Clin. exp. Immunol. (1975) 21, 109-114. THE INFLUENCE OF C3b INACTIVATOR (KAF) CONCENTRATION ON THE ABILITY OF SERUM TO SUPPORT COMPLEMENT ACTIVATION P. J. LACHMANN AND LISE HALBWACHS Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, London (Received 2 January 1975) SUMMARY Minor elevation of the concentration of the C3b inactivator (KAF) in whole serum (by 15-25%) markedly inhibits the capacity of the serum to support complement activation by inulin, aggregated IgG and by low concentrations of CVF. However, there was no effect when large concentrations of CVF were used nor was the spontaneous ageing of C3 slowed by increased KAF concentrations. These findings show that the activity of the C3b feedback cycle can be reduced by raising the KAF concentration above physiological levels. This finding may provide a mechanism for damping down complement activation locally in vivo. It seems that the spontaneous ageing of C3 in vitro does not involve a KAFinhibitable step and therefore cannot be involved to explain the 'C3 tickover' in vitro. INTRODUCTION The core of the alternative pathway of complement activation is the 'C3b feedback cycle' (see Fig. 1 for the reaction pathway). C3b-the principal product of C3 conversion-itself gives rise to the 'C3-convertase' of the alternative pathway as a result of its interaction with two further factors: factor B and factor D (Muller-Eberhard & Gotze, 1972; Lachmann & Nicol, 1973; Nicholson et al., 1974). This process is modulated in serum by the activity of the C3b inactivator (KAF) which acts enzymatically on C3b preventing, inter alia, its ability to trigger the C3b feedback cycle. It is known that absence of KAF either in vivo (Abramson et al., 1971; Alper, Rosen & Lachmann, 1973) or in vitro (Nicol & Lachmann, 1973) leads to the unrestrained activation of the feedback leading to the exhaustion of C3 and factor B. It is not, however, clear whether the physiological concentration of KAF is such that it is virtually in infinite excess or whether further increase in KAF level will influence the capacity of serum to sustain complement activation by substances known to activate the classical and properdin pathways. Correspondence: Dr Lise Halbwachs, Department of Immunology, Royal Postgraduate Medical School, Hammersmith Hospital, London, W12. 109 110 P. J. Lachmann and L. Halbwachs Factor B- CVF Mg2+ CVF-B FIG. 1. Diagram of the alternative pathway. The anticomplementary factor in cobra venom is now known to act as a C3b analogue which is KAF-resistant (Lachmann & Nicol, 1973). This material is unique in being able to activate the alternative pathway in the absence of C3b and this activity was therefore also investigated for its dependence on KAF concentration. A further puzzling feature of the apparently spontaneous activation of the alternative pathway by KAF depletion is the source of the initial C3b needed to trigger the pathway. It has been suggested that spontaneous catabolism of C3 might be the mechanism. For this reason the effect of increased KAF concentration was measured on the spontaneous conversion of C3 on ageing in serum. MATERIALS AND METHODS Inulin A standard suspension (50 mg/ml) in saline was sonicated. Dilutions of this suspension were made, the final concentration of inulin in the serum being given for each experiment. Cobra venom factor Cobra venom factor was purified from Naja naja venom (Sigma) by DEAE-cellulose and Sephadex G-200 chromatography (Ballow & Cochrane, 1969). CVF was measured by its anticomplementary activity. Serial dilutions of CVF samples were incubated with guinea-pig complement (diluted 1:50 in complement-fixation diluent). Sheep EA were added and the extent of lysis was measured by haemoglobin release. The CVF titre was the dilution which gave 50%o inhibition of the haemolytic activity of the guinea-pig complement alone. Aggregated human y-globulins These were obtained by heating a concentrated solution (27 mg/ml) of human y-globulins at 630C for 15 min. C3b inactivator effect on complement activation III KAF Functionally purified KAF was prepared from the euglobulin fraction of serum by DEAE-cellulose and Sephadex G-200 chromatography (Lachmann, Aston & Nicol, 1973). The titre of the purified KAF standard solution was measured and compared to the normal serum titre by its capacity of inducing EAC143 agglutination in presence of bovine conglutinin (Lachmann & Muller-Eberhard, 1968). Variations in KAF concentration in normal human serum were obtained by adding different dilutions of the purified KAF standard solution to the serum. C2-deficient and C4-deficient sera These were obtained from genetically C2- or C4-deficient patients. Factor B-depleted serum This was obtained by immunoabsorption. Normal human serum was precipitated at optimal proportion with F(ab')2 anti-human factor B. C3 conversion by inulin and CVF To one volume of normal human serum was added half a volume of inulin, y-globulin, or CVF solution, and two volumes of different KAF dilutions in phosphate-buffered saline. The mixture was incubated at 370C for 30 min in inulin and y-globulin experiments, and for 15 min in CVF experiments. The reaction was stopped by adding EDTA (0-01 M, pH 7 2) and cooling to 40C. 'Spontaneous' C3 conversion by ageing One volume of normal human serum was incubated at 37°C with two volumes of different KAF dilutions and 0.1% sodium azide. Samples were tested, at different times, for C3 conversion. The amount of C3 conversion in the serum was measured by two-dimensional Laurell electrophoresis (Laurell, 1965), in agarose gel (pH 8 6) using a sheep anti-human C3. Factor B conversion was observed by immunoelectrophoresis, using a rabbit anti-human factor B. RESULTS The effect of increased KAF concentration on complement activation by inulin (Fig. 2) KAF inhibits C3 conversion by inulin at all concentrations of inulin used. It also inhibits factor B conversion by inulin. Quite small amounts of KAF are sufficient for this inhibitory effect: an increase of only 15% of the normal KAF concentration in the serum results in 5000 inhibition of C3 conversion by inulin. The effects of increased KAF concentration on complement activation by aggregated y-globulins (Fig. 2) C3 conversion by aggregated y-globulins is also inhibited by increased KAF concentration, but in this case more than 20% increase is necessary in order to observe the inhibition. P. J. Lachmann and L. Halbwachs 112 .2 c 0 aU) 0 U) 1/4 1/8 KAF added ( unit=normal serum titre) 2/5 FIG. 2. Effect of increased KAF concentration on C3 conversion by inulin and by aggregated IgG. (0) Inulin (7 mg/ml). (A) Inulin (0 3 mg/ml). (A) Aggregated IgG. The effects of increased KAF concentration on complement activation by cobra venom factor (Fig. 3) At low concentrations of CVF (dilutions of CVF solution beyond 1:15), KAF inhibits C3 conversion by CVF as it does by inulin. At these CVF concentrations the C3b feedback cycle contributes to the C3 conversion (Lachmann & Nicol, 1973). Using higher concentrations of CVF however, all the C3 in the serum is converted after incubation for 15 min at 370C, and KAF has no effect on this reaction. At high CVF concentration the C3b feedback cycle fails and all the effects seen are due to CVF-Bb itself. * 100 A_ ~i 50 U KAF added (I unrtnorrnal serum 4itre) FIG. 3. Effect of KAF concentration on C3 conversion by CVF. CVF titre of: ()<s 1/15; (A) 1/3; (A) 1; (o)>2. C3b inactivator effect on complement activation 113 Effect of increased KAF concentration on C3 conversion by ageing (Fig. 4) When the serum is aged at 370C, the rate of C3 conversion is not affected by KAF concentration. C3 conversion is different in neither genetically C2-deficient or C4-deficient human sera nor in serum immunochemically depleted of Factor B. 100 * * A 0c v0 0 U /8 1/4 1/2 KAFadded (I unit= normal serum titre) FIG. 4. Effect of KAF concentration on C3 conversion by ageing. Incubation for: (0) 63 hr at 370C; (A) 48 hr at 370C; (0) 21 hr at 370C. After 16 hr at 370C, there was: 56-80O0 C3 conversion in normal human serum; 50-72O0 C3 conversion in C2-deficient serum; 48-74% C3 conversion in C4-deficient serum; 45500 C3 conversion in factor B-depleted serum. DISCUSSION It is clear from the experiments described that the KAF concentration in whole human serum is by no means so high that further elevation has no effect on complement activities. In fact, quite modest increases in the KAF concentration (1 5-25% ) markedly inhibit the capacity of a typical properdin pathway activator like inulin and (to a slightly lesser extent) of a typical classical pathway activator like aggregated human IgG to produce complement activation. This evidence suggests that variations in KAF concentration even within physiological limits may significantly modulate complement activation. The heterozygous relatives of the KAF-deficient subjects who have approximately half normal levels show no obvious clinical disease. However, the possibility of damping down complement activation by the artificial raising of KAF levels at local site (e.g. in an inflamed joint) deserves further investigation. The results with CVF show clearly that at low CVF concentrations, where C3b feedback activation plays an amplifying role, a raised KAF concentration is inhibitory. Where sufficiently large CVF concentrations are used to abolish C3b feedback cycle by consuming all the available factor B (Lachmann & Nicol, 1973), increased KAF concentrations are without effect. This confirms the view that the CVF-C3 convertase itself is not affected by KAF. The failure of increased KAF levels to influence spontaneous C3 conversion on ageing as H 114 P. J. Lachmann and L. Halbwachs well as the occurrence of typical C3 conversion on ageing of C4-deficient, C2-deficient and factor B-depleted human sera suggests that this conversion does not go via C3b, nor does it utilize either of the 'immunological' C3 convertases. Therefore this spontaneous conversion in vitro cannot be used to explain the 'C3-tickover'-the source of C3b needed to trigger the apparently spontaneous activation of the C3b feedback on KAF depletion. This is in accord with the recent observations of Williams et al., that KAF depletion will not lead to alternative pathway activation if both C2 and properdin are depleted from the serum, whereas in the absence of either of these components it is known that feedback activation does occur. It seems that the C3b-tickover must be explained in terms of the activation of one or other of the complement pathways. REFERENCES ABRAMSON, N., ALPER, C.A., LACHMANN, P.J., ROSEN, F.S. & JANDL, J.H. (1971) Deficiency of C3 inactivator in man. J. Immunol. 107, 19. ALPER, C.A., ROSEN, F.S. & LACHMANN, P.J. (1972) Inactivator of the third component of complement as an inhibitor in the properdin pathway. Proc. nat. Acad. Sci. (Wash.), 69, 2910. BALLOW, M., & COCHRANE, G.C. (1969) Two anticomplementary factors in cobra venom: hemolysis of guinea pig erythrocytes by one of them. J. Immunol. 103, 944. LACHMANN, P.J., ASTON, W.P. & NICOL, P.A.E. (1973) Further studies on the C3b inactivator or conglutinogen activating factor (KAF). Immunochemistry, 10, 695. LACHMANN, P.J. & MULLER-EBERHARD, H.J. (1968) The demonstration in human serum of 'conglutinogen activating factor' and its effect on the third component of complement. J. Immunol. 100, 691. LACHMANN, P.J. & NICOL, P.A.E. (1973) Reaction mechanism of the alternative pathway of complement fixation. Lancet, i, 465. LAURELL, C.B. (1965) Antigen-antibody crossed electrophoresis. Analyt. Biochem. 10, 358. MULLER-EBERHARD, H.J. & GOTZE, 0. (1972) C3 proactivator convertase and its mode of action. J. exp. Med. 135, 1003. NICHOLSON, A., BRADE, V., LEE, G.D., HYUN, S.S. & MAYER, M.M. (1974) Kinetic studies of the formation of the properdin system enzymes on zymozan evidence that nascent C3b controls the rate of assembly. J. Immunol. 112, 1115. NICOL, P.A.E. & LACHMANN, P.J. (1973). The alternate pathway of complement activation: the role of C3 and its inactivator (KAF). Immunology, 24, 259.