Cellular Immunology 289 (2014) 162–166
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Reactivity of autoantibodies against not only erythrocytes but also hepatocytes in sera of mice with malaria Yasuhiro Kanda a, Toshihiko Kawamura a,⇑, Takahiro Kobayashi a,b, Hiroki Kawamura a,c, Hisami Watanabe d, Toru Abo a a
Division of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan Department of Microbiology, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan Department of Clinical Engineering and Medical Technology, Niigata University of Health and Welfare, Niigata 950-3198, Japan d Division of Cellular and Molecular Immunology, Center of Molecular Biosciences, University of Ryukyus, Okinawa 903-0213, Japan b c
a r t i c l e
i n f o
Article history: Received 18 September 2013 Accepted 15 April 2014 Available online 24 April 2014 Keywords: Autoantibody Plasmodium yoelii Malaria Hepatocytes Erythrocytes
a b s t r a c t In order to further examine the reactivity of autoantibodies, mice were infected with a non-lethal strain of Plasmodium yoelii. Parasitemia appeared between days 10 and 21. During this period, hyperglycemia and hypothermia were serially obeserved and this phenomenon resembled stress-associated responses. In parallel with parasitemia, autoantibodies appeared against nucleus and double-stranded DNA in the sera. To examine further the reactivity of autoantibodies against tissues, immunohistochemical staining using sera from mice with or without malaria was conducted. Autoantibodies contained reactivity to erythrocytes in the spleen, bone marrow and peripheral blood, especially against tissues obtained from mice with malaria. In the liver and intestine, autoantibodies reacted with hepatocytes and intestinal epithelial cells, respectively. These results suggested that the reactivity of autoantibodies against erythrocytes and hepatocytes might be associated with the modulation of the disease course in malaria. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Protection against malaria was considered an event of the conventional immune system against malaria protozoa acting as foreign antigens [1–7]. Thymus-derived (T) lymphocytes and bone marrow-derived (B) lymphocytes play important roles in adoptive immune responses. This concept is the basis for malaria vaccination trials using antigens from the malaria protozoa [8–11]. It is considered that conventional B cells (B-2 cells) and helper T cells recognize protozoa antigens and produce protective antibodies [12–14]. However, malaria also induces innate immune responses and results in the activation of autoreactive extrathymic T cells [15– 19] and the production of autoantibodies by B-1 cells during infection [20–23]. Clinical studies have reported the detection of high titers of autoantibodies against human nucleus or double-stranded (ds) DNA in the sera of patients with malaria [24–29]. Thus, we have intensively characterized the properties of autoreactive extrathymic T cells and autoantibody-producing B-1 cells in mice with malaria. Simultaneous activation of extrathymic T
⇑ Corresponding author. Fax: +81 25 227 0766. E-mail address: [email protected] (T. Kawamura). http://dx.doi.org/10.1016/j.cellimm.2014.04.008 0008-8749/Ó 2014 Elsevier Inc. All rights reserved.
cells and B-1 cells is a common response in mice with malaria [30–33]. In the present study, we investigated immune responses during blood-stage malaria in mice and examined the reactivity of the autoantibodies in sera. The unique reactivity of autoantibodies to self-tissues during malaria (autoreactivity to erythrocytes and hepatocytes) suggested the beneficial function of autoantibodies for protection against malaria.
2. Materials and methods 2.1. Mice and parasites C57BL/6 (B6) mice at age 8–14 weeks were used. These mice were maintained at the animal facility of Niigata University (Niigata, Japan) under specific pathogen-free conditions. Plasmodium yoelii (P. yoelii) 17XNL (non-lethal strain), a generous gift from Dr. S. Waki (Gunma Prefectural College of Health Science, Maebashi, Japan) was used. Parasites were maintained by routine in vivo passage in mice [21]. Mice were infected by intraperitoneal injection of 104 parasitized erythrocytes per mouse. Parasitemia in the blood was observed by Giemsa staining every 2 or 3 days and mice were sacrificed at the indicated days after infection.
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2.2. Measurement of blood glucose and rectal temperature Blood glucose was measured from 3.5 ll blood from mice by venesection at the tail tip with Precision Xceed (Abbott Japan Co., Chiba, Japan). The rectal temperature of mice was measured every 2 or 3 days with a digital thermometer (TD-300, Shibaura Electronics Co. Ltd., Tokyo, Japan). 2.3. Identification of autoantibodies Autoantibodies were detected using a HEp-2 cell line in conjunction with an immunofluorescence test. Sera obtained from P. yoelii 17XNL infected or uninfected control mice were used after a 1/10 dilution. FITC-conjugated anti-mouse Ig (Dako, Glostrup, Denmark) was used as a secondary antibody for detection. The activity of such anti-ds DNA antibodies was also detected in the sera and supernatant using an anti-ds DNA mouse enzyme-linked immunosorbent assay (ELISA) kit (Shibayagi, Gunma, Japan). 2.4. Immnohistochemistry Tissues were removed from euthanized mice and fixed in formalin, dehydrated, embedded in paraffin and sectioned at 3 lm as previously described [34]. Blood smears were fixed with methanol. Endogenous peroxidase in the sections was blocked with 3% hydrogen peroxide in water for 5 min, and the Avidin/Biotin Blocking Kit (Vector, Burlingame, CA, USA) was used to block endogenous avidin and biotin. Mouse on Mouse (M.O.M.) Immunodetection Kit (Vector, Burlingame, CA, USA) was used for blocking non-specific mouse immunoglobulin (Ig) and reducing the background staining. The sections were incubated with Mouse Ig Blocking reagent for 1 h and M.O.M. Protein Concentrate solution. Sera of infected or uninfected mice at a 1/5 dilution were applied to the section for 30 min as primary autoantibodies. Subsequently, M.O.M. biotinylated anti-mouse IgG reagent was incubated for 10 min, followed by incubation with Vectastain Elite ABC Kit. Finally, the slides were stained with diaminobenzidine and counterstained with hematoxylin.
Fig. 1. Time kinetic study on parasitemia, blood glucose and rectal temperature. Four mice were used to determine the mean ± SD.
3. Results 3.1. Parasitemia and stress-associated responses A time-kinetic study on parasitemia was conducted after P. yoelii injection (Fig. 1). From day 7, erythrocytes infected with the protozoa were observed in the blood. Parasitemia gradually became prominent and continued up to day 21. In parallel, the levels of blood glucose and rectal temperature were examined. The level of blood glucose increased at days 10–17 and the rectal temperature decreased approximately at day 21. The presence of hyperglycemia and hypothermia resembled stress-associated responses. 3.2. Reactivity of autoantibodies Using HEp-2 cells, the reactivity of autoantibodies in sera from mice with malaria was examined (Fig. 2A). Sera of autoimmune prone MRL-lpr/lpr mice was used as a positive control (top of the figure). The low reactivity of sera (total Ig) was observed on day 14 but the highest reactivity of sera was present at day 28. This reactivity was confirmed by IgG and IgM isotypes. IgG autoantibodies had reactivity to the nucleus while IgM autoantibodies were reactive to a perinuclear area of cells. In the case of sera from MRLlpr/lpr mice, only IgG autoantibodies had specific reactivity to the nucleus. There was no reactivity of the IgM isotype in this case.
By ELISA, the titer of anti-ds DNA antibodies was examined (Fig. 2B). Reactivity appeared from day 14 after infection and increased up to day 28. Positive control sera from MRL-lpr/lpr mice had the highest titer of anti-ds DNA antibodies. 3.3. Reactivity of autoantibodies to the cell surface of erythrocytes and others Immunofluorescence and ELISA analysis suggested a wide reactivity of autoantibodies to the nucleus and cytoplasm (perinuclear area). We then determined whether the sera from mice with malaria had reactivity to tissues and erythrocytes (Fig. 3). First, the reactivity of sera against the spleen was examined. Sera from mice with malaria showed a positive reaction against the normal spleen, especially in the splenic medullary region where denatured erythrocytes were abundant. The reactivity of sera with follicular area was lower. In contrast, control sera did not have reactivity against the spleen. A similar reactivity was also observed using malaria-infected splenic tissue (right column). In this picture, dark spots in the spleen represent malarial pigments. Second, the reactivity of sera against bone marrow (BM) was examined. Some reactivity against BM cells (mainly erythroid cells) was observed even in normal BM.
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Fig. 2. Autoantibodies against intracellular components. (A) Immunofluorescence analysis using HEp-2 cells, (B) ELISA assay to detect the titers of anti-ds DNA antibodies. In experiment b, four mice were used to determine the mean ± SD.
This reactivity was confirmed using malaria-infected BM (right column). The reactivity of sera was examined in the smear of peripheral blood and was slightly positive against normal erythrocytes. Higher reactivity was seen against erythrocytes obtained from mice with malaria (day 28). The surface of erythrocytes was highly positive. The reactivity of sera against the liver (especially hepatocytes) was then examined using liver tissue. Control sera did not show reactivity against normal liver. In sharp contrast, sera from mice with malaria showed a positive reaction against normal liver and malaria-infected liver. In the right column, dark spots seen in the liver represent malarial pigments. Finally, the large intestine (Li) was used to examine the reactivity of sera. Considerable sera reactivity was observed in the epithelial region of the Li. This was apparent, irrespective of infection. 4. Discussion In the present study, we demonstrated that sera from mice with malaria contained autoantibodies with reactivity against erythrocytes and hepatocytes in tissue sections by immunohistochemical analysis. Although the reactivity was higher against infected cells compared with control cells, some antigens on normal erythro-
cytes and hepatocytes were targets of the autoantibodies in malaria. Therefore, the reactivity was not against malaria parasite-specific antigens but against erythrocytes and hepatocytes, irrespective of infection. Thus, malaria may increase the antigenicity of erythrocytes and hepatocytes. Immunofluorescence and ELISA assays demonstrated that autoantibodies were reactive with many cell components; not only nucleus and ds DNA, but also surface antigens of erythrocytes and hepatocytes. In humans and mice, malaria consistently induces serum autoantibodies [20–29]. In contrast, there is little evidence that conventional T cells (TCRhigh cells) and B cells (B-2 cells) are activated during malaria, especially in mice. Even if conventional immunity is activated, memory immune responses against malaria protozoa tend to disappear within 1 year [35,36]. However, extrathymic T cells (TCRint cells) are prominently activated in mice with malaria [15,16]. Previously, we demonstrated that autoantibody-producing B-1 cells were activated in parallel with the activation of extrathymic T cells [20,21]. In light of these findings, protection from malaria may be due to innate immunity mediated by unconventional extrathymic T cells and B-1 cells and is supported by the observation of reactive autoantibodies identified in this study. Malaria consists of a hepatic stage and blood stage during infection [37]. During the hepatic stage, intracellular malaria parasites invade hepatocytes whereas during the blood stage they invade erythrocytes. Therefore, conventional T cells and conventional antibodies against parasites may not react to parasitic antigens. Under this situation, autoantibodies against surface antigens on hepatocytes and erythrocytes may be important for protection from malaria. Previous studies demonstrated that autoantibodies against erythrocytes or their surface molecules were induced in malaria [38–43] (Table 1). In addition, the autoantibodies were found to react with intracellular components and many cell surface components, including those on neutrophils [44,45] and thrombocytes [46,47]. The immune status observed in malaria is summarized in Table 1. Since the antigenicity of such surface antigens (especially on erythrocytes) is increased by malaria, the autoreactivity of activated extrathymic T cells and the autoantibodies of activated B-1 cells might efficiently react with infected erythrocytes and hepatocytes. Finally, such denatured hepatocytes and erythrocytes are processed by macrophages in the spleen and Kupffer cells in the liver. Hepatosplenomegaly with malaria pigments is consistently observed in humans and mice in malaria [33]. Results from immunohistochemistry of tissue sections revealed that the most reactive sites of autoantibodies in mice with malaria were the splenic medulla, bone marrow and peripheral erythrocytes, especially in infected tissues. This reactivity supports the idea that autoantibodies preferentially attack infected erythrocytes. In addition to hepatocytes, epithelial cells in the intestine were reactive to the autoantibodies. Since hepatocytes were phylogenetically generated from intestinal epithelial cells [48], this suggests cross-reactivity between hepatocytes and intestinal epithelial cells. An immune status of activated extrathymic T cells and autoantibody-producing B-1 cells is common to aging [49,50], autoimmune diseases [51,52], chronic graft-vs-host disease [53,54] and malaria. During these immune responses, the thymus often becomes atrophied or involuted. Moreover, these immune states also resemble stress-associated responses [55,56]. This study confirmed that stress-associated responses such as hypothermia and hyperglycemia were observed in malaria. Taken together, this suggests that malaria induces an innate immune response rather than an adaptive immune response and that the reactivity of autoantibodies against the surface of erythrocytes and hepatocytes should be further studied.
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Fig. 3. Immunohistochemical staining of sera using various tissues. The spleen, bone marrow (BM), peripheral blood (PB), liver and large intestine (Li) were examined. Normal and malaria-infected tissues were used. Sections were stained with hematoxylin (blue) and diaminobenzidine (brown).
Table 1 Immunologic state seen in malarial infection. T cells
B cells (antibody production)
Thymic atrophy and suppression of conventional T cells [15,16]
Dysregulation of conventional B cells (B-2) and paucity of antibodies against Plasmodium [20,21] Activation of B-1 cells [20–23] and production of autoantibodies against:
Activation of unconventinal T cells, including NKT cells and extrathymic T cells in mice [15– 19] and in humans [32]
Intracellular components [24–29] e.g., nucleus, ds DNA, actin, etc. Cell surface components e.g., erythrocytes [38–43], neutrophils [44,45], thrombocytes [46,47], hepatocytesa, etc. [ ] References a This paper.
Acknowledgments We wish to thank Mrs. Yuko Kaneko for manuscript preparation. This work was supported by a grant-in-aid for scientific
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Reactivity of autoantibodies against not only erythrocytes but also hepatocytes in sera of mice with malaria Yasuhiro Kanda a, Toshihiko Kawamura a,⇑, Takahiro Kobayashi a,b, Hiroki Kawamura a,c, Hisami Watanabe d, Toru Abo a a
Division of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan Department of Microbiology, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan Department of Clinical Engineering and Medical Technology, Niigata University of Health and Welfare, Niigata 950-3198, Japan d Division of Cellular and Molecular Immunology, Center of Molecular Biosciences, University of Ryukyus, Okinawa 903-0213, Japan b c
a r t i c l e
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Article history: Received 18 September 2013 Accepted 15 April 2014 Available online 24 April 2014 Keywords: Autoantibody Plasmodium yoelii Malaria Hepatocytes Erythrocytes
a b s t r a c t In order to further examine the reactivity of autoantibodies, mice were infected with a non-lethal strain of Plasmodium yoelii. Parasitemia appeared between days 10 and 21. During this period, hyperglycemia and hypothermia were serially obeserved and this phenomenon resembled stress-associated responses. In parallel with parasitemia, autoantibodies appeared against nucleus and double-stranded DNA in the sera. To examine further the reactivity of autoantibodies against tissues, immunohistochemical staining using sera from mice with or without malaria was conducted. Autoantibodies contained reactivity to erythrocytes in the spleen, bone marrow and peripheral blood, especially against tissues obtained from mice with malaria. In the liver and intestine, autoantibodies reacted with hepatocytes and intestinal epithelial cells, respectively. These results suggested that the reactivity of autoantibodies against erythrocytes and hepatocytes might be associated with the modulation of the disease course in malaria. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Protection against malaria was considered an event of the conventional immune system against malaria protozoa acting as foreign antigens [1–7]. Thymus-derived (T) lymphocytes and bone marrow-derived (B) lymphocytes play important roles in adoptive immune responses. This concept is the basis for malaria vaccination trials using antigens from the malaria protozoa [8–11]. It is considered that conventional B cells (B-2 cells) and helper T cells recognize protozoa antigens and produce protective antibodies [12–14]. However, malaria also induces innate immune responses and results in the activation of autoreactive extrathymic T cells [15– 19] and the production of autoantibodies by B-1 cells during infection [20–23]. Clinical studies have reported the detection of high titers of autoantibodies against human nucleus or double-stranded (ds) DNA in the sera of patients with malaria [24–29]. Thus, we have intensively characterized the properties of autoreactive extrathymic T cells and autoantibody-producing B-1 cells in mice with malaria. Simultaneous activation of extrathymic T
⇑ Corresponding author. Fax: +81 25 227 0766. E-mail address: [email protected] (T. Kawamura). http://dx.doi.org/10.1016/j.cellimm.2014.04.008 0008-8749/Ó 2014 Elsevier Inc. All rights reserved.
cells and B-1 cells is a common response in mice with malaria [30–33]. In the present study, we investigated immune responses during blood-stage malaria in mice and examined the reactivity of the autoantibodies in sera. The unique reactivity of autoantibodies to self-tissues during malaria (autoreactivity to erythrocytes and hepatocytes) suggested the beneficial function of autoantibodies for protection against malaria.
2. Materials and methods 2.1. Mice and parasites C57BL/6 (B6) mice at age 8–14 weeks were used. These mice were maintained at the animal facility of Niigata University (Niigata, Japan) under specific pathogen-free conditions. Plasmodium yoelii (P. yoelii) 17XNL (non-lethal strain), a generous gift from Dr. S. Waki (Gunma Prefectural College of Health Science, Maebashi, Japan) was used. Parasites were maintained by routine in vivo passage in mice [21]. Mice were infected by intraperitoneal injection of 104 parasitized erythrocytes per mouse. Parasitemia in the blood was observed by Giemsa staining every 2 or 3 days and mice were sacrificed at the indicated days after infection.
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2.2. Measurement of blood glucose and rectal temperature Blood glucose was measured from 3.5 ll blood from mice by venesection at the tail tip with Precision Xceed (Abbott Japan Co., Chiba, Japan). The rectal temperature of mice was measured every 2 or 3 days with a digital thermometer (TD-300, Shibaura Electronics Co. Ltd., Tokyo, Japan). 2.3. Identification of autoantibodies Autoantibodies were detected using a HEp-2 cell line in conjunction with an immunofluorescence test. Sera obtained from P. yoelii 17XNL infected or uninfected control mice were used after a 1/10 dilution. FITC-conjugated anti-mouse Ig (Dako, Glostrup, Denmark) was used as a secondary antibody for detection. The activity of such anti-ds DNA antibodies was also detected in the sera and supernatant using an anti-ds DNA mouse enzyme-linked immunosorbent assay (ELISA) kit (Shibayagi, Gunma, Japan). 2.4. Immnohistochemistry Tissues were removed from euthanized mice and fixed in formalin, dehydrated, embedded in paraffin and sectioned at 3 lm as previously described [34]. Blood smears were fixed with methanol. Endogenous peroxidase in the sections was blocked with 3% hydrogen peroxide in water for 5 min, and the Avidin/Biotin Blocking Kit (Vector, Burlingame, CA, USA) was used to block endogenous avidin and biotin. Mouse on Mouse (M.O.M.) Immunodetection Kit (Vector, Burlingame, CA, USA) was used for blocking non-specific mouse immunoglobulin (Ig) and reducing the background staining. The sections were incubated with Mouse Ig Blocking reagent for 1 h and M.O.M. Protein Concentrate solution. Sera of infected or uninfected mice at a 1/5 dilution were applied to the section for 30 min as primary autoantibodies. Subsequently, M.O.M. biotinylated anti-mouse IgG reagent was incubated for 10 min, followed by incubation with Vectastain Elite ABC Kit. Finally, the slides were stained with diaminobenzidine and counterstained with hematoxylin.
Fig. 1. Time kinetic study on parasitemia, blood glucose and rectal temperature. Four mice were used to determine the mean ± SD.
3. Results 3.1. Parasitemia and stress-associated responses A time-kinetic study on parasitemia was conducted after P. yoelii injection (Fig. 1). From day 7, erythrocytes infected with the protozoa were observed in the blood. Parasitemia gradually became prominent and continued up to day 21. In parallel, the levels of blood glucose and rectal temperature were examined. The level of blood glucose increased at days 10–17 and the rectal temperature decreased approximately at day 21. The presence of hyperglycemia and hypothermia resembled stress-associated responses. 3.2. Reactivity of autoantibodies Using HEp-2 cells, the reactivity of autoantibodies in sera from mice with malaria was examined (Fig. 2A). Sera of autoimmune prone MRL-lpr/lpr mice was used as a positive control (top of the figure). The low reactivity of sera (total Ig) was observed on day 14 but the highest reactivity of sera was present at day 28. This reactivity was confirmed by IgG and IgM isotypes. IgG autoantibodies had reactivity to the nucleus while IgM autoantibodies were reactive to a perinuclear area of cells. In the case of sera from MRLlpr/lpr mice, only IgG autoantibodies had specific reactivity to the nucleus. There was no reactivity of the IgM isotype in this case.
By ELISA, the titer of anti-ds DNA antibodies was examined (Fig. 2B). Reactivity appeared from day 14 after infection and increased up to day 28. Positive control sera from MRL-lpr/lpr mice had the highest titer of anti-ds DNA antibodies. 3.3. Reactivity of autoantibodies to the cell surface of erythrocytes and others Immunofluorescence and ELISA analysis suggested a wide reactivity of autoantibodies to the nucleus and cytoplasm (perinuclear area). We then determined whether the sera from mice with malaria had reactivity to tissues and erythrocytes (Fig. 3). First, the reactivity of sera against the spleen was examined. Sera from mice with malaria showed a positive reaction against the normal spleen, especially in the splenic medullary region where denatured erythrocytes were abundant. The reactivity of sera with follicular area was lower. In contrast, control sera did not have reactivity against the spleen. A similar reactivity was also observed using malaria-infected splenic tissue (right column). In this picture, dark spots in the spleen represent malarial pigments. Second, the reactivity of sera against bone marrow (BM) was examined. Some reactivity against BM cells (mainly erythroid cells) was observed even in normal BM.
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Fig. 2. Autoantibodies against intracellular components. (A) Immunofluorescence analysis using HEp-2 cells, (B) ELISA assay to detect the titers of anti-ds DNA antibodies. In experiment b, four mice were used to determine the mean ± SD.
This reactivity was confirmed using malaria-infected BM (right column). The reactivity of sera was examined in the smear of peripheral blood and was slightly positive against normal erythrocytes. Higher reactivity was seen against erythrocytes obtained from mice with malaria (day 28). The surface of erythrocytes was highly positive. The reactivity of sera against the liver (especially hepatocytes) was then examined using liver tissue. Control sera did not show reactivity against normal liver. In sharp contrast, sera from mice with malaria showed a positive reaction against normal liver and malaria-infected liver. In the right column, dark spots seen in the liver represent malarial pigments. Finally, the large intestine (Li) was used to examine the reactivity of sera. Considerable sera reactivity was observed in the epithelial region of the Li. This was apparent, irrespective of infection. 4. Discussion In the present study, we demonstrated that sera from mice with malaria contained autoantibodies with reactivity against erythrocytes and hepatocytes in tissue sections by immunohistochemical analysis. Although the reactivity was higher against infected cells compared with control cells, some antigens on normal erythro-
cytes and hepatocytes were targets of the autoantibodies in malaria. Therefore, the reactivity was not against malaria parasite-specific antigens but against erythrocytes and hepatocytes, irrespective of infection. Thus, malaria may increase the antigenicity of erythrocytes and hepatocytes. Immunofluorescence and ELISA assays demonstrated that autoantibodies were reactive with many cell components; not only nucleus and ds DNA, but also surface antigens of erythrocytes and hepatocytes. In humans and mice, malaria consistently induces serum autoantibodies [20–29]. In contrast, there is little evidence that conventional T cells (TCRhigh cells) and B cells (B-2 cells) are activated during malaria, especially in mice. Even if conventional immunity is activated, memory immune responses against malaria protozoa tend to disappear within 1 year [35,36]. However, extrathymic T cells (TCRint cells) are prominently activated in mice with malaria [15,16]. Previously, we demonstrated that autoantibody-producing B-1 cells were activated in parallel with the activation of extrathymic T cells [20,21]. In light of these findings, protection from malaria may be due to innate immunity mediated by unconventional extrathymic T cells and B-1 cells and is supported by the observation of reactive autoantibodies identified in this study. Malaria consists of a hepatic stage and blood stage during infection [37]. During the hepatic stage, intracellular malaria parasites invade hepatocytes whereas during the blood stage they invade erythrocytes. Therefore, conventional T cells and conventional antibodies against parasites may not react to parasitic antigens. Under this situation, autoantibodies against surface antigens on hepatocytes and erythrocytes may be important for protection from malaria. Previous studies demonstrated that autoantibodies against erythrocytes or their surface molecules were induced in malaria [38–43] (Table 1). In addition, the autoantibodies were found to react with intracellular components and many cell surface components, including those on neutrophils [44,45] and thrombocytes [46,47]. The immune status observed in malaria is summarized in Table 1. Since the antigenicity of such surface antigens (especially on erythrocytes) is increased by malaria, the autoreactivity of activated extrathymic T cells and the autoantibodies of activated B-1 cells might efficiently react with infected erythrocytes and hepatocytes. Finally, such denatured hepatocytes and erythrocytes are processed by macrophages in the spleen and Kupffer cells in the liver. Hepatosplenomegaly with malaria pigments is consistently observed in humans and mice in malaria [33]. Results from immunohistochemistry of tissue sections revealed that the most reactive sites of autoantibodies in mice with malaria were the splenic medulla, bone marrow and peripheral erythrocytes, especially in infected tissues. This reactivity supports the idea that autoantibodies preferentially attack infected erythrocytes. In addition to hepatocytes, epithelial cells in the intestine were reactive to the autoantibodies. Since hepatocytes were phylogenetically generated from intestinal epithelial cells [48], this suggests cross-reactivity between hepatocytes and intestinal epithelial cells. An immune status of activated extrathymic T cells and autoantibody-producing B-1 cells is common to aging [49,50], autoimmune diseases [51,52], chronic graft-vs-host disease [53,54] and malaria. During these immune responses, the thymus often becomes atrophied or involuted. Moreover, these immune states also resemble stress-associated responses [55,56]. This study confirmed that stress-associated responses such as hypothermia and hyperglycemia were observed in malaria. Taken together, this suggests that malaria induces an innate immune response rather than an adaptive immune response and that the reactivity of autoantibodies against the surface of erythrocytes and hepatocytes should be further studied.
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Fig. 3. Immunohistochemical staining of sera using various tissues. The spleen, bone marrow (BM), peripheral blood (PB), liver and large intestine (Li) were examined. Normal and malaria-infected tissues were used. Sections were stained with hematoxylin (blue) and diaminobenzidine (brown).
Table 1 Immunologic state seen in malarial infection. T cells
B cells (antibody production)
Thymic atrophy and suppression of conventional T cells [15,16]
Dysregulation of conventional B cells (B-2) and paucity of antibodies against Plasmodium [20,21] Activation of B-1 cells [20–23] and production of autoantibodies against:
Activation of unconventinal T cells, including NKT cells and extrathymic T cells in mice [15– 19] and in humans [32]
Intracellular components [24–29] e.g., nucleus, ds DNA, actin, etc. Cell surface components e.g., erythrocytes [38–43], neutrophils [44,45], thrombocytes [46,47], hepatocytesa, etc. [ ] References a This paper.
Acknowledgments We wish to thank Mrs. Yuko Kaneko for manuscript preparation. This work was supported by a grant-in-aid for scientific
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