Faculty Profiles - MIZUNO, Masashi

Zymosan, but not lipopolysaccharide, triggers severe and progressive peritoneal injury accompanied by complement activation in a rat peritonitis model. Reviewed

Mizuno M, Ito Y, Hepburn N, Mizuno T, Noda Y, Yuzawa Y, Harris CL, Morgan BP, Matsuo S.

J Immunol Vol. 183 page： 1403-1412 2009

A protein toxin from the sea anemone Phyllodiscus semoni targets the kidney and causes a severe renal injury with predominant glomerular endothelial damage. Reviewed International coauthorship International journal

Mizuno, M; Nozaki, M; Morine, N; Suzuki, N; Nishikawa, K; Morgan, BP; Matsuo, S

AMERICAN JOURNAL OF PATHOLOGY Vol. 171 ( 2 ) page： 402 - 414 2007.8

High levels of complement C3a receptor in the glomeruli in lupus nephritis Reviewed International coauthorship International journal

Masashi Mizuno, Stephanie Blanchin, Philippe Gasque, Kazuhiro Nishikawa, Seiichi Matsuo

AMERICAN JOURNAL OF KIDNEY DISEASES Vol. 49 ( 5 ) page： 598 - 606 2007.5

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO-ELSEVIER INC

Background: Taking into consideration the key role of the complement system in renal diseases, we investigated molecular and cellular properties of the human complement C3a receptor (C3aR) in vitro and in situ, looking at its expression in several human renal pathological states.
Methods: Several antibodies were generated and used for immunohistochemistry and Western blot analyses to address C3aR expression and its regulation in vitro on cell lines of myeloid cells and nonmyeloid cell lineages. Furthermore, C3aR distribution was investigated in control nephrectomized kidneys and 116 biopsy specimens from patients with renal diseases, including lupus nephritis (lupus-N).
Results: C3aR is a highly N-glycosylated protein with an apparent molecular mass of 65 to 95 kd expressed by myeloid and endothelial cells. C3aR is particularly upregulated in response to interferon gamma treatment, but was unaffected by the other inflammatory cytokines, such as tumor necrosis factor alpha and transforming growth factor beta. In normal human kidney, C3aR staining was not observed. However, glomerular C3aR staining was detected in 42.9% of lupus-N specimens in association with immunoglobulin G immune-complex depositions. Staining intensity correlated with disease severity. C3aR was found in the endothelial area of 81.3% of samples classified as World Health Organization class IV with active lesions. Conversely, C3aR was not detected by means of immunohistochemistry in kidneys from patients with other renal diseases.
Conclusion: Our data indicate that C3aR expression is tightly regulated and altered in certain disease conditions. C3aR may be used as a unique biomarker of diagnosis and disease activity in patients with lupus-N.

DOI： 10.1053/j.ajkd.2007.02.271

Immunization with autologous CD46 generates a strong autoantibody response in rats that targets spermatozoa Reviewed International coauthorship International journal

Masashi Mizuno, Claire L. Harris, B. Paul Morgan

JOURNAL OF REPRODUCTIVE IMMUNOLOGY Vol. 73 ( 2 ) page： 135 - 147 2007.4

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

1CD46, a membrane complement regulator, has been implicated as pathogen receptor, T cell activator and contributor to spermatozoa-egg interactions. In man, a role in the fertilization process was suggested by its localization on the acrosome. In rodents, CD46 is expressed only on the spermatozoal acrosome, suggesting an essential role at this site. This restricted expression led us to ask whether immunization with CD46 would generate anti-CD46 antibody responses that might target spermatozoa and influence fertility. We immunized male and female rats with rat CD46. Strong immune responses were generated in all rats and immune sera stained CD46 in testis extracts and in situ in testis and sperm. Incubation of spermatozoa with immune sera caused deposition of immunoglobulin and C3b in an acrosome pattern and reduced motility. We mated immune male rats with naive females and female immune rats with naive males. The incidence of pregnancy and number of fetuses were not different in matings involving immune male or female rats compared to controls. Testis sections from immune rats revealed no immunoglobulin deposition on CD46-positive sperm precursors, suggesting that acrosomal CD46 was inaccessible in this location. A minority of spermatozoa harvested from epididymis of immune rats had immunoglobulin and C3b bound to the acrosome, suggesting that anti-CD46, present in genital tract fluids, bound after acrosome reaction. These data demonstrate that the restricted expression of CD46 allows strong anti-CD46 responses in rats that target spermatozoa in vitro and in vivo. The anti-CD46 response did not influence fertility, perhaps reflecting the considerable redundancy for fertilization in rodents. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.jri.2006.08.001

CD55 expression in rat male reproductive tissue: Differential expression in testis and expression of a unique truncated isoform on spermatozoa. Reviewed International coauthorship International journal

Mizuno M, Donev RM, Harris CL, Morgan BP

Mol. Immunol. Vol. 44 page： 1624-1633 2006

A review of current knowledge of the complement system and the therapeutic opportunities in inflammatory arthritis. Invited Reviewed

Mizuno M

Curr Med Chem Vol. 13 ( 14 ) page： 1707-1717 - 1717 2006

Authorship：Lead author,　Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：BENTHAM SCIENCE PUBL LTD

The complement activation system, a key component of the innate immune system, protects the host from microorganisms such as bacteria, and other foreign threats including abnormal cells. However, it is also double-edged in that it can have. negative effects in the host; excessive complement activation damages the host and can even kill in anaphylactic shock and septic shock. Regulation of the complement system is a useful strategy to control inflammatory diseases, including inflammatory arthritis. Rheumatoid arthritis is a common inflammatory disease worldwide. Many medicines are developed to control inflammation, including recently developed biological response modifiers such as anti-TNF and IL-6 agents. Nevertheless, in some patients disease remains difficult to control because of complications, side effects and tolerance of medicines. In inflammatory arthritis, including rheumatoid arthritis, there is abundant evidence implicating complement activation in humans and animal models. Therefore, anti-complement agents might be beneficial as part of clinical treatment. However, at present, there are still no applicable agents for therapeutic regulation of excessive complement activation in chronic disease. Novel agents in development might be useful as a strategy to control complement activation. Here I describe recent knowledge of the complement system in inflammatory arthritis, the recent developments in anti-complement agents and their considerable potential for the future.

CD46 plays a key role in tailoring innate immune recognition of apoptotic and necrotic cells Reviewed International coauthorship International journal

Elward, K; Griffiths, M; Mizuno, M; Harris, CL; Neal, JW; Morgan, BP; Gasque, P

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280 ( 43 ) page： 36342 - 36354 2005.10

Expression of CD46 in developing rat spermatozoa: Ultrastructural localization and utility as a marker of the various stages of the seminiferous tubuli Reviewed International coauthorship International journal

M Mizuno, CL Harris, N Suzuki, S Matsuo, BP Morgan

BIOLOGY OF REPRODUCTION Vol. 72 ( 4 ) page： 908 - 915 2005.4

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY REPRODUCTION

Identification of the various stages of the seminal tubule epithelium that are important in spermatogenesis in humans and rodents requires considerable expertise for analysis of ultrastructural appearance under light microscopy. Few good stage-specific markers have been reported to facilitate the process. We recently described characterization of the expression of CD46 (membrane cofactor protein) in the rat using a novel monoclonal antibody. Expression of CD46 was restricted to spermatozoa and their immediate precursors in the testis. In the present study, we used a combination of morphological analyses, known acrosome markers, actin staining, direct nuclear staining, and staining for CD46 to delineate precisely the subcellular location of CD46. Staining of CD46 colocalized with known acrosome markers in late spermatids and mature spermatozoa and was confirmed by electron microscopy to be acrosome-restricted. Expression was first detected in step 7 spermatids, whereas known markers were not expressed until step 9. The CD46 staining pattern differed through spermatid development, and distinct patterns of staining could be identified that, when combined with 4 '-6-diamino-2-phenylindole-2HCl nuclear staining, enabled the accurate staging of the seminiferous tubule epithelium in different profiles. This detailed description of the spatiotemporal expression patterns of CD46 provides a valuable tool for analysis of spermatogenesis in the rat. Furthermore, this information will aid ongoing studies regarding the roles of CD46 in acrosome-related spermatozoal functions.

DOI： 10.1095/biolreprod.104.035485

Nucleic acids and CD46 are key regulators in innate immune recognition and the finely tuned removal of “danger-self". Reviewed

Elward K, Griffiths M., Mizuno M., Harris CL., Neal JW., Morgan BP., Gasque P.

J Biol Chem Vol. 280 page： 36342-36354 2005

Rat membrane cofactor protein (MCP; CD46) is expressed only in the acrosome of developing and mature spermatozoa and mediates binding to immobilized activated C3 Reviewed International coauthorship International journal

M Mizuno, CL Harris, PM Johnson, BP Morgan

BIOLOGY OF REPRODUCTION Vol. 71 ( 4 ) page： 1374 - 1383 2004.10

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：SOC STUDY REPRODUCTION

The rat analogue of the complement regulator membrane cofactor protein (MCP; CD46) was recently cloned and analysis at the mRNA level suggested that expression was restricted to testis. In light of the proposed roles of human MCP in sperm-egg interaction, we undertook to analyze rat MCP expression at the protein level in order better to address its putative role in fertilization. Recombinant fusion proteins comprising antibody Fc and specific domains of rat MCP were generated and used to develop a monoclonal antibody, MM.1, specific for rat MCP. Immunohistochemistry using these reagents confirmed the reported testis-specific expression of MCP in sexually mature rats and demonstrated that MCP was expressed only by spermatozoa and their immediate precursors in spermiogenesis, spermatids. Prepubertal male rats did not express MCP, and there was no evidence of MCP expression at any site in the embryo. Spermatozoal MCP expression was restricted to the inner acrosomal membrane, exposed only after fixation or induction of the acrosome reaction. Acrosome-reacted but not unreacted spermatozoa bound methylamine-activated C3 immobilized on plastic. The retention of MCP at this subcellular site, which is probably crucial to sperm-egg interaction, and the functional demonstration of binding to activated C3 strengthen suggestions from human studies that MCP may play an important role in fertilization. The reagents and results described here will enable studies of the role of spermatozoal MCP in sperm-egg interaction using a relevant animal model system.

DOI： 10.1095/biolreprod.104.030114

Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis Reviewed International coauthorship International journal

AS Williams, M Mizuno, PJ Richards, DS Holt, BP Morgan

ARTHRITIS AND RHEUMATISM Vol. 50 ( 9 ) page： 3035 - 3044 2004.9

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Objective. To investigate the roles of CD59a in the protection of joint tissue in the context of murine antigen-induced arthritis (AIA).
Methods. AIA was triggered in CD59a-deficient (CD59a(-/-)) mice and in CD59a-sufficient (CD59a(+/+)) controls; the course and severity of disease were compared between groups. The effects on arthritis of restoring CD59 to the joint in CD59a(-/-) mice by use of a membrane-targeted recombinant CD59 were also explored.
Results. Disease, as assessed clinically by measurement of joint swelling on day I (P < 0.0001), day 2 (P < 0.01), and day 7 (P < 0.02) and histologically from indicators of joint damage on day 21 (P < 0.02), was significantly enhanced in CD59a(-/-) mice compared with CD59a(+/+) wild-type controls. Membrane attack complex (MAC) deposition in the arthritic joints of CD59a(-/-) mice was also increased compared with that in the joints of CD59a(+/+) controls. Restitution of CD59 activity in joints of CD59a(-/-) mice was attempted with soluble recombinant rat CD59 (sCD59) or with a novel membrane-targeted rat CD59 derivative (sCD59APT542). Strong immunohistochemical staining of the synovial membrane and subsynovial tissue was apparent in sCD59-APT542-injected joints, but not in joints injected with untargeted sCD59. Intraarticular administration of sCD59-APT542 markedly ameliorated disease severity in CD59a(-/-) mice, knee swelling was significantly reduced over the time course of the disease, and joint damage, assessed histologically, was significantly milder on day 21 (P < 0.05).
Conclusion. These data firmly implicate the MAC of complement as a major effector of joint damage in the murine AIA model of rheumatoid arthritis (RA), and they provide a rationale for the inhibition of MAC assembly as a therapeutic strategy for RA.

DOI： 10.1002/art.20478

CD59a is the primary regulator of membrane attack complex assembly in the mouse Reviewed International coauthorship International journal

S Baalasubramanian, CL Harris, RM Donev, M Mizuno, N Omidvar, WC Song, BP Morgan

JOURNAL OF IMMUNOLOGY Vol. 173 ( 6 ) page： 3684 - 3692 2004.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC IMMUNOLOGISTS

Gene-deleted mice have provided a potent tool in efforts to understand the roles of complement and complement-regulating proteins in vivo. In particular, mice deficient in the membrane regulators complement receptor 1-related gene/protein y, decay-accelerating factor, or CD59 have demonstrated homeostatic relevance and backcrossing between the strains has revealed cooperativity in regulation. In mouse, genes encoding decay-accelerating factor and CD59 have been duplicated and show differential expression in tissues, complicating interpretation and extrapolation of findings to man. The first described form of CD59, CD59a, is broadly distributed and deletion of the cd59a gene causes a mild hemolytic phenotype with increased susceptibility in complement-mediated disease models. The distribution of the second form, CD59b, was originally described as testis specific, but later by some as widespread. Deletion of the cd59b gene caused a severe hemolytic and thrombotic phenotype. To apply data from these mouse models to man it is essential to know the relative distribution and functional roles of these two forms of CD59. We have generated new specific reagents and used them in sensitive quantitative analyses to comprehensively characterize expression of mRNA and protein and functional roles of CD59a and CD59b in wild-type (wt) and CD59a-negative mice. cd59b mRNA was detected only in testis and, at very low levels, in bone marrow. CD59b protein was present on mature spermatozoa and precursors and, in trace amounts, erythrocytes. Erythrocyte CD59b did not inhibit complement lysis except when CD59a was absent or blocked. These data confirm that CD59a is the primary regulator of complement membrane attack in mouse.

The possibilities and pitfalls for anti-complement therapies in inflammatory diseases. Invited Reviewed International coauthorship International journal

Mizuno M, Morgan BP

Current Drug Targets Inflammation & Allergy Vol. 3 page： 85-94 2004

Generation of a recombinant, membrane-targeted form of the complement regulator CD59 - Activity in vitro and in vivo Reviewed International coauthorship International journal

DA Fraser, CL Harris, AS Williams, M Mizuno, S Gallagher, RAG Smith, BP Morgan

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278 ( 49 ) page： 48921 - 48927 2003.12

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Inappropriate activation of complement contributes to pathology in diverse inflammatory diseases. Soluble recombinant forms of the natural cell membrane regulators of complement are effective in animal models and some human diseases. However, their use is limited for reasons related to cost, short half lives, and propensity to cause unwanted systemic effects. Some of these limitations may be overcome by use of bacterial expression systems, specific targeting moieties, and judicious choice of regulator. Here we describe the application of these strategies to the generation of a membrane-targeted form of CD59. A recombinant soluble form of rat CD59, comprising the first 71 residues of the mature protein and missing the membrane-anchoring signal, was expressed in bacteria, purified, and refolded in a fully active form. The protein was coupled through its carboxyl terminus to a short, synthetic address tag that confers membrane binding activity. Attachment of the membrane address tag markedly increased complement-inhibitory activity assessed in vitro in hemolysis assays. Intra-articular administration of the tagged agent markedly suppressed disease in a model of rheumatoid arthritis in Lewis rats. This novel type of agent, termed sCD59-APT542, offers for the first time the prospect of efficient and specific inhibition of membrane attack complex activity in vivo.

DOI： 10.1074/jbc.M302598200

Generation of a recombinant, membrane-targeted form of the complement regulator, CD59: activity in vitro and in vivo. Reviewed

Fraser DA., Harris CL., Williams AS., Mizuno M., Gallagher S., Smith RAG., Morgan BP.

J. Biol. Chem. Vol. 278 page： 48921-48927 2003

Membrane complement regulators protect against the development of type II collagen-induced arthritis in rats Reviewed International coauthorship International journal

M Mizuno, K Nishikawa, OB Spiller, BP Morgan, N Okada, H Okada, S Matsuo

ARTHRITIS AND RHEUMATISM Vol. 44 ( 10 ) page： 2425 - 2434 2001.10

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Objective. To investigate changes in the distribution patterns of membrane complement regulators (MCRs) during the development of type II collagen-induced arthritis (CIA) and to examine the protective effects of these molecules against the augmentation of CIA in the knee joint.
Methods. Immunohistochemistry was used to examine the distribution of the MCRs Crry, DAF, and CD59 in the synovium of knee joints before and 2, 4, and 10 weeks after induction of CIA by immunization with type II collagen. In addition, at 2 or 10 weeks after induction of CIA, rats were injected intraarticularly with anti-Crry and/or anti-CD59 as the F(ab')(2) fraction of monoclonal antibodies (mAb). Knee joint swelling and histologic changes in the synovium were examined 2 weeks after mAb injection.
Results. Synovial expression of Crry, DAF, and CD59 decreased in parallel with increased inflammation. When Crry and CD59 were functionally blocked at 2 weeks after the induction of CIA, swelling of the knee joints was markedly increased. Blocking of either regulator alone had no effect on swelling. Thickening of the synovial surface and proliferation of subsynovial tissue were all increased after blocking Crry and CD59, whereas blocking of either MCR alone had no effect. When both Crry and CD59 were blocked, deposits of membrane attack complex were found in the synovium.
Conclusion. Our findings indicate that in rats with CIA and severely inflamed synovium, local expression of MCR is reduced. The MCRs Crry and CD59 appear to suppress the development of CIA.

DOI： 10.1002/1529-0131(200110)44:10<2425::AID-ART407>3.0.CO;2-4

CD59 protects rat kidney from complement mediated injury in collaboration with Crry Reviewed International coauthorship International journal

M Watanabe, Y Morita, M Mizuno, K Nishikawa, Y Yuzawa, N Hotta, BP Morgan, N Okada, H Okada, S Matsuo

KIDNEY INTERNATIONAL Vol. 58 ( 4 ) page： 1569 - 1579 2000.10

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE INC

Background. As previously reported, the membrane-bound complement regulator at the C3 level (Crry/p65) is important in maintaining normal integrity of the kidney in rats. However, the role of a complement regulator at the C8/9 level (CD59) is not clear, especially when activation of complement occurs at the C3 level. The aim of this work was to elucidate the in vivo role of CD59 under C3 activating conditions.
Methods. Two monoclonal antibodies, 5I2 and 6D1, were used to suppress the function of Crry and CD59, respectively. In order to activate alternative the pathway of complement, the left kidney was perfused with 5I2 and/or 6D1 and was recirculated.
Results. In the kidneys perfused with 5I2 alone, deposition of C3 and membrane attack complex (MAC) was observed in the peritubular capillaries, vasa recta, and tubular basement membranes. Cast formation, tubular dilation and degeneration, and cellular infiltration were observed at days 1 and 4, and they recovered by day 7. Further suppression of CD59 by 6D1 significantly enhanced the deposition of MAC and worsened the already exacerbated tubulointerstitial injury. These effects of 6D1 were dose dependent. Perfusion with 6D1 alone did not induce histologic damage or MAC deposition in the tubulointerstitium.
Conclusions. In rats, CD59 maintains normal integrity of the kidney in collaboration with Crry in rats against complement-mediated damage in vivo.

DOI： 10.1046/j.1523-1755.2000.00318.x

Acute renal failure after a sea anemone sting Reviewed International journal

M Mizuno, K Nishikawa, Y Yuzawa, T Kanie, H Mori, Y Araki, N Hotta, S Matsuo

AMERICAN JOURNAL OF KIDNEY DISEASES Vol. 36 ( 2 ) page： art. no. - E10 2000.8

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：W B SAUNDERS CO

A 27-year-old man suffering from severe swelling and pain in his right arm was referred to our hospital. He showed signs of acute renal failure (ARF) with severe dermatitis of his right arm. Three days before being admitted, he accidentally touched some kind of marine organism with his right hand while snorkeling in the Sulu Sea around Cebu Island. Within a few minutes, he was experiencing severe pain in his right hand. Then his right hand gradually became swollen. The marine creature responsible for this injury was thought to have been a sea anemone, which is a type of coelenterate. Histologic findings of a renal biopsy indicated that acute tubular necrosis (ATN) had caused ARF in this patient's case. Supportive therapies improved renal function of this patient, and steroid pulse therapy attenuated the severe skin discoloration. The ATN was thought to have been caused by the poison from a sea anemone because there were no other conceivable reasons for the patient's condition. This is the first time that a marine envenomation case has been reported in which the sting of a sea anemone has caused ATN without the failure of any other organs. (C) 2000 by the National Kidney Foundation, Inc.

DOI： 10.1053/ajkd.2000.9006