The IgG-specific endoglycosidase EndoS inhibits both cellular and complement-mediated autoimmune hemolysis.

EndoS from Streptococcus pyogenes is an immunomodulating enzyme that specifically hydrolyzes glycans from human immunoglobulin G and thereby affects antibody effector functions. Autoimmune hemolytic anemia is caused by antibody-mediated red blood cell (RBC) destruction and often resists treatment with corticosteroids that also cause frequent adverse effects. We show here that anti-RhD (anti-D) and rabbit anti-human-RBC antibodies (anti-RBC) mediated destruction of RBC, ie, phagocytosis, complement activation, and hemolysis in vitro and in vivo was inhibited by EndoS. Phagocytosis by monocytes in vitro was inhibited by pretreatment of anti-D with EndoS before sensitization of RBCs and abrogated by direct addition of EndoS to blood containing sensitized RBCs. The toxic effects of monocytes stimulated with anti-D-sensitized RBCs, as measured by interleukin-8 secretion and oxygen metabolite production, was restrained by EndoS. Agglutination of RBCs and complement-mediated hemolysis in vitro in whole human blood caused by rabbit anti-RBCs was inhibited by EndoS. Development of anemia in mice caused by a murine anti-RBC immunoglobulin G2a monoclonal autoantibody and complement activation and erythrophagocytosis by Kupffer cells in the liver were reduced by EndoS. Our data indicate that EndoS is a potential therapeutic agent that might be evaluated as an alternative to current treatment regimens against antibody-mediated destruction of RBCs.

[1]  G. Daniels.  Naming blood groups and the genes that control them , 2009 .

[2]  J. Ravetch,et al.  In vivo enzymatic modulation of IgG glycosylation inhibits autoimmune disease in an IgG subclass-dependent manner , 2008, Proceedings of the National Academy of Sciences.

[3]  L. Björck,et al.  IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions , 2008, Proceedings of the National Academy of Sciences.

[4]  J. Ravetch,et al.  Differential Contribution of Three Activating IgG Fc Receptors (FcγRI, FcγRIII, and FcγRIV) to IgG2a- and IgG2b-Induced Autoimmune Hemolytic Anemia in Mice1 , 2008, The Journal of Immunology.

[5]  Maria Allhorn,et al.  Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis , 2008, PloS one.

[6]  Henrik Clausen,et al.  Modifying the red cell surface: towards an ABO‐universal blood supply , 2007, British journal of haematology.

[7]  A. Blom,et al.  Endoglycosidase treatment abrogates IgG arthritogenicity: Importance of IgG glycosylation in arthritis , 2007, European journal of immunology.

[8]  S. Rogerson,et al.  A novel flow cytometric phagocytosis assay of malaria-infected erythrocytes. , 2007, Journal of immunological methods.

[9]  Maria Kavai,et al.  Immune complex clearance by monocytes and macrophages in systemic lupus erythematosus. , 2007, Autoimmunity reviews.

[10]  J. Ravetch,et al.  Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity , 2007, Proceedings of the National Academy of Sciences.

[11]  William Arbuthnot Sir Lane,et al.  Bacterial glycosidases for the production of universal red blood cells , 2007, Nature Biotechnology.

[12]  S. Holland,et al.  Persistent bacterial infections and primary immune disorders. , 2007, Current opinion in microbiology.

[13]  J. Ravetch,et al.  Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation , 2006, Science.

[14]  J. Lundahl,et al.  Characterization of red cell autoantibodies in consecutive DAT-positive patients with relation to in vivo haemolysis , 2005, Annals of Hematology.

[15]  Mattias Collin,et al.  A Novel Secreted Endoglycosidase from Enterococcus faecalis with Activity on Human Immunoglobulin G and Ribonuclease B* , 2004, Journal of Biological Chemistry.

[16]  G. Garratty,et al.  Immune Hemolytic Anemias , 2004 .

[17]  J. Jensenius,et al.  EndoS and SpeB from Streptococcus pyogenes Inhibit Immunoglobulin-Mediated Opsonophagocytosis , 2002, Infection and Immunity.

[18]  M. Walport,et al.  Complement Activation Selectively Potentiates the Pathogenicity of the IgG2b and IgG3 Isotypes of a High Affinity Anti-Erythrocyte Autoantibody , 2002, The Journal of experimental medicine.

[19]  A. Olsén,et al.  Effect of SpeB and EndoS from Streptococcus pyogenes on Human Immunoglobulins , 2001, Infection and Immunity.

[20]  M. Walport,et al.  Reconstitution of the Complement Function in C1q-Deficient (C1qa−/−) Mice with Wild-Type Bone Marrow Cells1 , 2001, The Journal of Immunology.

[21]  B. Flesch,et al.  Fcγ receptor‐mediated immune phagocytosis depends on the class of FcγR and on the immunoglobulin‐coated target cell , 2001, Vox sanguinis.

[22]  A. Olsén,et al.  EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG , 2001, The EMBO journal.

[23]  Poole,et al.  A chemiluminescence test for predicting the outcome of transfusing incompatible blood , 1999, Transfusion medicine.

[24]  R. Schmidt,et al.  FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia , 1998 .

[25]  R. Sokol,et al.  The glycosylation of red cell autoantibodies affects their functional activity in vitro , 1995, British journal of haematology.

[26]  M. Goodrick,et al.  Human Rh D monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D+ red blood cells and suppression of Rh D immunization in Rh D- volunteers. , 1995, Blood.

[27]  J. Ravetch,et al.  Cytotoxic antibodies trigger inflammation through Fc receptors. , 1995, Immunity.

[28]  R. Davenport.  Cytokines and erythrocyte incompatibility. , 1994, Current opinion in hematology.

[29]  M. Burdick,et al.  In vitro production of interleukin‐1 receptor antagonist in IgG‐ mediated red cell incompatibility , 1994, Transfusion.

[30]  G. Szücs,et al.  Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes. , 1991, Immunology.

[31]  M. Frank,et al.  High doses of intravenous immunoglobulin do not affect the recognition phase of the classical complement pathway , 1991 .

[32]  T. Standiford,et al.  Interleukin-8 production in red blood cell incompatibility. , 1990, Blood.

[33]  T. Berney,et al.  Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause autoimmune hemolytic anemia by two distinct pathogenic mechanisms. , 1990, International immunology.

[34]  M. Malaise,et al.  Effects of mannose and mannose derivatives on the clearance of IgG antibody-coated erythrocytes in the rat. , 1989, Immunology.

[35]  M. Malaise,et al.  The ability of normal human monocytes to phagocytose IgG-coated red blood cells is related to the number of accessible galactosyl and mannosyl residues in the Fc domain of the anti-red blood cell IgG antibody molecules. , 1989, Journal of immunological methods.

[36]  G. Garratty,et al.  Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay , 1987, Transfusion.

[37]  P. Arese,et al.  Measurement of phagocytosis utilizing [14C]cyanate‐labelled human red cells and monocytes , 1987, British journal of haematology.

[38]  M. Frank,et al.  Role of antibody and complement in the immune clearance and destruction of erythrocytes. I. In vivo effects of IgG and IgM complement-fixing sites. , 1972, The Journal of clinical investigation.

[39]  P. Valent,et al.  Diagnosis and treatment of autoimmune haemolytic anaemias in adults: a clinical review , 2008, Wiener klinische Wochenschrift.

[40]  J. Ravetch,et al.  Differential contribution of three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) to IgG2a- and IgG2b-induced autoimmune hemolytic anemia in mice. , 2008, Journal of immunology.

[41]  J. Ravetch,et al.  Fc-receptors as regulators of immunity. , 2007, Advances in immunology.

[42]  M. Uchikawa,et al.  Gene organization and rearrangements at the human Rhesus blood group locus revealed by fiber-FISH analysis , 2000, Human Genetics.

[43]  R. Schmidt,et al.  FcgammaRIII (CD16)-deficient mice show IgG isotype-dependent protection to experimental autoimmune hemolytic anemia. , 1998, Blood.

[44]  B. Kumpel,et al.  The biological activity of human monoclonal IgG anti-D is reduced by beta-galactosidase treatment. , 1995, Human antibodies and hybridomas.

[45]  A. Tarentino,et al.  Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. , 1994, Methods in enzymology.

[46]  M. Baggiolini,et al.  Neutrophil activation and the effects of interleukin-8/neutrophil-activating peptide 1 (IL-8/NAP-1). , 1992, Cytokines.

[47]  D. Wakefield,et al.  The role of cytokines in the pathogenesis of inflammatory eye disease. , 1992, Cytokine.

[48]  A. Bradwell,et al.  Rat Monoclonal Antibodies against Mouse Immunoglobulins , 1988 .

[49]  M. Pierres,et al.  Rat monoclonal antibodies to mouse IgG1, IgG2a, IgG2b, and IgG3 subclasses, and kappa chain isotypic determinants. , 1984, Hybridoma.