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The isolated active complex of C5 and C6, C56, was found to bind to EAC142 in the absence of C3 or C7, and to form a unique intermediate, EAC14256, which is susceptible to lysis by the addition of C7, C8 and C9. Further studies revealed that C56 alone could bind to EAC142 but not to E, EA, EAC1 or EAC4, nor to EAC14 in the absence of C7, that the C56 binding to EAC142 was highly dependent on temperature and on the ionic strength of the buffer, and that the degree of EAC14256 formation from EAC142 and C56 depended on the amount of C2 on EAC142 and on the amount of added C56. These findings suggest that C2 or C42 on EAC142 may be an acceptor for C56. In addition, C56 appears to bind to EAC142 much more efficiently than to unsensitized erythrocytes, even in the presence of C7. Thus, binding of C56 to EAC142 is likely to be an initial step of membrane attack complex formation in C3-independent immune haemolysis.

We observed two sisters with lupus-like syndrome with homozygous C3 deficiencies. A 19-year-old woman and her 15-year-old sister developed malar rash, arthralgia, and photosensitivity, but antinuclear antibodies and LE cell preparations were negative. The older sister experienced recurrent bronchitis in her childhood, but the younger sister had no recurrent infections. Serum C3 was not detected immunochemically in either sister, and total complement activity and C3 hemolytic activity were extremely low.

C3-independent immune haemolysis was studied using EAC14oxy2 cells and purified C5, C6, C7, C8 and C9. We have found that EAC14oxy2 cells were lysed by C5-C9 and that haemolysis occurs, even after pretreatment of the cells and the C5-C9 preparation with anti-C3. This indicates that EAC14oxy2 can be lysed by C5-C9 without any participation of C3. In contrast, EAC1 and EAC14 cells are not lysed by C5-C9, suggesting that our C5-C9 preparation lacks activated complement components, such as C3bBb, C5b6 or C(56)a. Based on our study of the haemolysis of EAC14oxy2, we have determined that: (i) EAC14oxy2 cells are lysed by a preparation of C5, C6, C7, C8 and C9, but no lysis occurs when any one of these complement is absent, (ii) for significant haemolysis of EAC14oxy2, a higher concentration of C5 is necessary as compared to the C5 requirement when haemolysis occurs in the presence of C3, (iii) the degree of haemolysis is linearly related to the concentration of C5 and does not reach a plateau, despite the addition of as much as 3,200 U of C5, and (iv) the degree of haemolysis is linearly related to the concentration of cell bound C42. These observations suggest that, in the absence of C3, the C3 convertase C42 can activate C5 directly, resulting in the formation of the membrane attack complex, C5b-9.

Incubation os zymosan (Z) with normal human serum led to the formation of ZXhu, which had the abilities to cleave C3 and C5. Kinetic studies on ZXhu formation revealed that the amount of inactivated C5 by ZXhu, when expressed in site-forming unit (SFU), was much greater than that of inactivated C3. When ZXhu having limited C3 and C5-cleaving was incubated at 37 degrees C it decayed with a first order reaction completely in 120 min. At any stage of decay of ZXhu, the activities could be restored by the addition of both B and D, but not by the addition of B or D alone. Not D but B could be bound to the completely decayed ZXhu, and activation of bound B by D led to the regeneration of ZXhu.

Analysis of the serum of an individual having a deficiency of C3 (C3D) revealed that despite the fact that the C3 could not be detected immunochemically, a small amount of C3 haemolytic activity and total complement activity was indeed found to be present. The present study was performed to analyse the C3-like factor, responsible for this C3 activity in C3D serum. It was found that the C3-like factor was not neutralized by anti-C3 but was neutralized by anti-C5. The electrophoretic mobility of the C3-like factor corresponded to that of purified C5. Thus, C3-like factor was shown to have the antigenicity and electrophoretic mobility of C5. This factor was found in the sera of three other C3D subjects and even in pooled normal human serum (NHS). These findings suggest that, in cases of C3D, C5 compensates for the genetic lack of C3 and serves a protective function and that sensitized erythrocytes (EA) may be lysed by complement components without participation of C3.

Unsensitized guinea-pig erythrocytes (Egp) were lysed by a combination of eight isolated, human-derived complement components, Cls, C4, C2, C5, C6, C7, C8 and C9 (Cls-C9exC3), even in the presence of anti-C3. It was determined that a factor was generated in the reaction mixture of Cls, C4, C2, C5 and C6, which had a lytic activity against Egp when C7, C8 and C9 were added. The lytic factor was similar to C56 in the following properties: the activity of the lytic factor decreased when incubated with C7 prior to its reaction with Egp, the lytic factor did not bind to Egp by itself but it did bind in the presence of C7, EDTA did not have any inhibitory effect on the lytic factor, and the activity of the lytic factor was lost by treatment with anti-C5 or anti-C6 but not by treatment with anti-C4. Furthermore, C5a, a cleavage product of C5, was clearly detected in the reaction mixture of Cls, C4, C2 and C5. These findings indicate that C5 can be activated proteolytically into C5a and C5b in the fluid phase solely by the classical pathway C3 convertase, C42, without any participation of C3.