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Hemolysis of Sheep Erythrocytes in Guinea Pig Serum Deficient in the Fourth Component of Complement

II. Evidence for Involvement of C1 and Components of the Alternate Complement Pathway

From the Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014

Abstract

Sheep erythrocytes sensitized with 20 to 40 times the quantity of IgG or IgM antibody required for lysis via the classical complement pathway undergo lysis in C4-deficient guinea pig serum (C4D) or C2-deficient human serum (C2D). The mechanism of this lysis was analyzed in detail, and was found to have an absolute requirement for a Cl-like protein and components of the alternate complement pathway.

Three independent lines of investigation suggested the requirement of C1 for lysis in the deficient sera: 1) C4D serum depleted of C1 by low ionic strength precipitation sustained a marked loss of lytic activity which was overcome by the use of the complement-cell intermediate EAC1. 2) C2D serum depleted of C1 by use of a monospecific anti-C1q immunoabsorbent sustained marked loss of hemolytic activity. Addition of properdin to such sera did not restore hemolytic activity. These absorbed sera were able to lyse the cellular intermediate EAC1. 3) Mg-EGTA completely inhibited lysis in C2D and C4D serum by this pathway while allowing normal function of the alternate complement pathway. Function of C4 and C2 in this lytic mechanism was further excluded by the absence of C2 consumption in C4D serum incubated with heavily sensitized RBC, the failure of anti-C4 to inhibit the lytic reaction, and the inability to lyse heavily sensitized RBC by the sequential addition of purified C1, C2, and C EDTA. Participation of components of the alternate complement pathway was suggested by: 1) conversion of the C3 proactivator by heavily sensitized RBC, 2) abolition or marked reduction of the cytotoxic capacity of C4D and C2D serum heated at 50°C for 30 min, conditions which did not deplete C1, and 3) abolition of lysis in C4D or C2D treated with 0.03 M hydrazine. This lytic mechanism utilizes the later acting complement components as shown by complete absence of lysis in rabbit serum lacking C6.

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