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

Abstract

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.

Figure 1

Treatment of anti-D with EndoS does not affect the binding of anti-D to RBCs. (A) Western blot analysis of anti-D on RBCs using peroxidase-conjugated anti–human IgG. (B) Glycan hydrolysis of anti-D on RBCs determined by lectin ELISA (optical density [OD] = 450 nm). (C) Quantification of RBC-bound anti-D by ELISA (OD = 450 nm) using antiserum against human IgG.

Figure 2

RBCs sensitized with anti-D IgG pretreated with EndoS are not attached to, or phagocytosed by, monocytes. (A) The degree of adherence/phagocytosis of RBCs sensitized with anti-D determined by monocyte monolayer assay and expressed as a number of monocytes with one or more adherent/phagocytosed RBCs of 100 monocytes detected. (B) The appearance of “rosettes” showed using Grünwald-Giemsa staining of monocyte monolayers incubated with RBCs sensitized with anti-D (top panel) and anti-D pretreated with EndoS (bottom panel; original magnification ×60). (C) Phagocytosis of sensitized RBCs by THP-1 cells and blood monocytes with or without treatment of anti-D with EndoS. The results are presented as the relative amount of internalized hemoglobin in cells detected by reactivity with 2,7-diaminofluorene. (D) Resuspended monocytes after incubation with RBCs sensitized with anti-D followed by the removal of noningested RBCs. (Left) RBCs sensitized with anti-D. (Right) Anti-D pretreated with EndoS. (E). Purified blood monocytes were incubated with FITC-labeled RBCs sensitized with anti-D and the degree of ingested RBCs determined by measuring the fluorescence.

Figure 3

EndoS inhibits metabolic oxygen responses induced by RBCs sensitized with anti-D immune plasma. Metabolic oxygen responses from monocytes were analyzed by measuring the chemiluminescence after addition of RBC-sensitized anti-D-positive plasma (with or without treatment with EndoS) and luminol, at 5-minute intervals for 1 hour. y-axis represents opsonic index, ie, the ratio of monocyte responses to sensitized RBCs versus unsensitized RBCs. control RBC indicates RBCs sensitized with nonimmune plasma.

Figure 4

EndoS inhibits secretion of IL-8 by monocytes induced by RBCs sensitized with anti-D. Relative quantification of IL-8 secretion in supernatants from THP-1 cells or blood monocytes incubated with RBCs sensitized with anti-D for 3 hours, with or without pretreatment of anti-D with EndoS. (Inset) IL-8 incubated with EndoS followed by separation on SDS-PAGE and staining with Coomassie Blue.

Figure 5

Hydrolysis of anti–human RBC-IgG by EndoS. (A) Rabbit antiserum against human RBCs was treated with EndoS or PBS and used for sensitization of RBCs. RBC-bound IgG was determined by solubilization of sensitized RBCs followed by Western blot using antiserum rabbit IgG. (B) Glycan hydrolysis of RBC-bound IgG determined by lectin ELISA (OD = 450 nm). (C) RBC-bound antibody was quantified by ELISA (OD = 450 nm) using antiserum against rabbit IgG.

Figure 6

EndoS inhibits hemolysis and agglutination of sensitized RBCs and prevents binding of C1q to sensitized RBCs. (A) Relative amount of hemoglobin quantified by 2,7-diaminofluorene in plasma after incubation of human blood with antiserum against human RBCs, pretreated with EndoS or not. ⁽¹⁾Direct addition of EndoS to blood containing RBC antiserum. ⁽²⁾EndoS alone added to blood. NS indicates not significant. (B) Blood smears. (Top panel) A total of 80 μg of anti-RBC IgG added to 700 μL human blood, diluted 35 times in PBS, and incubated 1 hour at 37°C. (Middle panel) Anti-RBC treated with EndoS before addition to blood. (Bottom panel) No addition of antibodies. (C) Relative C1q deposition onto IgG-sensitized RBCs detected using FITC-conjugated anti–human C1q.

Figure 7

EndoS decreases the development of AIHA in BALB/c mice pretreated with 34-3C anti-RBC mAb. BALB/c mice were intravenously injected with 34-3C anti-RBC mAb; and 24 hours later, 20 μg of EndoS was intravenously injected. (A) Hematocrit values of individual mice treated with EndoS (○) or PBS (●) were measured 1, 2, 4, 6, and 8 days after injection of 200 μg 34-3C IgG2b mAb. Horizontal lines indicate mean values. (B). Representative histologic appearance of iron deposits in Kupffer cells from mice injected with 34-3C IgG2b anti-RBC mAb and then with EndoS. Note substantial reduction of iron deposits in livers from mice treated with EndoS, compared with completely untreated mice (original magnification ×200). (Top right panel) Higher magnification of iron deposits as indicated by arrows. (C) BALB/c mice were intravenously injected with 300 μg of 34-3C IgG1 or 200 μg of 34-3C IgG2a class-switch variant; and 24 hours later, 20 μg of EndoS was intravenously injected. Hematocrit values of individual mice treated with EndoS (○) or PBS (●) were measured 4 days after injection of 34-3C mAb. Horizontal lines indicate mean values. The normal range of hematocrit values (mean ± 3 SD) of 2- to 3-month-old BALB/c mice is represented as a shaded area.