Contributions
Abstract: S1586
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 08:15 - 08:30
Location: Room A9
Background
Autoimmune hemolytic anemia (AIHA) is a rare disease characterized by autoantibodies against erythrocytes. These autoantibodies may activate the classical complement pathway leading to opsonization by complement proteins C3b and C4b. This results in increased clearance of erythrocytes by phagocytes, called extravascular hemolysis. Occasionally, complement activation results in formation of the membrane attack complex (MAC), causing intravascular hemolysis. C3-inhibitor compstatin inhibits C3 activation and thus it is expected to reduce both, C3b deposition as well as well as formation of the MAC. Therefore, compstatin is a potentially efficient inhibitor of extra- and intravascular hemolysis in AIHA.
Aims
The current aim of this research is to investigate in vitro whether compstatin would be a suitable drug for AIHA treatment.
Methods
Healthy donor erythrocytes treated with bromelin were incubated with AIHA patient serum together with anti-C5 so that opsonization could be analyzed by FACS using anti-C3-FITC and anti-C4-APC antibodies. In the absence of eculizumab, hemolysis of RBCs was tested by measuring the extinction at 412/690 nm. The percentage hemolysis was calculated relative to the 100% water control. To assess the effect of compstatin cp40 on uptake of erythrocytes by phagocytes, erythrocytes were fluorescently labeled with PKH26 before opsonization. Monocytes isolated by leukapheresis were cultured for 8 or 9 days in the presence of 10 ng/mL GM-CSF to obtain M1-like macrophages or 50 ng/mL M-CSF to obtain M2-like macrophages. Opsonized erythrocytes were then incubated with healthy monocyte derived macrophages and phagocytosis of erythrocytes was measured with ImageStream after lysing the non-phagocytosed erythrocytes.
Results
Complement deposition and MAC formation was induced by AIHA serum via the classical pathway of complement, while this activation was efficiently inhibited by EDTA and other inhibitors, e.g. C1-inhibitors. In these systems, it was shown that compstatin completely inhibited C3 deposition on erythrocytes, while unexpectedly C4 deposition appeared to be increased. Regardless of the increase in C4, compstatin prevented formation of the MAC and RBC lysis. In addition, compstatin inhibited complement-mediated phagocytosis of erythrocytes by M1 macrophages while the effect on phagocytosis by M2 macrophages was less pronounced. When erythrocytes opsonized with both complement and IgG phagocytosis inhibition with compstatin was less efficient as compared to the inhibition of phagocytosis of erythrocytes opsonized with complement only.
Conclusion
We demonstrate that compstatin inhibits C3b deposition, C3-mediated phagocytosis of opsonized RBCs as well as MAC formation. Therefore, compstatin is a potential candidate for therapeutical application in AIHA patients to inhibit both intra- and extravascular hemolysis.
Session topic: 32. Transfusion medicine
Keyword(s): AIHA (see autoimmune hemolytic anemia), Complement, transfusion
Abstract: S1586
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 08:15 - 08:30
Location: Room A9
Background
Autoimmune hemolytic anemia (AIHA) is a rare disease characterized by autoantibodies against erythrocytes. These autoantibodies may activate the classical complement pathway leading to opsonization by complement proteins C3b and C4b. This results in increased clearance of erythrocytes by phagocytes, called extravascular hemolysis. Occasionally, complement activation results in formation of the membrane attack complex (MAC), causing intravascular hemolysis. C3-inhibitor compstatin inhibits C3 activation and thus it is expected to reduce both, C3b deposition as well as well as formation of the MAC. Therefore, compstatin is a potentially efficient inhibitor of extra- and intravascular hemolysis in AIHA.
Aims
The current aim of this research is to investigate in vitro whether compstatin would be a suitable drug for AIHA treatment.
Methods
Healthy donor erythrocytes treated with bromelin were incubated with AIHA patient serum together with anti-C5 so that opsonization could be analyzed by FACS using anti-C3-FITC and anti-C4-APC antibodies. In the absence of eculizumab, hemolysis of RBCs was tested by measuring the extinction at 412/690 nm. The percentage hemolysis was calculated relative to the 100% water control. To assess the effect of compstatin cp40 on uptake of erythrocytes by phagocytes, erythrocytes were fluorescently labeled with PKH26 before opsonization. Monocytes isolated by leukapheresis were cultured for 8 or 9 days in the presence of 10 ng/mL GM-CSF to obtain M1-like macrophages or 50 ng/mL M-CSF to obtain M2-like macrophages. Opsonized erythrocytes were then incubated with healthy monocyte derived macrophages and phagocytosis of erythrocytes was measured with ImageStream after lysing the non-phagocytosed erythrocytes.
Results
Complement deposition and MAC formation was induced by AIHA serum via the classical pathway of complement, while this activation was efficiently inhibited by EDTA and other inhibitors, e.g. C1-inhibitors. In these systems, it was shown that compstatin completely inhibited C3 deposition on erythrocytes, while unexpectedly C4 deposition appeared to be increased. Regardless of the increase in C4, compstatin prevented formation of the MAC and RBC lysis. In addition, compstatin inhibited complement-mediated phagocytosis of erythrocytes by M1 macrophages while the effect on phagocytosis by M2 macrophages was less pronounced. When erythrocytes opsonized with both complement and IgG phagocytosis inhibition with compstatin was less efficient as compared to the inhibition of phagocytosis of erythrocytes opsonized with complement only.
Conclusion
We demonstrate that compstatin inhibits C3b deposition, C3-mediated phagocytosis of opsonized RBCs as well as MAC formation. Therefore, compstatin is a potential candidate for therapeutical application in AIHA patients to inhibit both intra- and extravascular hemolysis.
Session topic: 32. Transfusion medicine
Keyword(s): AIHA (see autoimmune hemolytic anemia), Complement, transfusion