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Becker J, Wilting J. Molecules That Have Rarely Been Studied in Lymphatic Endothelial Cells. Int J Mol Sci 2024; 25:12226. [PMID: 39596293 PMCID: PMC11594919 DOI: 10.3390/ijms252212226] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2024] [Revised: 11/05/2024] [Accepted: 11/10/2024] [Indexed: 11/28/2024] Open
Abstract
A number of standard molecules are used for the molecular and histological characterization of lymphatic endothelial cells (LECs), including lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), Podoplanin (D2-40), VEGFR3, Prospero homeobox protein 1 (PROX1), and CD31. The number of molecules whose mutations cause lymphatic malformations or primary congenital lymphedema is considerable, but the majority of these diseases have not yet been characterized at the molecular level. Therefore, there is still considerable scope for molecular and functional studies of the lymphatic vasculature. Using RNASeq, we have previously characterized lymphatic endothelial cells (LECs) under normoxic and hypoxic conditions. We used this information to compare it with immunohistochemical data. We carried out some of the immunohistology ourselves, and systematically studied the Human Protein Atlas, a cell and tissue database based in Sweden. Here we describe molecules that are expressed at RNA and protein levels in LECs, hoping to stimulate future functional studies of these molecules.
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Affiliation(s)
| | - Jörg Wilting
- Institute of Anatomy and Cell Biology, University Medical Center Goettingen, Georg-August-University Goettingen, Kreuzbergring 36, 37075 Göttingen, Germany
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Müller GA, Müller TD. (Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins I: Localization at Plasma Membranes and Extracellular Compartments. Biomolecules 2023; 13:biom13050855. [PMID: 37238725 DOI: 10.3390/biom13050855] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2023] [Revised: 05/11/2023] [Accepted: 05/13/2023] [Indexed: 05/28/2023] Open
Abstract
Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).
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Affiliation(s)
- Günter A Müller
- Institute for Diabetes and Obesity (IDO), Helmholtz Diabetes Center (HDC) at Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstraße 1, 85764 Oberschleissheim, Germany
- German Center for Diabetes Research (DZD), 85764 Oberschleissheim, Germany
| | - Timo D Müller
- Institute for Diabetes and Obesity (IDO), Helmholtz Diabetes Center (HDC) at Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstraße 1, 85764 Oberschleissheim, Germany
- German Center for Diabetes Research (DZD), 85764 Oberschleissheim, Germany
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Kumar-Singh R. The role of complement membrane attack complex in dry and wet AMD - From hypothesis to clinical trials. Exp Eye Res 2019; 184:266-277. [PMID: 31082363 DOI: 10.1016/j.exer.2019.05.006] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 05/06/2019] [Accepted: 05/09/2019] [Indexed: 12/12/2022]
Abstract
Data from human dry and wet age-related macular degeneration (AMD) eyes support the hypothesis that constant 'tickover' of the alternative complement pathway results in chronic deposition of the complement membrane attack complex (MAC) on the choriocapillaris and the retinal pigment epithelium (RPE). Sub-lytic levels of MAC lead to cell signaling associated with tissue remodeling and the production of cytokines and inflammatory molecules. Lytic levels of MAC lead to cell death. CD59 is a naturally occurring inhibitor of the assembly of MAC. CD59 may thus be therapeutically efficacious against the pathophysiology of dry and wet AMD. The first gene therapy clinical trial for geographic atrophy - the advanced form of dry AMD has recently completed recruitment. This trial is studying the safety and tolerability of expressing CD59 from an adeno-associated virus (AAV) vector injected once into the vitreous. A second clinical trial assessing the efficacy of CD59 in wet AMD patients is also under way. Herein, the evidence for the role of MAC in the pathophysiology of dry as well as wet AMD and the scientific rationale underlying the use of AAV- delivered CD59 for the treatment of dry and wet AMD is discussed.
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Affiliation(s)
- Rajendra Kumar-Singh
- Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, 02111, USA.
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Karbian N, Eshed-Eisenbach Y, Tabib A, Hoizman H, Morgan BP, Schueler-Furman O, Peles E, Mevorach D. Molecular pathogenesis of human CD59 deficiency. NEUROLOGY-GENETICS 2018; 4:e280. [PMID: 30533526 PMCID: PMC6244018 DOI: 10.1212/nxg.0000000000000280] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/21/2018] [Accepted: 08/07/2018] [Indexed: 11/15/2022]
Abstract
Objective To characterize all 4 mutations described for CD59 congenital deficiency. Methods The 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality. Results Immunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation. Conclusions All 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non-MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.
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Affiliation(s)
- Netanel Karbian
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - Yael Eshed-Eisenbach
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - Adi Tabib
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - Hila Hoizman
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - B Paul Morgan
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - Ora Schueler-Furman
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - Elior Peles
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
| | - Dror Mevorach
- Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel
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Schnabolk G, Beon MK, Tomlinson S, Rohrer B. New Insights on Complement Inhibitor CD59 in Mouse Laser-Induced Choroidal Neovascularization: Mislocalization After Injury and Targeted Delivery for Protein Replacement. J Ocul Pharmacol Ther 2017; 33:400-411. [PMID: 28333572 DOI: 10.1089/jop.2016.0101] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
PURPOSE The membrane attack complex (MAC) in choriocapillaris (CC) and retinal pigment epithelium (RPE) increase with age and disease (age-related macular degeneration). MAC assembly can be inhibited by CD59, a membrane-bound regulator. Here we further investigated the role of CD59 in murine choroidal neovascularization (CNV), a model involving both CC and RPE, and tested whether CR2-CD59, a soluble targeted form of CD59, provides protection. METHODS Laser-induced CNV was generated in wild type and CD59a-deficient mice (CD59-/-). CNV size was measured by optical coherence tomography, and CR2-CD59 was injected intraperitoneally. Endogenous CD59 localization and MAC deposition were identified by immunohistochemistry and quantified by confocal microscopy. Cell-type-specific responses to MAC were examined in retinal pigment epithelial cells (ARPE-19) and microvascular endothelial cells (HMEC-1). RESULTS CD59 levels were severely reduced and protein was mislocalized in the RPE surrounding the lesion. CNV lesion size and subretinal fluid accumulation were exacerbated in CD59-/- when compared with those in WT mice, and an increase in MAC deposition was noted. In contrast, CR2-CD59 significantly reduced both structural features of CNV severity. In vitro, MAC inhibition in ARPE-19 cells prevented barrier function loss and accelerated wound healing and cell adhesion, whereas in HMEC-1 cells, CR2-CD59 decelerated wound healing and cell adhesion. CONCLUSION These data further support the importance of CD59 in controlling ocular injury responses and indicate that pharmacological inhibition of the MAC with CR2-CD59 may be a viable therapeutic approach for reducing complement-mediated ocular pathology.
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Affiliation(s)
- Gloriane Schnabolk
- 1 Division of Research, Ralph H. Johnson VA Medical Center , Charleston, South Carolina.,2 Department of Opthalmology, Medical University of South Carolina , Charleston, South Carolina
| | - Mee Keong Beon
- 2 Department of Opthalmology, Medical University of South Carolina , Charleston, South Carolina
| | - Stephen Tomlinson
- 1 Division of Research, Ralph H. Johnson VA Medical Center , Charleston, South Carolina.,3 Department of Microbiology and Immunology, Medical University of South Carolina , Charleston, South Carolina
| | - Bärbel Rohrer
- 1 Division of Research, Ralph H. Johnson VA Medical Center , Charleston, South Carolina.,2 Department of Opthalmology, Medical University of South Carolina , Charleston, South Carolina.,4 Department of Neurosciences Division of Research, Medical University of South Carolina , Charleston, South Carolina
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Mevorach D. Paroxysmal nocturnal hemoglobinuria (PNH) and primary p.Cys89Tyr mutation in CD59: Differences and similarities. Mol Immunol 2015; 67:51-5. [DOI: 10.1016/j.molimm.2015.03.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2015] [Accepted: 03/03/2015] [Indexed: 11/29/2022]
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Kimbara N, Dohi N, Miyamoto M, Asai S, Okada H, Okada N. Diagnostic Surface Expression of SWAP-70 on HIV-1 Infected T Cells. Microbiol Immunol 2013; 50:235-42. [PMID: 16547421 DOI: 10.1111/j.1348-0421.2006.tb03790.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Following immunization with HIV-1 infected cells, a hybridoma cell line termed 9F11 was established from the P3U-1 myeloma line fused with lymphocytes from a trans-chromosome (TC) mouse, that harbors human chromosomes containing immunoglobulin genes. The 9F11 human IgM monoclonal antibody (9F11 Ab) reacts with HIV-1 infected MOLT4 cells but not with uninfected MOLT4 cells, and causes immune cytolysis with homologous human complement at a concentration as low as 0.4 microg/ml. This Ab was used to perform immunoscreening of a cDNA expression library derived from HIV-1 infected cells. All positive cDNA clones contained SWAP-70 cDNA. SWAP-70 RNA and protein expression are much stronger in HIV-1 infected cells. SWAP-70 was also detected on the surface of HIV-1 infected cells by flow cytometric analysis. The monocyte cell line U937 cells expresses SWAP-70 on its cell surface regardless of whether it was infected with HIV-1. Furthermore, among PBMCs surface expression of SWAP-70 was detected on CD21+, CD56+ and CD14+ cells. Although CD3+ cells scarcely express SWAP-70 on their surface, once activated, they become positive. SWAP-70 may therefore serve as a marker for T cell differentiation as well as for HIV-1 infection.
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Affiliation(s)
- Noriaki Kimbara
- Department of Biodefense Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601, Japan
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Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy. Transplantation 2013; 95:1242-8. [PMID: 23635876 DOI: 10.1097/tp.0b013e318291a298] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
BACKGROUND Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. METHODS We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. RESULTS Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. CONCLUSIONS The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.
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Membrane-bound complement regulatory proteins as biomarkers and potential therapeutic targets for SLE. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2013; 735:55-81. [PMID: 23402019 DOI: 10.1007/978-1-4614-4118-2_4] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
For the last two decades, there had been remarkable advancement in understanding the role of complement regulatory proteins in autoimmune disorders and importance of complement inhibitors as therapeutics. Systemic lupus erythematosus is a prototype of systemic autoimmune disorders. The disease, though rare, is potentially fatal and afflicts women at their reproductive age. It is a complex disease with multiorgan involvement, and each patient presents with a different set of symptoms. The diagnosis is often difficult and is based on the diagnostic criteria set by the American Rheumatology Association. Presence of antinuclear antibodies and more specifically antidouble-stranded DNA indicates SLE. Since the disease is multifactorial and its phenotypes are highly heterogeneous, there is a need to identify multiple noninvasive biomarkers for SLE. Lack of validated biomarkers for SLE disease activity or response to treatment is a barrier to the efficient management of the disease, drug discovery, as well as development of new therapeutics. Recent studies with gene knockout mice have suggested that membrane-bound complement regulatory proteins (CRPs) may critically determine the sensitivity of host tissues to complement injury in autoimmune and inflammatory disorders. Case-controlled and followup studies carried out in our laboratory suggest an intimate relation between the level of DAF, MCP, CR1, and CD59 transcripts and the disease activity in SLE. Based on comparative evaluation of our data on these four membrane-bound complement regulatory proteins, we envisaged CR1 and MCP transcripts as putative noninvasive disease activity markers and the respective proteins as therapeutic targets for SLE. Following is a brief appraisal on membrane-bound complement regulatory proteins DAF, MCP, CR1, and CD59 as biomarkers and therapeutic targets for SLE.
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Endo Y, Iwaki D, Ishida Y, Takahashi M, Matsushita M, Fujita T. Mouse ficolin B has an ability to form complexes with mannose-binding lectin-associated serine proteases and activate complement through the lectin pathway. J Biomed Biotechnol 2012; 2012:105891. [PMID: 22523468 PMCID: PMC3306798 DOI: 10.1155/2012/105891] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2011] [Accepted: 11/08/2011] [Indexed: 11/18/2022] Open
Abstract
Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.
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Affiliation(s)
- Yuichi Endo
- Department of Immunology, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1295, Japan.
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Kerr H, Richards A. Complement-mediated injury and protection of endothelium: lessons from atypical haemolytic uraemic syndrome. Immunobiology 2012; 217:195-203. [PMID: 21855165 PMCID: PMC4083254 DOI: 10.1016/j.imbio.2011.07.028] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2011] [Revised: 07/22/2011] [Accepted: 07/24/2011] [Indexed: 12/25/2022]
Abstract
The complement system provides a vital defence against invading pathogens. As an intrinsic system it is always 'on', in a state of constant, low level activation. This activation is principally mediated through the deposition of C3b on to pathogenic surfaces and host tissues. C3b is generated by spontaneous 'tick over' and formal activation of the alternative pathway, and by activation of the classical and lectin pathways. If the deposited C3b is not appropriately regulated, there is progression to terminal pathway complement activation via the C5 convertases, generating the potent anaphylotoxin C5a and the membrane attack complex C5b-9. Unsurprisingly, these highly active components have the potential to cause injury to bystander host tissue, including the vascular endothelium. As such, complement activation on endothelium is normally tightly controlled by a large number of fluid-phase and membrane bound inhibitors, in an attempt to ensure that propagation of complement activation is appropriately restricted to invading pathogens and altered 'self', e.g. apoptotic and necrotic cells. The kidney is increasingly recognised as a site at particular risk from complement-mediated endothelial injury. Both genetic and acquired defects which impact on complement regulation predispose to this susceptibility. The thrombotic microangiopathy, haemolytic uraemic syndrome (HUS), will be used to illustrate the mechanisms by which the endothelial cell injury occurs. Finally, the underlying rationale for current and future potential therapeutic interventions in HUS and also the opportunities for enhancing endothelial defence to prevent relapsing disease through increased complement cytoprotective strategies will be summarised.
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Affiliation(s)
- Heather Kerr
- Department of Nephrology, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, UK
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An G, Miwa T, Song WL, Lawson JA, Rader DJ, Zhang Y, Song WC. CD59 but not DAF deficiency accelerates atherosclerosis in female ApoE knockout mice. Mol Immunol 2009; 46:1702-9. [PMID: 19297024 PMCID: PMC2705121 DOI: 10.1016/j.molimm.2009.02.009] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2008] [Revised: 02/04/2009] [Accepted: 02/10/2009] [Indexed: 11/22/2022]
Abstract
Although the complement system has been implicated in atherosclerosis, the influence of membrane-bound complement regulators in this process has not been well understood. We studied the role of two membrane complement regulators, decay-accelerating factor (DAF) and CD59, in a murine model of atherosclerosis. DAF(-/-) and CD59(-/-) mice were crossed with apolipoprotein E (ApoE)-deficient mice to generate DAF(-/-)ApoE(-/-) and CD59(-/-)ApoE(-/-) mice. Mice were fed a high fat diet (HFD) for 8 or 16 weeks. En face analysis showed that CD59 deficiency led to more extensive lesions in female ApoE(-/-) mice both at 8 weeks (2.07+/-0.27% vs.1.34+/-0.21%, P=0.06) and 16 weeks (17.13+/-1.14% vs. 9.72+/-1.14%, P<0.001). Similarly, lesions measured by aortic root sectioning were larger in female CD59(-/-)ApoE(-/-) mice than in controls at 8 weeks of HFD feeding (20.74+/-1.33% vs. 13.12+/-1.46%, P<0.005). On the other hand, DAF deficiency did not significantly influence atherosclerosis in ApoE(-/-) mice. Immunohistochemistry revealed more abundant membrane attack complex (MAC) deposition and more collagen staining in the aortic roots of CD59(-/-)ApoE(-/-) mice. Unexpectedly, total plasma cholesterol levels in female CD59(-/-)ApoE(-/-) mice were found to be elevated compared with CD59(+/+)ApoE(-/-) mice. We conclude that CD59 but not DAF offered protection in atherosclerosis in the context of ApoE deficiency. The protective role of CD59 was gender-biased and most likely involved prevention of MAC-mediated vascular injury, with possible contribution from an undefined effect on plasma cholesterol homeostasis.
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Affiliation(s)
- Guipeng An
- Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Room 1254 BRBII/III, 421 Curie Blvd, Philadelphia, PA 19104, USA
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Abe K, Endo Y, Nakazawa N, Kanno K, Okubo M, Hoshino T, Fujita T. Unique Phenotypes of C1s Deficiency and Abnormality Caused by Two Compound Heterozygosities in a Japanese Family. THE JOURNAL OF IMMUNOLOGY 2009; 182:1681-8. [DOI: 10.4049/jimmunol.182.3.1681] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
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Kinderlerer AR, Ali F, Johns M, Lidington EA, Leung V, Boyle JJ, Hamdulay SS, Evans PC, Haskard DO, Mason JC. KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature. J Biol Chem 2008; 283:14636-44. [PMID: 18362151 DOI: 10.1074/jbc.m800362200] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.
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Affiliation(s)
- Anne R Kinderlerer
- Cardiovascular Sciences, Bywaters Center for Vascular Inflammation, National Heart and Lung Institute, Imperial College London, London, UK
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15
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Parker CJ. Isolation and functional assay of the membrane complement inhibitors CD55 (DAF) and CD59 (MIRL). CURRENT PROTOCOLS IN IMMUNOLOGY 2008; Chapter 13:13.5.1-13.5.18. [PMID: 18432718 DOI: 10.1002/0471142735.im1305s11] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The complement system is the primary effector of humoral immunity. Because of its enormous destructive capacity, mechanisms for confining the activity of the system to the desired target and elaborate safeguards for protecting self against complement-mediated injury have evolved. Human cells, particularly those found at sites of inflammation (e.g., hematopoietic and endothelial cells), express highly specialized membrane constituents that act independently or in concert with plasma regulatory proteins to inhibit the functional activity of complement. Decay-accelerating factor (DAF), or CD55, directly inhibits the formation and stability of the amplification C3 and C5 convertases of both the classical and the alternative pathways. Failure of a cell to regulate the amplification C3 and C5 convertases allows the generation of the potentially cytolytic membrane attack complex (MAC), or C5b-9 (consisting of the complement components C5b, C6, C7, C8, and C9). The primary cellular regulator of the MAC is the membrane inhibitor of reactive lysis (MIRL), or CD59, which restricts complement-mediated lysis by blocking assembly of the MAC (primarily at the stage of C9 binding and polymerization). This unit provides a basic protocol for isolating CD55 and CD59, along with two support protocols describing separate functional assays for CD59 and CD55.
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Affiliation(s)
- C J Parker
- University of Utah School of Medicine and The Veterans Affairs Medical Center, Salt Lake City, Utah, USA
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16
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Molecular cloning and expression of channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59. Vet Immunol Immunopathol 2007; 120:246-53. [DOI: 10.1016/j.vetimm.2007.07.016] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2006] [Revised: 07/11/2007] [Accepted: 07/17/2007] [Indexed: 11/23/2022]
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Kim DD, Miwa T, Song WC. Retrovirus-mediated over-expression of decay-accelerating factor rescues Crry-deficient erythrocytes from acute alternative pathway complement attack. THE JOURNAL OF IMMUNOLOGY 2007; 177:5558-66. [PMID: 17015743 DOI: 10.4049/jimmunol.177.8.5558] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry(-/-)) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 +/- 127 DAF molecules/cell (DAF(high)) compared with 249 +/- 94 DAF molecules/cell (DAF(low)) and 774 +/- 135 Crry molecules/cell on control mouse erythrocytes. DAF(high)-Crry(-/-) erythrocytes were significantly more resistant than either DAF(low)-Crry(-/-), DAF(-/-) -Crry(+/+) or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry(-/-) erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAF(high)-Crry(-/-) erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.
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Affiliation(s)
- David D Kim
- Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
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18
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Abstract
The complement system is known to be involved in autoimmunity at several levels. Activated complement contributes to the inflammatory tissue injury characteristic of many autoimmune disease settings. On the other hand, early components of the classical pathway, including C1q, C4 and C2, are thought to be important for disposing apoptotic cellular autoantigens and/or the induction of B cell tolerance in the bone marrow, and their deficiency is a strong risk factor for systemic autoimmunity. Recent studies using transgenic mice have revealed membrane complement regulatory proteins as important modulators in the pathogenesis and manifestation of autoimmune injury. Available evidence suggests that these regulatory proteins may act to suppress autoimmunity via both complement-dependent and -independent mechanisms.
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Affiliation(s)
- Wen-Chao Song
- Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Rm 1254 BRBII/III, 421 Curie Blvd, Philadelphia, PA 19104, USA.
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Wakabayashi M, Ohi H, Tamano M, Onda K, Fujita T, Tomino Y. Acquired loss of erythrocyte complement receptor type 1 in patients with diabetic nephropathy undergoing hemodialysis. Nephron Clin Pract 2006; 104:e89-95. [PMID: 16837818 DOI: 10.1159/000094547] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2005] [Accepted: 04/26/2006] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND Complement receptor type 1 on erythrocytes (E-CR1) plays important roles not only in the regulation of complement activation, but also the clearance of immune complexes. Reduced E-CR1 was previously found in patients undergoing hemodialysis (HD). We investigated whether the E-CR1level in HD patients with diabetic nephropathy (DMN) is decreased. The levels of decay accelerating factor (DAF) and CD59 on erythrocytes (E) were also determined to ascertain whether the loss of CR1 is a specific phenomenon or other complement regulatory proteins are also affected. METHODS The levels of CR1, DAF, and CD59 on E were analyzed in 176 HD patients with DMN, 101 HD patients with non-diabetes mellitus renal diseases (non-DMN), and 108 healthy individuals. Hind III restriction fragment length polymorphism of intron 27 of the CR1 gene was analyzed. The serum-soluble CR1 levelwas measured by ELISA. RESULTS The E-CR1 level was significantly lower in the DMN group than the non-DMN group (p < 0.0001) and healthy individuals (p < 0.05). The E-CR1 level was significantly higher in the non-DMN group than in healthy individuals (p < 0.01). The levels of E-DAF and E-CD59 were significantly lower in the DMN group than non-DMN group (DAF, p < 0.01; CD59, p < 0.0001). Within each genotype of the CR1 gene, the E-CR1 level was significantly lower in the DMN group than in the non-DMN group and healthy individuals (non-DMN, p < 0.01; healthy individuals, p < 0.05). The serum-soluble CR1 level was significantly higher in the DMN group than non-DMN group and control group (p < 0.01 each). However, soluble CR1 did not correlate with E-CR1. CONCLUSION Acquired loss of E-CR1 was found among HD patients with DMN. From the viewpoint of host defense, it may be a prognostic factor.
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Affiliation(s)
- Michiro Wakabayashi
- Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan
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20
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Abstract
Complement activity was first described in the 1890s and the characterisation of this highly complex system has continued ever since. This review traces the history of complement research from its beginnings until it was transformed by the advent of molecular biology in the 1980s. It takes as a focus point the CIBA symposium on Complement held in London in May 1964 and reflects-and is slanted by-the views and research experience of the author.
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Affiliation(s)
- Peter Lachmann
- Emeritus Sheila Joan Smith Professor of Immunology, University of Cambridge, Centre for Veterinary Science, Madingley Road, Cambridge CB3 0ES, UK.
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21
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Okada N, Yin S, Asai S, Kimbara N, Dohi N, Hosokawa M, Wu X, Okada H. Human IgM monoclonal antibodies reactive with HIV-1-infected cells generated using a trans-chromosome mouse. Microbiol Immunol 2005; 49:447-59. [PMID: 15905607 DOI: 10.1111/j.1348-0421.2005.tb03749.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The trans-chromosome (TC) mouse that we used harbors human chromosomes 2, 14 and/or 22, and has undergone knock-out of its endogeneous genes coding for mu-and kappa-chains of immunoglobulin. One of these TC mice was immunized with HIV-1-infected U937 cells, and spleen cells from the immunized animal were fused with the mouse myeloma cell line to generate hybridoma cells. We selected hybridomas that produce human IgM antibodies (Abs) reactive with HIV-1-infected MOLT4 cells but not with uninfected MOLT4 cells. Two hybridoma cell lines were established termed 9F11 and 2G9. Although 0.4 mug/ml of 9F11 was able to induce complement-mediated cytolysis of the infected cells in the presence of fresh human serum, 2G9 could not. There was no difference between the two monoclonal Abs in the base sequences of cDNAs coding for the constant regions of mu-and kappa-chains. Therefore, we speculate that the ability to activate complement on homologous cell membranes might reflect the structural presentation of antigenic molecules, which could facilitate the binding of an IgM Ab to multiple binding sites resulting in escape from restriction by species-specific inhibitors of complement such as DAF (CD55) and CD59. On the other hand, 2G9 induced apoptosis of HIV-1-infected cells, including latently infected OM10.1 cells, although the Ag for 2G9 remains to be identified. Since both of the Abs had reduced reactivity toward HIV-1-infected MOLT4 cells following cultivation in the presence of tunicamycin, the responsible antigens would involve a sugar moiety.
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Affiliation(s)
- Noriko Okada
- Department of Biodefense, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan.
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22
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Zhang C, Ge CL, Guo RX, He SG. Effect of IL-4 on altered expression of complement activation regulators in rat pancreatic cells during severe acute pancreatitis. World J Gastroenterol 2005; 11:6770-4. [PMID: 16425382 PMCID: PMC4725027 DOI: 10.3748/wjg.v11.i43.6770] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of IL-4 on the altered expression of complement activation regulators in pancreas and pancreatic necrosis during experimental severe acute pancreatitis (SAP).
METHODS: SAP model of rats was established by retrograde injection of 5% sodium taurocholate (1 mL/kg) into the pancreatic duct. We immunohistochemically assayed the expression of three complement activation regulators: decay accelerating factor (DAF; CD55), 20 ku homologous restriction factor (HRF20; CD59) and membrane cofactor protein (MCP; CD46), in the pancreatic acinar cells of rats at 0, 3, 6, 12, and 24 h after the induction of SAP model. Meanwhile the levels of amylase and lipase were determined, and morphological examination was performed. Then, 61 rats were randomly divided into three groups. Group A (n = 21) received no treatment after the SAP model was established; group B (n = 20) was given IL-4 (8 µg/animal) intraperitoneally 0.5 h before the SAP model was established; group C (n = 20) was given IL-4 (8 µg/animal) intraperitoneally 0.5 h after the SAP model was established. Plasma amylase and lipase, extent of pancreatic necrosis and expression of complement activation regulators were investigated 6 h after the induction of SAP model.
RESULTS: Three complement activation regulators were all expressed in pancreatic acinar cells. MCP was not found on the basolateral surface as reported. Contrary to the gradually increasing plasma level of amylase and lipase, expression of complement activation regulators decreased after SAP model was set up. At the same time, the severity of pancreatic necrosis was enhanced. A strong negative correlation was found between the expression of MCP, DAF, CD59 in pancreatic acinar cells and the severity of pancreatic necrosis (r = –0.748, –0.827, –0.723; P<0.01). In the second series of experiments, no matter when the treatment of IL-4 was given (before or after the induction of SAP model), the serum level of amylase or lipase was decreased and the extent of pancreatic necrosis was ameliorated significantly. Compared to SAP control group, the expression of DAF and CD59 in pancreas was reinforced when IL-4 was given before the induction of SAP model (P<0.01, P<0.05), but the expression of MCP was not influenced (P>0.05). The expression of DAF was enhanced, when IL-4 was given after the induction of SAP model (P<0.05), but the expression of CD59 and MCP did not change (P>0.05).
CONCLUSION: Complement activation regulators may participate in the pathogenesis of pancreatic inflammation. Downregulation of complement activation regulators expression may be one of the causes of pancreatic necrosis. IL-4 treatment may control SAP aggravation by enhancing expression of DAF and CD59 in pancreas and decreasing pancreatic necrosis. Moreover, DAF and CD59 may play an important role in the regulation of complement activation regulators during SAP.
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Affiliation(s)
- Cheng Zhang
- Department of General Surgery, 201 Hospital of Chinese PLA, Liaoyang 111000, Liaoning Province, China.
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Joh T, Sasaki M, Kataoka H, Tanida S, Itoh K, Kondo Y, Ogasawara N, Oshima T, Okada N, Ohara H, Sano H, Nakao H, Sobue S, Itoh M. Helicobacter pylori eradication decreases the expression of glycosylphosphatidylinositol-anchored complement regulators, decay-accelerating factor and homologous restriction factor 20, in human gastric epithelium. J Gastroenterol Hepatol 2005; 20:1344-51. [PMID: 16105119 DOI: 10.1111/j.1440-1746.2005.03876.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
BACKGROUND It has previously been reported that there is a strong correlation between the expression of glycosylphosphatidylinositol (GPI)-anchored complement membrane inhibitor in gastric epithelium and the severity of inflammation of gastric mucosa. To investigate the regulation of complement activity in gastric epithelium during Helicobacter pylori (H. pylori)-associated gastritis, the expression of GPI-anchored complement membrane inhibitors, decay-accelerating factor (DAF) and 20-kDa homologous restriction factor 20 (HRF20), and membrane cofactor protein (MCP), which is a transmembrane protein, were evaluated after removal of the H. pylori stimulus. Furthermore, the expression of the complement fragment, C3c, was also investigated. METHODS Forty-six patients with epigastric symptoms and endoscopically confirmed peptic ulcer or gastritis who had H. pylori infection of the gastric mucosa were enrolled in the present study. Biopsy specimens were obtained from the gastric antrum and corpus 1 month before and after eradication. Helicobacter pylori infection was determined by the rapid urease test, histology, and culture before eradication, and by histology, culture, and urea breath test after eradication. Gastric biopsy specimens obtained before and after eradication were evaluated for infiltration by neutrophils and mononuclear cells. The expression of complement membrane inhibitors, DAF, HRF20, and MCP and that of the main complement fragment, C3c, was immunohistochemically evaluated. RESULTS One month after the eradication of H. pylori, the infiltration by neutrophils and mononuclear cells in the gastric mucosa decreased significantly (P < 0.0001) as compared with that before eradication. The expression of DAF, HRF20, and C3c on gastric mucosal epithelium also significantly decreased in both the antrum and the corpus (P < 0.05) 1 month after eradication. However, no change was observed in the expression of MCP. CONCLUSIONS The decrease in the expression of GPI-anchored complement regulator and the complement after removal of a chronic microbial stimulus suggests that the gastric epithelium appears to undergo an aggressive stress of complement during H. pylori infection. Conclusively, DAF and HRF20 may play an important protective role against complement-mediated damage induced by chronic microbial stimuli in such a pathological condition.
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Affiliation(s)
- Takashi Joh
- Department of Internal Medicine and Bioregulation, Nagoya City University Graduate School of Medical Sciences, Japan
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Morgan BP, Berg CW, Harris CL. ''Homologous restriction'' in complement lysis: roles of membrane complement regulators. Xenotransplantation 2005; 12:258-65. [PMID: 15943774 DOI: 10.1111/j.1399-3089.2005.00237.x] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
The complement system is a powerful bactericidal immune defence with the potential to damage self cells. Protection of self is provided by expression on cells of a battery of membrane regulators that inhibit activation of complement. Roles of complement in the rejection of transplanted organs have long been recognized, and are particularly relevant in xenotransplantation, where hyperacute rejection is complement-driven. Inhibiting complement was therefore considered early in the history of xenografting, and the use of membrane complement regulators to this end was proposed more than two decades ago. For each of the membrane regulators in humans, early studies implied a species-specificity of action, inhibiting human complement but not that from other species. The dogma of species-specificity dictated strategies for inhibiting complement in xenografts and drove the creation of donor transgenic pigs expressing human regulators. Here we critically evaluate the evidence for species-specificity in membrane complement regulators from humans and other animals. We challenge the dogma and show that there is considerable cross-species activity for each of the membrane regulators of complement. Acceptance of the fact that species selectivity is not a limitation will open new avenues for protection of the xenograft from complement damage.
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Affiliation(s)
- B Paul Morgan
- Complement Biology Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, UK.
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25
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Antic Stankovic J, Vucevic D, Majstorovic I, Vasilijic S, Colic M. The role of rat Crry, a complement regulatory protein, in proliferation of thymocytes. Life Sci 2004; 75:3053-62. [PMID: 15474557 DOI: 10.1016/j.lfs.2004.06.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2003] [Accepted: 06/14/2004] [Indexed: 10/26/2022]
Abstract
In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 microg/ml and 20 microg/ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6+ antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far.
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Affiliation(s)
- Jelena Antic Stankovic
- Institute for Microbiology and Immunology, Faculty of Pharmacy, Vojvode Stepe 450, 11 000 Belgrade, Serbia and Montenegro.
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26
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Ohta R, Kondor N, Dohi N, Tomlinson S, Imai M, Holers VM, Okada H, Okada N. Mouse Complement Receptor-Related Gene y/p65-Neutralized Tumor Vaccine Induces Antitumor Activity In Vivo. THE JOURNAL OF IMMUNOLOGY 2004; 173:205-13. [PMID: 15210776 DOI: 10.4049/jimmunol.173.1.205] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Two mouse tumor cell lines, Meth A (BALB/c mouse-derived fibrosarcoma) and MM46 (C3H/He mouse-derived mammary tumor), were shown to express high levels of complement receptor-related gene y/p65 (Crry/p65), a membrane-bound complement-regulatory protein. Inhibiting the complement-regulatory activity of Crry/p65 with mAb 5D5 induced high levels of C3 deposition on in vivo tumor-derived Meth A and MM46 cells. To determine the effect of Crry/p65 blockade and increased C3 deposition on in vivo tumor growth, Meth A and MM46 cells were treated with 5D5 mAb and injected into BALB/c and C3H/He mice, respectively. Pretreating MM46 cells with 5D5 mAb significantly suppressed their tumorigenicity when injected s.c. Pretreatment with 5D5 mAb had a modest effect on Meth A s.c. tumor growth. Because complement is involved in the induction of an immune response, we investigated the effect of Crry/p65 blockade and increased C3 deposition on the immunogenicity of the tumor cells in a vaccination protocol. Vaccination of mice with irradiated Meth A cells pretreated with 5D5 mAb protected mice from subsequent challenge. In contrast, vaccination with irradiated Meth A cells without pretreatment was not protective. Survival was correlated with a high titer IgM response and specific CTL activity. These data demonstrate that the functional inhibition of Crry/p65 on tumor cells affects tumor growth and immunogenicity, and that the complement deposition resulting from this inhibition can act in concert with antitumor effector mechanisms to elicit potent antitumor immunity in vivo.
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Affiliation(s)
- Rieko Ohta
- Department of Biodefense, Nagoya City University Graduate School of Medical Sciences, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
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Shimo K, Mizuno M, Nasu J, Hiraoka S, Makidono C, Okazaki H, Yamamoto K, Okada H, Fujita T, Shiratori Y. Complement regulatory proteins in normal human esophagus and esophageal squamous cell carcinoma. J Gastroenterol Hepatol 2004; 19:643-7. [PMID: 15151618 DOI: 10.1111/j.1440-1746.2003.03328.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
BACKGROUND Altered expression of three complement regulatory proteins, decay-accelerating factor (CD55), membrane cofactor protein (CD46) and homologous restriction factor 20 (CD59) has been identified in human gastrointestinal malignancies, but their expression in esophageal cancer has not been described. Therefore the purpose of the present paper was to study the distribution of these proteins in human normal and malignant esophageal mucosa. METHODS AND RESULTS In the normal esophageal mucosa, CD55 predominantly stained on the cell membrane of squamous epithelium in the superficial and prickle cell layers, whereas CD46 most intensely stained on the cell membrane in the basal and parabasal cell layers. In contrast to this reciprocal expression of CD55 and CD46, CD59 was broadly distributed on the cell membrane in all layers. In the esophageal squamous cell carcinoma, CD55 staining was intense in the stroma but was negligible in the cancer cells. In contrast, CD46 and CD59 stained almost uniformly on the tumor cell membrane. There was a significant difference in the intensity of the staining of CD55 and CD46 among cells in various layers of normal esophageal mucosa and esophageal carcinoma cells, but not in the staining of CD59. Similar expression patterns of the three complement regulatory proteins in carcinoma cells and in normal epithelium in the basal and parabasal cell layers were observed. CONCLUSIONS These observations on the expression of the three complement regulatory proteins would help understanding of the host immune responses involving the complement system against esophageal squamous cell carcinoma.
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Affiliation(s)
- Kimihiro Shimo
- Department of Medicine and Medical Science (Medicine 1), Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
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Hiraoka S, Mizuno M, Nasu J, Okazaki H, Makidono C, Okada H, Terada R, Yamamoto K, Fujita T, Shiratori Y. Enhanced expression of decay-accelerating factor, a complement-regulatory protein, in the specialized intestinal metaplasia of Barrett's esophagus. ACTA ACUST UNITED AC 2004; 143:201-6. [PMID: 15085078 DOI: 10.1016/j.lab.2003.12.013] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Intestinal-type epithelium in Barrett's esophagus, so-called specialized intestinal metaplasia (SIM), is a risk factor for the development of esophageal adenocarcinoma. Surface expression of decay-accelerating factor (DAF), a complement-regulatory protein, is markedly enhanced in intestinal metaplasia of the gastric mucosa. We therefore examined DAF expression in areas of SIM in Barrett's esophagus in an attempt to determine whether DAF is a biomarker of SIM. We obtained 53 endoscopic biopsy specimens from the esophageal columnar mucosae of 45 patients. We immunohistochemically examined the distribution of DAF and 2 other complement-regulatory proteins: homologous restriction factor-20 and membrane cofactor protein. We also examined the expression of DAF messenger RNA in SIM with the use of laser-capture microdissection and reverse transcription-polymerase chain reaction. Of the 53 specimens, 10 were found histologically to involve areas of SIM, 41 were SIM-negative epithelium, and 2 comprised areas of SIM and SIM-negative epithelium. DAF staining was negligible in 35 of 43 specimens of the SIM-negative columnar epithelium, but DAF was strongly stained on the apical surface in all 12 SIM-positive specimens (P <.0001). In the 2 biopsy specimens in which both SIM and SIM-negative columnar epithelium were present, DAF staining was confined to the area of SIM. The expression of DAF messenger RNA was detected significantly more often in SIM than in SIM-negative columnar epithelium (P =.022). We conclude that DAF may be a surface marker for SIM and therefore useful in the identification of areas of the mucosa at risk for the development of adenocarcinoma in Barrett's esophagus.
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Affiliation(s)
- Sakiko Hiraoka
- Department of Medicine and Medical Science (Medicine 1), Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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Ahmad SR, Lidington EA, Ohta R, Okada N, Robson MG, Davies KA, Leitges M, Harris CL, Haskard DO, Mason JC. Decay-accelerating factor induction by tumour necrosis factor-alpha, through a phosphatidylinositol-3 kinase and protein kinase C-dependent pathway, protects murine vascular endothelial cells against complement deposition. Immunology 2003; 110:258-68. [PMID: 14511240 PMCID: PMC1783036 DOI: 10.1046/j.1365-2567.2003.01733.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have shown that human endothelial cells (EC) are protected against complement-mediated injury by the inducible expression of decay-accelerating factor (DAF). To understand further the importance of DAF regulation, we characterized EC DAF expression on murine EC in vitro and in vivo using a model of glomerulonephritis. Flow cytometry using the monoclonal antibody (mAb) Riko-3 [binds transmembrane- and glycosylphosphatidylinositol (GPI)-anchored DAF], mAb Riko-4 (binds GPI-anchored DAF) and reverse transcription-polymerase chain reaction (RT-PCR), demonstrated that murine EC DAF is GPI-anchored. Tumour necrosis factor-alpha (TNF-alpha) increased EC DAF expression, detectable at 6 hr and maximal at 24-48 hr poststimulation. DAF upregulation required increased steady-state DAF mRNA and protein synthesis. In contrast, no increased expression of the murine complement receptor-related protein-Y (Crry) was seen with TNF-alpha. DAF upregulation was mediated via a protein kinase C (PKC)alpha, phosphoinositide-3 kinase (PI-3 kinase), p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB)-dependent pathway. The increased DAF was functionally relevant, resulting in a marked reduction in C3 deposition following complement activation. In a nephrotoxic nephritis model, DAF expression on glomerular capillaries was significantly increased 2 hr after the induction of disease. The demonstration of DAF upregulation above constitutive levels suggests that this may be important in the maintenance of vascular integrity during inflammation, when the risk of complement-mediated injury is increased. The mouse represents a suitable model for the study of novel therapeutic approaches by which vascular endothelium may be conditioned against complement-mediated injury.
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Affiliation(s)
- Saifur R Ahmad
- British Heart Foundation Cardiovascular Medicine Unit, The Bywaters Centre, Imperial College London, Hammersmith Hospital, London, UK
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Kawai M, He L, Kawamura T, Omoto S, Fujii YR, Okada N. Chimeric human/murine monoclonal IgM antibodies to HIV-1 Nef antigen expressed on chronically infected cells. Microbiol Immunol 2003; 47:247-53. [PMID: 12725296 DOI: 10.1111/j.1348-0421.2003.tb03384.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV-1-infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV-1 infected cells could cause complement-mediated cytolysis. The trans-chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human mu-chain-positive Abs reactive with Nef fixed on an ELISA plate. However, the L-chain of the monoclonal Abs (mAbs) were murine lambda in type and were chimeric, and we could not succeed in obtaining mAb with human mu- and human kappa-chains. The chimeric mAbs reacted with the HIV-1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV-1-infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti-Nef IgM mAb with other IgM mAbs reactive with the surface of HIV-1-infected cells may induce a synergistic effect in complement mediated cytolysis.
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Affiliation(s)
- Masahiro Kawai
- Department of Biodefense, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601, Japan
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Iwagaki N, Mizuno M, Nasu J, Mizuno M, Okazaki H, Hori S, Yamamoto K, Okada H, Tsuji T, Fujita T, Shiratori Y. Advances in the development of a reliable assay for the measurement of stool decay-accelerating factor in the detection of colorectal cancer. J Immunoassay Immunochem 2003; 23:497-507. [PMID: 12458732 DOI: 10.1081/ias-120015480] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
We have previously shown that stool concentrations of decay-accelerating factor (DAF; CD55), a membrane-bound complement-regulatory protein, are significantly elevated in patients with colorectal cancer and that the measurement of stool DAF may be a valuable test for the detection of colorectal cancer. Accordingly, we are working to develop a clinically useful immunoassay for fecal DAF. A requirement for such assay is a plentiful and reliable supply of anti-DAF antibodies. We developed a sandwich enzyme-linked immunosorbent assay (ELISA) for DAF in stool specimens, using two monoclonal anti-DAF antibodies recognizing different epitopes on the DAF molecule. When we first used a biotin-labeled antibody and enzyme-linked streptavidin method, we often observed stool interference, probably due to the presence of a substance(s) with biotin activity which non-specifically bound to the Fc portion of IgG of the first anti-DAF antibody on the ELISA wells. By the use of inorganic salts in the sample-dilution buffer and HRP-labeled anti-DAF as second antibody, we circumvented the stool interference and established that the new ELISA system could reliably measure DAF at low concentrations in stool specimens. Because the new assay system uses only monoclonal antibodies, we can now consistently supply ample amounts of antibodies for routine measurement of stool DAF.
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Affiliation(s)
- Naofumi Iwagaki
- Department of Medicine and Medical Science (Medinen 1). Okayama University Graduate School of Medicine and Dentistry, 2-5-1, Shikata-cho, Okayama 700-8558, Japan
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Kakinuma Y, Endo Y, Takahashi M, Nakata M, Matsushita M, Takenoshita S, Fujita T. Molecular cloning and characterization of novel ficolins from Xenopus laevis. Immunogenetics 2003; 55:29-37. [PMID: 12679857 DOI: 10.1007/s00251-003-0552-2] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2002] [Revised: 02/03/2003] [Indexed: 12/01/2022]
Abstract
Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins and are thought to play crucial roles in host defense through opsonization and complement activation. To elucidate the evolution of ficolins and the primordial complement lectin pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its lectin activity.
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Affiliation(s)
- Yuji Kakinuma
- Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikarigaoka, 960-1295, Fukushima, Japan
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Pausa M, Pellis V, Cinco M, Giulianini PG, Presani G, Perticarari S, Murgia R, Tedesco F. Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2003; 170:3214-22. [PMID: 12626580 DOI: 10.4049/jimmunol.170.6.3214] [Citation(s) in RCA: 76] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.
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Affiliation(s)
- Mario Pausa
- Department of Physiology and Pathology, University of Trieste, Trieste, Italy
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Ohi H, Tamano M, Sudo S, Okada N. Recombinant EPO therapy increases erythrocyte expression of complement regulatory proteins. Am J Kidney Dis 2003; 41:179-85. [PMID: 12500235 DOI: 10.1053/ajkd.2003.50002] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
BACKGROUND One of the complications of hemodialysis (HD) therapy is anemia caused by erythropoietin (EPO) deficiency. Recombinant EPO (rEPO) has been used routinely as a supplemental treatment. Erythrocyte expression of the complement regulatory proteins decay accelerating factor (DAF) and CD59 restricts complement activation and inhibits hemolysis. We hypothesized that the efficacy of rEPO treatment may be caused in part by the ability of rEPO to increase erythrocyte expression of DAF and CD59. METHODS DAF, CD59, and complement receptor 1 (CR1) levels were analyzed for a group of 95 HD patients and compared with those of a control group. To evaluate effects of discontinuation of rEPO therapy, rEPO therapy was stopped for 12 HD patients until hematocrits decreased to less than 25%. DAF and CD59 levels then were reanalyzed. RESULTS In the 95 HD patients, three factors correlated significantly: DAF and CD59 (r = 0.642), DAF and CR1 (r = 0.503), and CD59 and CR1 (r = 0.324), whereas no correlations were found in the group of 42 healthy controls. In the experiment in which rEPO therapy was discontinued, 8 of 12 patients reached the defined level of anemia 4 to 7 weeks after rEPO treatment had been withheld. Both DAF and CD59 levels decreased significantly after discontinuation of rEPO therapy (P < 0.01). DAF and CD59 levels increased in all 8 patients after rEPO treatment was reinitiated (P < 0.01), and CR1 levels increased in 5 of 8 patients. Four of 12 patients showed no evidence of anemia after discontinuation of rEPO treatment. In these patients, DAF, CD59, and CR1 levels did not change before or after withholding rEPO therapy. CONCLUSION One of the mechanisms mediating the efficacy of EPO therapy is increased DAF and CD59 expression.
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Affiliation(s)
- Hiroyuki Ohi
- Department of Internal Medicine II, Nihon University School of Medicine, Tokyo, Japan.
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Miwa T, Zhou L, Hilliard B, Molina H, Song WC. Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack. Blood 2002; 99:3707-16. [PMID: 11986227 DOI: 10.1182/blood.v99.10.3707] [Citation(s) in RCA: 67] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Decay-accelerating factor (DAF) and CD59 are 2 glycosylphosphatidylinositol-anchored membrane proteins that inhibit complement activation at the C3 and C5b-9 step, respectively. CD59 is considered critical for protecting erythrocytes from spontaneous complement attack, as deficiency of CD59 or CD59/DAF, but not of DAF alone, on human erythrocytes renders them sensitive to complement lysis in paroxysmal nocturnal hemoglobinuria syndrome. To evaluate the relative roles of CD59 and DAF in vivo, we have generated and studied a CD59 knockout and a CD59/DAF double-knockout mouse. CD59-deficient and CD59/DAF-double-deficient mouse erythrocytes were highly sensitive to antibody-induced complement lysis in vitro, yet neither CD59 knockout nor CD59/DAF double-knockout mouse developed spontaneous hemolytic anemia. Consistent with the latter observation, erythrocytes from the 2 strains of mutant mice were shown to have a normal lifespan in vivo. In contrast, mouse erythrocytes deficient in complement receptor 1 (CR1)-related gene y (Crry), a membrane C3 inhibitor with DAF and membrane cofactor protein activities, were rapidly eliminated from the circulation by a complement-dependent mechanism. Compared with DAF-deficient erythrocytes, Crry-deficient erythrocytes incurred higher levels of spontaneous C3 deposition in vivo. These findings demonstrate that CD59 and DAF are not indispensable on murine erythrocytes. Rather, effective C3 regulation on the cell surface, provided by Crry rather than DAF, is necessary for mouse erythrocytes to resist spontaneous complement attack. Our results raise the possibility that proper control of C3 activation may also be critical on human erythrocytes, where CR1 but not DAF could be the principal regulator of spontaneous C3 activation.
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Affiliation(s)
- Takashi Miwa
- Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA
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Lin F, Emancipator SN, Salant DJ, Medof ME. Decay-accelerating factor confers protection against complement-mediated podocyte injury in acute nephrotoxic nephritis. J Transl Med 2002; 82:563-9. [PMID: 12003997 DOI: 10.1038/labinvest.3780451] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
SUMMARY Decay-accelerating factor (DAF or CD55) is one of a set of regulators that function to protect self cells from deposition of autologous C3b on their surfaces. Its relative importance in vivo, however, is incompletely understood. As one approach to address this issue, we induced nephrotoxic serum (NTS) nephritis in wild-type mice and Daf1 gene-floxed mice devoid of renal DAF expression. For these experiments NTS IgG was administered at a dose (0.5 mg iv) that requires complement for glomerular injury. After 18 hours, renal injury was assessed by proteinuria and by histologic, immunohistochemical, and electron microscopic analyses of kidneys. Fifteen normal and 15 DAF-deficient mice were studied. Baseline albuminuria in the Daf1(-/-) mice was 115.9 +/- 41.4 microg/mg creatinine as compared with 85.7 +/- 32.3 microg/mg creatinine in their Daf1(+/+) littermates (p = 0.075). After administration of NTS IgG, albuminuria increased to 2001.7 +/- 688.7 microg/mg creatinine as compared with 799.7 +/- 340.5 microg/mg creatinine in the controls (p = 0.0003). Glomerular histology was similar in Daf1(-/-) and Daf1(+/+) mice, with essentially no infiltrating leukocytes. In contrast, electron microscopy revealed severe podocyte fusion in the Daf1(-/-) mice but only mild focal changes in the controls. Immunohistochemical staining showed equivalent deposition of the administered (sheep) NTS IgG in the Daf1(-/-) and Daf1(+/+) animals. This contrasted with marked deposition of autologous murine C3 in the former and minimal deposition in the latter. The results show that DAF is essential physiologically for protecting glomeruli against autologous complement attack initiated by the classical pathway.
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Affiliation(s)
- Feng Lin
- Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA
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37
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Kiso T, Mizuno M, Nasu J, Shimo K, Uesu T, Yamamoto K, Okada H, Fujita T, Tsuji T. Enhanced expression of decay-accelerating factor and CD59/homologous restriction factor 20 in intestinal metaplasia, gastric adenomas and intestinal-type gastric carcinomas but not in diffuse-type carcinomas. Histopathology 2002; 40:339-47. [PMID: 11943018 DOI: 10.1046/j.1365-2559.2002.01350.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
AIMS Variable expression of the complement regulatory proteins, decay-accelerating factor, CD59/homologous restriction factor 20 (HRF20) and membrane cofactor protein has been shown in human gastrointestinal malignancies, but their expression in gastric cancer has not been fully described. Thus, we immunohistochemically defined the distribution of these proteins in human normal gastric mucosa, intestinal metaplasia, adenomas and gastric cancers. METHODS AND RESULTS Gastric tissues were obtained by endoscopic biopsy or surgical resection and stained with mouse monoclonal antibodies to decay-accelerating factor, CD59/HRF20, and membrane cofactor protein. In the normal gastric mucosa, membrane cofactor protein was diffusely stained on the basolateral surface of epithelial cells, whereas the expression of decay-accelerating factor and CD59/HRF20 was inconspicuous. In intestinal metaplasia, adenoma and intestinal-type gastric carcinoma cells, decay-accelerating factor and HRF20 were intensely stained on the apical surface; membrane cofactor protein retained its location on the basolateral surface. In diffuse-type gastric carcinomas, the expression of decay-accelerating factor, CD59/HRF20 was lost, but membrane cofactor protein was present on the tumour cell surface. CONCLUSIONS These findings suggest that membrane cofactor protein plays a primary role in the regulation of complement activation in normal and neoplastic gastric cells and that the expression pattern of the complement regulatory proteins is closely related to gastric carcinoma development.
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Affiliation(s)
- T Kiso
- Department of Medicine and Medical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
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Maeda K, Hayashi S, Tanioka Y, Matsumoto Y, Otsuka H. Pseudorabies virus (PRV) is protected from complement attack by cellular factors and glycoprotein C (gC). Virus Res 2002; 84:79-87. [PMID: 11900841 DOI: 10.1016/s0168-1702(01)00417-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Swine kidney derived CPK cells were resistant to swine complement attack in vitro while rabbit kidney derived RK13 cells were destroyed by swine complement. To rabbit complement, RK13 cells were resistant but CPK cells were sensitive. This phenomenon was known as homologous restriction (Proc. Natl. Acad. Sci. USA 78 (1981) 5118). The gC deletion mutant of pseudorabies virus (PRVdlgC) grown in CPK cells was resistant to swine complement while the same virus grown in RK13 cells was neutralized by swine complement. PRVdlgC grown in RK13 cells was more resistant to rabbit complement than the virus grown in CPK cells. Hence, the sensitivity of PRVdlgC to swine or rabbit complement was similar to that of the cells in which the virus was grown. It would appear that cell derived factors were present on the virion and they were protective against homologous complement but not against heterologous complement. The expression of gC rendered PRV more resistant to swine or rabbit complement, but the protective effect of gC was much less than that of cell derived factors. The best protection against complement was obtained when gC and cell derived factors were coexistent on the virion.
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Affiliation(s)
- Kohshi Maeda
- Department of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
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Farkas I, Baranyi L, Ishikawa Y, Okada N, Bohata C, Budai D, Fukuda A, Imai M, Okada H. CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9. J Physiol 2002; 539:537-45. [PMID: 11882685 PMCID: PMC2290142 DOI: 10.1113/jphysiol.2001.013381] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59(-) human B-cells, which formed non-selective ion channels with a total conductance of 360 +/- 24 pS as measured at the beginning of the steady-state phase of the inward currents. C5b-8 and small-size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59(+) human B-cells in spite of small-size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59(-) cells inhibited the serum-evoked inward current. The ion channels formed by the small-size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b-8 as well as that of small-size C5b-9. These results offer an explanation as to why CD59-expressing cells are not leaky in spite of a buildup of homologous C5b-8 and small-size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.
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Affiliation(s)
- Imre Farkas
- Department of Molecular Biology, Nagoya City University School of Medicine, Nagoya 467-8601, Japan
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Sampaziotis F, Kokotas S, Gorgoulis VG. P53 possibly upregulates the expression of CD58 (LFA-3) and CD59 (MIRL). Med Hypotheses 2002; 58:136-40. [PMID: 11812190 DOI: 10.1054/mehy.2001.1476] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
P53 is an oncosuppressor protein which acts via transcriptional and non-transcriptional mechanisms. The transcriptional function of p53 is mediated by specific responsive elements (RE). In the present study we found one perfect p53 RE located in the first intron of the gene encoding for CD58 (lymphocyte function Antigen-3/LFA-3) membrane protein. Additionally, we detected one perfect p53 RE within the promoter region and one imperfect p53 RE placed in the first intron of the gene encoding for CD59 (membrane inhibitor of reactive lysis/MIRL). The results of our investigation suggest that p53 may enhance the transcription of both CD59 and CD58 and imply a novel role for p53 as a direct regulator of the immune response.
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Affiliation(s)
- F Sampaziotis
- Department of Histology and Embryology, Medical School, University of Athens, Greece
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Bardenstein DS, Cheyer CJ, Lee C, Cocuzzi E, Mizuno M, Okada N, Medof ME. Blockage of complement regulators in the conjunctiva and within the eye leads to massive inflammation and iritis. Immunology 2001; 104:423-30. [PMID: 11899428 PMCID: PMC1783320 DOI: 10.1046/j.1365-2567.2001.01316.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The open environment of the eye is continuously subject to an influx of foreign agents that can activate complement. Decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 are regulators that protect self-cells from autologous complement activation on their surfaces. They are expressed in the eye at unusually high levels but their physiological importance in this site is unstudied. In the rat, a structural analogue termed 5I2 antigen (5I2 Ag) has actions overlapping DAF and MCP. In this investigation, we injected F(ab')2 fragments of 5I2 mAb into the conjunctiva and aqueous humor, in the latter case with and without concomitant blockage of CD59. Massive neutrophilic infiltration of the stroma and iris resulted upon blocking 5I2 Ag activity. Frank necrosis of the iris occurred upon concomitant intraocular blockage of CD59. C3b was identified immunohistochemically, and minimal effects were seen in complement-depleted animals and in those treated with non-relevant antibody. The finding that blockage of 5I2 Ag function in periocular tissues and within the eye causes intense conjunctival inflammation and iritis demonstrates the importance of intrinsic complement regulators in protecting ocular tissues from spontaneous or bystander attack by autologous complement.
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Affiliation(s)
- D S Bardenstein
- Institute of Pathology, Center for Vision Research, Case Western Reserve University School of Medicine, Cleveland, OH, USA
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42
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Huang J, Gou D, Zhen C, Jiang D, Mao X, Li W, Chen S, Cai C. Protection of xenogeneic cells from human complement-mediated lysis by the expression of human DAF, CD59 and MCP. FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 2001; 31:203-9. [PMID: 11720816 DOI: 10.1111/j.1574-695x.2001.tb00521.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.
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Affiliation(s)
- J Huang
- College of Life Science, Wuhan University, Wuhan439972, PR China.
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Lin F, Fukuoka Y, Spicer A, Ohta R, Okada N, Harris CL, Emancipator SN, Medof ME. Tissue distribution of products of the mouse decay-accelerating factor (DAF) genes. Exploitation of a Daf1 knock-out mouse and site-specific monoclonal antibodies. Immunology 2001; 104:215-25. [PMID: 11683962 PMCID: PMC1783297 DOI: 10.1046/j.1365-2567.2001.01287.x] [Citation(s) in RCA: 79] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2001] [Revised: 05/29/2001] [Accepted: 06/04/2001] [Indexed: 11/20/2022] Open
Abstract
Decay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembrane-anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock-out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure.
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Affiliation(s)
- F Lin
- Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA
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Lin F, Immormino RM, Shoham M, Medof ME. Bulk production and functional analyses of mouse CD55's native and deglycosylated active domains. Arch Biochem Biophys 2001; 393:67-72. [PMID: 11516162 DOI: 10.1006/abbi.2001.2488] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
We report the use of methylotrophic yeast Pichia pastoris as a host to efficiently express complement control protein repeats (CCPs) 1-4 of mouse decay accelerating factor (DAF, CD55) as a soluble protein. With this system, the mouse DAF CCP1-4-active-domain-containing module linked to a 6x His tag at its C terminus was secreted into the culture supernatant at 15 mg/L after 24 h of induction with methanol. A mouse DAF CCP1-4 mutant protein in which its two potential N-glycosylation sites were deleted by changing Asn(187) and Asn(262) to Gln was also produced. Using Ni(2+)-immobilized agarose affinity chromatography, the recombinant mouse DAF modules with their 6x His tags could be one-step isolated to SDS-PAGE purity. Polyclonal antibody against native mouse DAF CCP1-4 was raised by immunizing NZW rabbits with the purified product. Measurements of the bioactivities of the wild-type and mutant mouse DAF proteins in C3b uptake assays showed no differences in regulatory activities in either the classical or the alternative pathways. With the use of the mutant DAF protein, small rod-shaped crystals were produced and preliminary data obtained. The production of large quantities of functional recombinant mouse DAF CCP1-4 modules and their antibody offers the opportunity to study DAF structure and DAF function in vivo.
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Affiliation(s)
- F Lin
- Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
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Sogabe H, Nangaku M, Ishibashi Y, Wada T, Fujita T, Sun X, Miwa T, Madaio MP, Song WC. Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2001; 167:2791-7. [PMID: 11509624 DOI: 10.4049/jimmunol.167.5.2791] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 +/- 25.0 vs 10.0 +/- 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 +/- 68 vs 325 +/- 18 x 10(3) microm(3) per glomerulus; p < 0.0001) and cellularity (47.1 +/- 8.9 vs 32.0 +/- 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 +/- 0.7 vs 0.02 +/- 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.
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Affiliation(s)
- H Sogabe
- Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan
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Miwa T, Song WC. Membrane complement regulatory proteins: insight from animal studies and relevance to human diseases. Int Immunopharmacol 2001; 1:445-59. [PMID: 11367529 DOI: 10.1016/s1567-5769(00)00043-6] [Citation(s) in RCA: 115] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The complement system plays an important role in host defense. However, if not properly regulated, activated complement can also cause significant damage to host tissues. To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement regulatory proteins. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59. Recent studies of membrane complement regulatory proteins from various animal species have revealed similarities as well as significant differences from the corresponding human proteins. In this review, we summarize recent advances in this area and contrast the structure, function and tissue distribution of membrane complement regulatory proteins in human and nonprimate mammalian species. We also discuss how the characterization of the animal proteins has provided important clues and might continue to show relevance to the pathogenesis and therapeutics of a number of human diseases.
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Affiliation(s)
- T Miwa
- Centre for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, 1351 BRBII-III, 421 Curie Blvd., Philadelphia, PA 19104, USA
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Arora M, Arora R, Tiwari SC, DAS1 N, SRIVASTAVA1 LM. Expression of complement regulatory proteins on erythrocytes from patients with idiopathic focal segmental glomerulosclerosis. Nephrology (Carlton) 2001. [DOI: 10.1046/j.1440-1797.2001.00020.x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Affiliation(s)
- Meenakshi Arora
- Department of Biochemistry, All India Institute of Medical Sciences, School of Life Sciences, Jawahar Lal University and Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India
| | - Reetakshi Arora
- Department of Biochemistry, All India Institute of Medical Sciences, School of Life Sciences, Jawahar Lal University and Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India
| | - S C Tiwari
- Department of Biochemistry, All India Institute of Medical Sciences, School of Life Sciences, Jawahar Lal University and Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India
| | - Nibhriti DAS1
- Department of Biochemistry, All India Institute of Medical Sciences, School of Life Sciences, Jawahar Lal University and Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India
| | - L M SRIVASTAVA1
- Department of Biochemistry, All India Institute of Medical Sciences, School of Life Sciences, Jawahar Lal University and Department of Nephrology, All India Institute of Medical Sciences, New Delhi, India
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Watanabe M, Morita Y, Mizuno M, Nishikawa K, Yuzawa Y, Hotta N, Morgan BP, Okada N, Okada H, Matsuo S. CD59 protects rat kidney from complement mediated injury in collaboration with crry. Kidney Int 2000; 58:1569-79. [PMID: 11012891 DOI: 10.1046/j.1523-1755.2000.00318.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
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.
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Affiliation(s)
- M Watanabe
- Internal Medicine III, Nagoya University School of Medicine, Nagoya, Japan
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Qian YM, Qin X, Miwa T, Sun X, Halperin JA, Song WC. Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2000; 165:2528-34. [PMID: 10946279 DOI: 10.4049/jimmunol.165.5.2528] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.
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Affiliation(s)
- Y M Qian
- Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
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Arora M, Arora R, Tiwari SC, Das N, Srivastava LM. Expression of complement regulatory proteins in diffuse proliferative glomerulonephritis. Lupus 2000; 9:127-31. [PMID: 10787010 DOI: 10.1191/096120300678828154] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
This study assessed the expression of complement receptor 1 (CR1), decay accelerating factor (DAF) and membrane inhibitor of reactive lysis (CD59) on the erythrocytes and glomerulus of diffuse proliferative glomerulonephritis (DPGN) of systemic lupus erythematosus (SLE) patients using flow cytometry and immunofluorescence techniques to elucidate their role in the pathogenesis of DPGN. Expression of CR1 on the erythrocytes and glomerulus of DPGN patients was reduced compared with expression in normal subjects. However, expression of DAF and CD59 was increased on both erythrocytes and glomerulus of DPGN patients, suggesting the generation of a protective response against complement-mediated injury.
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Affiliation(s)
- M Arora
- Department of Biochemistry, All India Institute of Medical Sciences, New Delhi.
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