Addition Of C2 Research Articles

Modulation of the classical pathway C3 convertase by plasma proteins C4 binding protein and C3b inactivator.

We recently described the isolation from human serum of a serum protein (C4 binding protein) that functions as an essential cofactor for C3b inactivator in the proteolysis of fluid-phase C4b and to a much lesser extent, C3b. We show here the role of C4 binding protein in the formation and function of the classical pathway C3 convertase (C42). C4 binding protein interferes with the assembly of the membrane-bound C3 convertase of the classical pathway and accelerates the decay of C42 in a dose-dependent fashion. Its removal from serum by means of specific immune absorption promotes the vigorous consumption of C3 after addition of C1; this effect is abolished by reconstitution with purified C4 binding protein. Although C4 binding protein inhibits the hemolytic function of cell-bound C4b, we did not detect any change in the structure of C4b even after prolonged incubations of EAC14 with C4 binding protein. For this reason, and on the basis of studies of the time required for maximal reactivity (Tmax) of cellular intermediates generated in the presence of C4 binding protein and limited amounts of C2, we conclude that the effects of C4 binding protein are probably mediated by displacing C2a from specific binding sites on C4b. In addition, C4 binding protein enhances the cleavage by C3b inactivator of the alpha' chain of cell-bound C4b. When EAC14 cells were incubated with both control proteins, the Tmax of the cells was prolonged and the lysis was markedly diminished. We conclude that C4 binding protein and C3b inactivator control the C3 convertase of the classical pathway in a fashion similar to that described for beta 1H and C3b inactivator in the alternative pathway.

Demonstration of a Non Osmotic Swelling and of a Three Step Mechanism During the Immune Lysis of Erythrocytes by Antibody and Complement

Abstract E to EAC1-9 cell intermediates were sized electrically with a hydrodynamic focusing system which is a modified Coulter counter method that avoids the methodologically right skewed volume distribution curves of the original method. No changes in the volume distribution curves of the erythrocytes were observed up to the EAC1-8 state. On addition of C9, a dose-dependent part of the cells did swell and three steps could be distinguished in the lytic mechanism: 1) fixation of C9 to EAC1-8 cells, a process which proceeds also well at 0°C; 2) a temperature-dependent swelling of the erythrocytes to their maximal volume in an all or nothing fashion; and 3) lysis of the cell. The swelling of the erythrocytes to spheroids is of a non-osmotic nature because it occurs also in highly concentrated 36% albumin solutions where the colloid osmotic forces of the intracellular protein are balanced on a molar basis. It persists into the ghost phase.

The computer analysis of volume distribution curves. Demonstration of two erythrocyte populations of different size in the young guinea pig and analysis of the mechanism of immune lysis of cells by antibody and complement.

Guinea pig, rat and sheep erythrocytes were sized electrically using the hydrodynamic focusing technique. The experimental curves were approximated with a computer by linear and logarithmic normal distributions. Rat and guinea pig erythrocytes from adult animals were best approximated by one linear normal distribution. Two populations (I, II) of erythrocytes with different mean volume could be demonstrated in young guinea pigs by this analysis. Population I erythrocytes are small, have a lower electrophoretic mobility and are mainly present at birth. They are gradually replaced by the larger population II erythrocytes. Both types of erythrocytes are probably the result of separate differentiation pathways. The analysis of erythrocyte volume distribution curves during immune lysis by antibody and complement shows that intact and ghost erythrocytes are measured by electrical sizing. No volume changes were observed up to the EAC1-8 intermediate. After the addition of C9, a C9 dose-dependent part of the erythrocytes swell permanently to spheroids. The spheroid transformation is a temperature-dependent, all or nothing reaction which is independent of protein osmotic forces from the interior of the cell.

Coliphage T2 neutralization by 7S antibody and C1

Neutralization of bacteriophage T2 by 19S and 7S fractions or rabbit antisera was studied in the presence and absence of isolated human C1. Neutralization by 19S antibody is not effected by addition of C1. Neutralization of the phage by 7S antibody, however, is markedly increased by addition of C1. Ammonia treatment of both the 7S antibody and the C1 preparations to inactive C4 did not reduce the ability of C1 to enhance neutralization. Heat treatment of C1 at 56°C for 30 min, however, did inactivate the enhancing factor as did treatment with EDTA. When added individually in the presence of antibody, the other isolated C components (C4, 2, 3, 5, 6, 7, 8, 9) were ineffective in the neutralization of T2.

Requirement for an Extra Factor from Serum for Bacterial Killing by Nascent C5̄6̄7̄, C8 and C9

Abstract An activated complex of C5 and C6 (C) appears in the supernatants of reaction mixtures containing purified C5 and C6 and sheep erythrocytes which have been reacted with antibody and the first four C components (EAC1423). Addition of C7 to C yields the trimolecular complex C which, for a short time, can affix to the surface of an unsensitized erythrocyte rendering it susceptible to lysis upon the further addition of C8 and C9 (reactive lysis). Lysis is readily accomplished when sheep erythrocytes are the target cells, however, killing of rough Escherichia coli cannot be accomplished with the same reagents over a wide range of concentrations, even though the bacteria are highly susceptible to killing by whole serum. The addition of an extra factor present in a pH 7.5 pseudoglobulin fraction of human serum does lead to killing of the bacteria.