990 URBAN ET AL. INFECT. IMMUN.

6 VOL. 73, 2005 FATAL MALARIA CAUSES CHANGES IN SPLENIC ARCHITECTURE 1991 FIG. 2. Immunostaining of spleen sections for B cells. Three representative spleen sections from P. falciparum malaria cases and from control cases (normal) are shown. In normal cases, B cells are densely packed in B-cell follicles. B cells are also abundant in the surrounding marginal zone but are more loosely packed. In malaria cases, B cells could be identified in B-cell follicles, but the number of cells was significantly reduced (P 0.001, as determined by Mann-Whitney U test). All sections were counterstained with hematoxylin. Magnification, 100. higher than the percentage in the peripheral circulation just before death. Many of the irbc contained trophozoites or mature schizonts and were knob positive (K ), indicating that there was expression of variant surface antigens mediating cytoadherence (Fig. 4a and b). In one case, a high proportion of the irbc were knob negative (K ), but these irbc contained large parasites consistent with gametocyte formation. It was possible to identify irbc pushing through fenestration between endothelial cells (Fig. 4a and c). In addition, a number of irbc exhibited constrictions that appeared to isolate part of the cytoplasm containing the parasite (Fig. 4d). A number of macrophages were observed to contain irbc, pigment crystals, and irbc ghosts (Fig. 4d). DISCUSSION The spleen plays a central role in the control of malaria infection. Here, we describe for the first time, to our knowledge, an integrated histological, immunohistochemical, and ultrastructural analysis of the architecture and distribution of leukocytes in spleens from patients who died from P. falciparum malaria and a comparison of sections from these patients and sections from patients who died with systemic sepsis. Several pathological changes in the spleen were specific for malaria. We observed depletion of B cells from the marginal zone, which gave the appearance of complete dissolution of the marginal zone. However, we could not establish whether other FIG. 1. Histology and immunostaining of spleen sections. Representative sections of spleens from patients who had their spleens removed during surgery (normal) or who died with P. falciparum malaria (malaria) or with disseminated sepsis (sepsis) are shown. Sections were stained with a standard silver stain (a) to reveal reticular fibers or were immunostained with anti-cd20 (b) or anti-cd3 (c) to demonstrated segregation of B-cell and T-cell compartments in the white pulp. In normal and sepsis cases, T- and B-cell areas are clearly distinguished (dark staining) from the marginal zone (light staining in panel b) surrounding B-cell follicles and the periarteriolar lymphatic sheath. In malaria cases, the B-cell follicle (dark staining in panel b) remains clearly separated, but T cells (dark staining in panel c) appear to be scattered around arterioles in the B-cell follicle and throughout the white pulp. In malaria cases, the marginal zone has a ragged appearance and is almost completely devoid of B cells (light blue staining in panel b surrounding B-cell follicle). (d) The anti-cd68 antibody identifies cordal macrophages in the red pulp cords and tingable body macrophages in the germinal center. Again, in normal and sepsis cases, B-cell follicles (dark blue areas containing germinal centers in the section from a normal case) can be easily distinguished from the surrounding marginal zone (light blue), whereas in malaria cases the marginal zone is apparently absent. (e) HLA-DR-positive cells were scattered through the red pulp and in the marginal zone. In malaria cases, the white pulp and the marginal zone contain only a few HLA-DR-positive cells. (f) Strongly staining cells are present in the white pulp and near central arterioles. These cells have large cell bodies and cytoplasmic protrusions between neighboring cells typical of interdigitating dendritic cells. Note the strong expression of HLA-DR on sinusoidal lining cells (d) but the almost complete absence of strongly HLA-DR-positive cells in the white pulp (f) in spleen sections from malaria patients. (g and h) Immunostaining for neutrophils (elastase) (g) and PrP-positive myeloid dendritic cells (h). Neutrophils are enriched in the perifollicular zone just adjacent to the white pulp, as well as scattered throughout the red pulp. The staining pattern for PrP-positve myeloid dendritic cells is clearly distinct from that for neutrophils and macrophages. All sections were counterstained with hematoxylin. (a, b, and d to h) Magnification, 90. (c) Magnification, 360.

7 1992 URBAN ET AL. INFECT. IMMUN. FIG. 3. Box plots of frequencies of myeloid dendritic cells in different spleen compartments. PrP-positive myeloid dendritic cells (DC) were counted in the marginal zone (open bars) and red pulp (shaded bars) in control, malaria, and sepsis spleen sections. The boxes indicate the median and 25th and 75th percentiles. Outliers are indicated by circles. There is a highly significant increase in the number of dendritic cells in both compartments in malaria cases compared to sepsis and control cases (P 0.001, as determined by Mann-Whitney U test). TABLE 3. Parasitemia in different organs as determined by electron microscopy Case Diagnosis a Spleen Total K K Parastemia (%) Peripheral blood (total) b Brain (total) Malaria 16 CM Malaria 17 CM Malaria 18 CM Malaria 19 NCM Malaria 20 NCM Malaria 21 NCM a CM, cerebral malaria; NCM, noncerebral malaria (other severe complications). b Peripheral blood parasite count within 4 h before death. FIG. 4. Electron micrographs of the spleens of patients dying of malaria. (a) Low-power micrograph showing part of a sinus within the spleen containing a number irbc (IR), some of which appear to be passing through fenestration between endothelial cells (En). Bar 5 m. (b) Detail of one irbc containing a mature schizont with merozoites (M). Note the electron-dense knobs (K) at the surface of the red cell. Bar 1 m. (c) Higher-power micrograph showing three infected red blood cells (arrowheads) passing through small gaps between endothelial cells (En). P, parasite. Bar 5 m. (d) Infected knobpositive red blood cell (K) showing constriction of the cell cytoplasm (arrow), which appears to isolate the portion of the cell containing the parasite (P). Bar 1 m. (e) Section through a macrophage which has phagosomes containing infected red blood cells (IR), ghosts of infected cells (G), and pigment granules (Pi). Bar 5 m. cell types were depleted from the marginal zone, as has been observed for marginal zone macrophages in the spleens of mice infected with rodent malaria species (39). Within the white pulp the T-cell areas were not clearly separated from B-cell areas, and T cells frequently could be observed in B-cell follicles. In a recent study on the phenotype and localization of B cells in mice during infection with the rodent parasite P. chabaudi chabaudi the workers reported changes similar to those which we describe here; Achtman et al. (1) showed that there was depletion of B cells from the marginal zone, as well as profound disruption of the T-cell areas. In contrast to our study, they found vigorous germinal center formation and extrafollicular B-cell proliferation in the red pulp. These observations are not necessarily contradictory because P. chabaudi chabaudi infection in C57BL/6 mice is self-limiting and therefore is a good model for mild malaria. In our study, the depletion of B cells from the marginal zone and the lack of germinal center formation could indicate that B-cell maturation and migration are more perturbed in severe forms of human falciparum malaria. Humoral immune responses play an important part in the development of clinical immunity to malaria (7, 24). Although exposure may lead to the accumulation of a repertoire of antibodies to variant and conserved parasite antigens, these responses are often short lived and depend on the presence of parasites, suggesting that there are inadequate memory B-cell responses or T-cell help (8, 10, 17, 30). Our observations of the depletion of B cells from the marginal zone and the lack of germinal center formation indicate that B-cell maturation and migration are perturbed in malaria, and the causes of this are unclear. Interestingly, adult mice treated with LT- immunoglobulin fusion protein (11) have a similar phenotype, and in a recent report Engwerda and colleagues suggested that lymphotoxin (LT)- may play a key role in severe mouse malaria (16). Therefore, it is important to examine the effect of LT- on B-cell migration and maturation in vitro and by analyzing frozen spleen sections from patients who died with falciparum malaria ex vivo. In sepsis cases, we observed only a few germinal centers and a general depletion of B cells from the red pulp, as has been described previously (5). Possibly, T-dependent B cells were activated early in the disease, and the resulting plasma cells left the spleen and migrated into the bone marrow, where they secreted antibodies. Alternatively, activated B-cell populations could have undergone apoptosis, a phenomenon that has been described both in disseminated sepsis and in malaria (23, 45). Acute malaria is characterized by a vigorous acute-phase response with high levels of inflammatory and anti-inflammatory cytokines, and it is increasingly evident that the ratio of these two types of cytokines contributes to the clinical syndrome and the severity of infection (21, 29). It was therefore

8 VOL. 73, 2005 FATAL MALARIA CAUSES CHANGES IN SPLENIC ARCHITECTURE 1993 relevant that the expression of HLA-DR on sinusoidal lining cells was increased in the red pulp of malaria cases, indicating that there were acute inflammatory responses. In marked contrast, macrophages in the red pulp and interdigitating dendritic cells in the white pulp showed only weak or no expression of HLA-DR in malaria cases compared with good expression levels in control and sepsis cases. We have shown previously that peripheral blood dendritic cells from Kenyan children suffering from acute P. falciparum malaria have reduced HLA-DR expression levels ex vivo compared to healthy children (43). In addition, monocyte-derived dendritic cells fail to upregulate surface expression of MHC class II molecules in response to inflammatory signals when they are exposed to intact irbc in vitro (42), and ingestion of malarial pigment renders macrophages refractory to activating signals (27, 36). Together, these observations suggested that the antigen-presenting function of dendritic cells and macrophages might be profoundly altered during acute P. falciparum malaria, with likely consequences for the induction and maintenance of T- and B-cell responses. Myeloid dendritic cells in the red pulp and in the marginal zone express the normal form of the prion protein (9). Our observations confirm that these PrP-positive cells can be distinguished from neutrophils or macrophages, as judged by the differences in distribution of these cell types in consecutive sections. The number of the PrP-expressing myeloid dendritic cells were increased in the red pulp and marginal zone of malaria cases compared to sepsis cases and controls. These results indicate that these cells were recruited into the spleen during acute malarial disease but failed to migrate into the white pulp, where they can activate T cells. However, migration of dendritic cells into the white pulp is a transient process. The use of autopsy pathology inevitably gives a limited temporal picture of a dynamic process. While the patients who died within 24 h of admission to the hospital (n 5) showed similar distributions of dendritic cells in the red pulp and in the marginal zone, we cannot exclude the possibility that the limitations of autopsy pathology and sample size might have missed such a transient process. Although a few reports suggested that intact irbc can be found in the spleen (34, 41), we analyzed spleen sections from patients who died with P. falciparum malaria by electron microscopy in order to estimate the number of mature, intact irbc in the peripheral circulation within 4 h before death and in the brain. Indeed, mature parasite stages were present in the spleen, and sometimes they were even enriched compared to the peripheral circulation, although parasitemia tended to be highest in the brain. We do not know whether parasites sequestering in the spleen survive or whether they are destroyed by macrophages in the splenic cords. However, mature, K parasites in the spleen could potentially interact directly with host cells, including dendritic cells in the perifollicular zone and in the marginal zone. P. falciparum malaria is characterized by acute inflammatory immune responses, followed by adaptive humoral immune responses. The early innate inflammatory response is probably important for initial control of the parasitemia but can also result in immunopathology depending on previous exposure to the parasite, the host, and the parasite genotype (40). In addition, the mature forms of irbc and hemozoin modulate the function of antigen-presenting cells (36, 42, 43), which may have consequences for the initiation of effective adaptive immune responses. The amount of hemozoin in peripheral blood monocytes and neutrophils is correlated with disease severity and fatal outcome (31). In our study, we found evidence for the induction of inflammatory responses, as shown by the induction of HLA-DR molecules on sinusoidal lining cells, as well as downregulation of HLA-DR expression on macrophages and dendritic cells in the red pulp and in the white pulp, respectively. In parallel, the marginal zone was devoid of B cells, and the distribution of T cells within the white pulp was perturbed. Whether there is a causative link between these observations cannot be established by immunohistochemistry. However, the results are consistent with the possibility that in these malaria cases, adaptive B- and T-cell responses were not sufficiently induced by antigen-presenting cells and subsequently parasitemia was not controlled. Sequestration of irbc in vital organs together with unregulated inflammatory responses could then result in irreparable organ damage and death. Of course, the malaria cases which we analyzed represent extremes, in which the immune defense mechanisms of the human hosts failed to prevent fatal infection. It is likely that changes in the histology of the spleen in patients surviving blood stage malaria or suffering from asymptomatic infection are quantitatively different. Nevertheless, disruption of the architecture of the white pulp, even if it is mild and temporarily, could impair the induction or the maintenance of effective and long-lasting immune responses. The marginal zone and the inner white pulp are interdependent in generating effective immune responses because antigen-presenting cells are located in the marginal zone. However, B-cell follicles, as well as T-cell areas, send signals in the form of chemokines toward which activated antigen-presenting cells can migrate (18, 32). What causes the disruption in splenic architecture, whether it is the adhesion of irbc and subsequent signaling events in host leukocytes, a defective chemokine and/or cytokine response, or both, requires examination by a combination of in vitro studies and more detailed immunohistology. ACKNOWLEDGMENTS This work was supported by the Wellcome Trust of Great Britain as part of the Oxford-Vietnam-Mahidol Tropical Network and by the Sir E. P. Abraham Trust, University of Oxford. D.J.R. is a Howard Hughes International Research Scholar, and B.C.U. holds a Wellcome Trust Career Development Fellowship in Basic Biomedical Research. We acknowledge the staff on the Acute Malaria Ward, Centre for Tropical Diseases, Ho Chi Minh City, Vietnam, for their help with the clinical care of the malaria patients. 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