Clin. exp. Immunol. (1979) 36, 140-144.
Activation of the alternative pathway of human complement by haemoglobin W. A. WILS ON & E. JEA N THOMA S Department of Medicine, University of the West Indies, Kingston 7, Jamaica
(Accepted for publication 26 October 1978)
SUMMARY
Haemoglobin solutions (concentration> 1 5 mg/ml), prepared from lysates of erythrocytes from a normal subject and from a patient with sickle cell anaemia, caused factor B and C3 cleavage and loss of haemolytic activity of factor B when incubated with fresh autologous serum. Under the same experimental conditions, preparations of erythrocyte stroma or of buffy coat lysates did not produce factor B and C3 cleavage. This reaction required Mg' + but not Clq or C4, indicating that the alternative complement pathway was activated.
INTRODUCTION The recruitment of complement and its biological sequelae occurs by two pathways-the classical and the alternative (Gotze & Muller-Eberhard, 1976). A large number of substances is known to activate the alternative pathway independently of the classical route, including aggregated IgA and IgG under special circumstances. Polysaccharides, e.g. from pneumococci or gram-negative organisms, and also inulin, zymosan, rabbit erythrocytes and certain polyanionic substances, e.g. dextran sulphate, can also activate the alternative pathway. Of these, perhaps only the first three could be said to have much in vivo immunological significance in man. The possibility that red cell constituents might produce activation of the complement system in man was suggested by the association of haemolytic disorders, specifically sickle cell anaemia and ,3 thalassaemia major, with evidence of hypercatabolism of complement proteins (Wilson, Hughes & Lachmann, 1976; Auderset, Casali & Lambert, 1977; Wilson, Thomas & Sissons, 1979). For this reason we investigated the complement-activating properties of various components of human blood and of haemoglobin and erythrocyte stroma in particular. MATERIALS AND METHODS In order to test their ability to activate complement in autologous human serum, haemoglobin solutions and erythrocyte stroma were prepared as follows. Blood was anti-coagulated with acid citrate dextrose, centrifuged at 500 g, the plasma discarded, and the buffy coat (with upper 5% of erythrocytes) removed. The remaining erythrocytes were washed thrice in 20 volumes of phosphate buffered saline (PBS, 0*15 M pH 7-4) and the upper 5% of cells discarded after each wash. The packed erythrocytes were lysed in 50 volumes of hypotonic PBS (0-02 M pH 7.4) at 4°C for 1 hr, and centrifuged for 1 hr at 25,000 g. The supernatant haemoglobin was pressure filtered through an Amicon XM 100 membrane. The haemoglobin containing filtrate was concentrated and filtered through a Sephadex G200 column in PBS; haemoglobin was the only component found. It was re-concentrated to 50 mg/ml and used in the incubation experiments described below. The residue from the centrifugation of the haemolysate (erythrocyte stroma) was diluted to a concentration equivalent (with respect to whole blood) to that present in the haemoglobin solution, using a dilution factor calculated by the formula: Hb concentration in whole blood (mg/ml) x 100 percentage haematocrit x 50 (mg/ml) The haemoglobin concentration in erythrocyte stroma prepared in this way was approximately 2.0 mg/ml. The buffy coat removed initially was also washed thrice in 20 volumes of isotonic PBS, incubated for 1 hr at 4°C in hypotonic PBS, Correspondence: Dr W. A. Wilson, Department of Medicine, University of the West Indies, Kingston 7, Jamaica. 0099-9104/79/0040-0140$02.00 (© 1979 Blackwell Scientific Publications
140
Complement activation by haemoglobin
141
centrifuged at 25,000 g for 1 hr, and the supernatant re-concentrated by the same factor as described above for the haemolysate. The anti-coagulant and all the buffers or solutions used had sodium azide 2-5 mm added to prevent bacterial contamination. Purified C3 and factor B, and partly purified factor D0 for use in the incubation experiments described below were obtained from human blood as described previously (Lachmann, Hobart & Aston, 1973; Martin et al., 1976). Purified factor B and semi-purified factor D were tested for functional activity in haemolytic plate assays (Martin et al., 1976). Factors B and ID, and C3 contained no Clq or C4 as detected by the Ouchterlony assay, and the haemolytic plate assay for C4 (Lachmann et al., 1973). Incubation experiments. (a) Solutions of haemoglobin of varying concentration, or suspensions of erythrocyte stroma of concentration equivalent (with respect to whole blood) to the haemoglobin solutions (see above) were incubated with fresh autologous serum at 370C for 30 min in glass tubes, then EDTA 0-01 M was added to stop the reaction. Control tubes contained serum and PBS alone. C3 conversion to C3c was measured by crossed immunoelectrophoresis (Laurell, 1965). Percentage conversion of C3 to C3c in test and control mixtures was calculated as follows: area under C3c precipitin arc x 100 area under C3c precipitin arc+area under C3 precipitin arc
Mean percentage C3 conversion in control tubes was 23% (s.d. 5%, n = 4) and was subtracted from the percentage C3 conversion in the test mixtures to obtain the net percentage C3 conversion given in Fig. 1 and Table 1. Results of these experiments are shown in Fig. 1. 50 40-
0 C
c30-
20 10
O
0
-I
4 8 12 16 Haemoglobin concentration (mg/ml)
I
20
FIG. 1. Net immunochemical C3 conversion (calculated as stated in the Materials and Methods section) in autologous human serum produced by normal (o 0) haemoglobin, and by equivo) and sickle ( alent concentrations (see the Materials and Methods section) of normal erythrocyte stroma (oL a) and erythrocyte stroma from a patient with sickle cell anaemia (U U).
(b) In order to determine requirements for Ca+ + and Mg+ +, and whether C4 was consumed in the reaction mixtures, the following procedure was adopted, the results of which are shown in Table 1. Ethyleneglycoltetraacetic acid (EGTA) 0 01 M, pH 7-2, which chelates both Ca+ + and Mg+ +, or EGTA 0 01 M pH 7-2 containing Mg 0-0035 M (MgEGTA) were included in some incubation mixtures. Immediately after incubation at 37°C for 30 min, factor B in the incubation mixtures was measured by haemolytic plate assays (Martin et al., 1976), and C4 by haemolytic assay using C4-deficient guinea-pig serum (Lachmann et al., 1973). Standard curves for the haemolytic assays were produced by using dilutions of pooled normal human serum, and loss of haemolytic activity was expressed as a percentage of the haemolytic activity present in the control tubes (serum and PBS alone). EDTA (0-01 M) was added to the incubation mixture to stop the reaction (after removal of samples for factor B and C4 haemolytic assay), and the percentage immunochemical conversion of C3 to C3c and of factor B to Bb was measured by crossed immunoelectrophoresis (see above). Mean factor B conversion in the control tubes was 9% ks.d. 3%, n = 4) and was subtracted from the percentage conversion in each test mixture to give the percentage immunochemical conversion given in Table 1. (c) In order to determine requirements for Clq and C4 in haemoglobin-induced C3 conversion, incubation experiments were carried out using purified C3 and factor B, and partly purified factor 1D. Factor B and ID were diluted to haemolytic concentrations of 100% of pooled normal serum, and C3 was diluted to a concentration of 1-2 mg/ml. Equal volumes of the three proteins and a four-fold concentrated complement fixation diluent (CFD, Oxoid) were mixed, and haemoglobin AA at a final concentration of 5*0 mg/ml or 10 mg/ml was added. After incubation at 37°C for 15 min, C3 conversion was assessed by crossed immunoelectrophoresis.
142
W.A. Wilson & E. Jean Thomas TABLE 1. Effect of haemoglobin, erythrocyte stroma and buffy coat lysate on C3 and factor B in fresh autologous serum Percentage immunochemical conversion
Incubation mixture
C3
Factor B
HS+PBS-+ MgEGTA HS + PBS A- inulin HS + PBS + MgEGTA+ inulin HS - IgG HS +MgEGTA+ IgG
2 42 34 41 7 44 0 39 40 0 35 3 2 3
0 20 25 0 0 21 0 24 23 0 20 0 0 0
HS+HbAA HS +EGTA+ HbAA HSI-MgEGTA+HbAA HS+HbSS
HS+EGTA+ HbSS HS + MgEGTA 4 HbSS HS+stroma AA HS4-stroma SS HS+buffy coat lysate
Percentage loss of haemolytic activity C4
Factor B
-
-
7
43
-
-
51
15
-
-
0
30
-
-
-
7
27
-
--
5 6 0
0 3 4
Requirements for Ca+ + and Mg+ +, and for Ca+ + were assessed by using EGTA and MgEGTA, respectively. Loss of C4 and factor B haemolytic activity was measured simultaneously. HS = autologous serum, PBS = phosphate buffered saline 0 015 M pH 7.4, IgG = human heat-aggregated IgG at a final concentration of 1 0 mg/ml. Inulin = inulin suspension (sonicated) at a final concentration of 100 pg/ml. HbAA = normal haemoglobin at a final concentration of 10 mg/ml, HbSS = sickle haemoglobin at a final concentration of 10 mg/ml. Percentage immunochemical conversion and percentage loss of haemolytic activity were calculated as stated in the Materials and Methods section.
RESULTS Fig. 1 shows that haemoglobin in solution produced C3 conversion and that the percentage of C3 conversion increased with the increasing haemoglobin concentration. Haemoglobin which had not been subjected to Sephadex G200 or pressure filtration gave similar results, as did preparations of haemoglobin from subjects with homozygous sickle cell (HbSS) disease (Fig. 1). As shown in Table 1, haemo-
C3 +
B:0+:D + CFU
C31:C\ ..
.
HB(B)
+
C3 I
C3
B+
D+CFD+HBA)
C3+B+D+CFD C3
C3
C3c .
..
,
:
F .Tl"..
FIG. 2. Immunochemical C3 conversion (assessed by crossed immunoelectrophoresis) by haemoglobin AA in incubation mixtures containing purified C3 and factor B, and semi-purified factor ID, and in the absence of Clq and C4. HB(A) = Normal adult haemoglobin at a final concentration of 50 mg/ml. HB(s) = Normal adult haemoglobin at a final concentration of 10 mg/ml.
Complement activation by haemoglobin
143
globin produced C3 and factor B cleavage and loss of haemolytic activity of factor B. EGTA 0-01 M pH 7-2, which chelates both Ca' + and Mg' +, thereby blocking both complement pathways, inhibited C3 and factor B cleavage, but EGTA 0 01 M pH 7-2 containing Mg"+ 00035 M (MgEGTA) (Bryant & Jenkins, 1968; Platts-Mills & Ishizaka, 1974) did not. Aggregated IgG produced C3 conversion which was blocked by Mg EGTA, whereas the conversion produced by inulin was not; MgEGTA, under these conditions, therefore inhibited the classical pathway, but provided enough Mg' + for alternative pathway activation to proceed. Table 1 also shows that C4 consumption did not occur when C3 and factor B activation were produced by haemoglobin or inulin, but did so when it was produced by aggregated IgG. Experiments using haemoglobin solutions and purified factors B, C3, and 17 showed (Fig. 2) that C3 conversion was produced by haemoglobin in the absence of antigenically detectable Clq or C4 and of functionally detectable C4.
DISCUSSION In these experiments, haemoglobin, whether normal (HbAA) or sickle (HbSS), produced C3 and factor B conversion in autologous serum; this reaction was blocked by EGTA, but permitted by MgEGTA which in our experiments was shown to selectively block classical pathway activity. This conversion did not consume C4 and occurred in its absence, as well as in the absence of Clq. These features indicate that haemoglobin in solution activates the alternative pathway and that the activity was present in haemoglobin solutions of a concentration of 1-5 mg/ml or greater. Compared with inulin, haemoglobin is a weak alternative pathway activator. It seems unlikely that activation was due to a trace contaminant present in the haemoglobin, as no complement-activating properties were found in our preparations of erythrocyte stroma (Fig. 1), nor in our lysates of the buffy coat (Table 1), thus excluding a possible contribution by platelet and leucocyte components which may have been present in the haemolysate. The precautions taken against contamination with endotoxin and the method of treating the haemolysate are further arguments against the presence of a non-haemoglobin activator. The absence of factor B splitting activity in our preparations of erythrocyte stroma is in contrast with a previous report (Poskitt, Fortwengler & Lunskis, 1973), in which it is unclear by which pathway factor B was split and special precautions were apparently not taken against contamination with endotoxin. It should also be noted that our preparations of haemoglobin and erythrocyte stroma were fresh (less than 5 days old), as it is possible that storage may alter the properties of stroma. Glutaraldehyde-treated erythrocytes, for example, have been shown to activate the classical complement pathway (Hugh-Jones, Gardner & Rowlands, 1977), and loss of sialic acid from sheep red cell membranes converts these membranes to an activator of the alternative pathway (Fearon, 1978). This study does not indicate how haemoglobin activates the alternative pathway. C3 conversion by this pathway occurs mainly by a positive feed-back cycle (Gotze & Muller-Eberhard, 1976) in which factor B binds to the major cleavage fragment of C3, C3b, and is then split by factor 17 to give the C3 converting enzyme C3bBb. Properdin delays the intrinsic decay of this otheru-ise labile enzyme, which is also inactivated by the plasma protein ,B1H and by C3b inactivator. The initial C3b needed for this cycle may be generated by the classical pathway or independently of Cl, C4 and C2 by the alternative pathway. There is still some uncertainty about the way in which the alternative pathway is initiated independently of the classical pathway (Fearon & Austen, 1977). Haemoglobin may conceivably act either by initiating the alternative pathway proper, or by potentiating the feed-back cycle. It may also possibly prevent the decay of C3b or C3bBb, or interact with ,B1H or C3b INA, as the concentration of these inhibitors may also be of importance in allowing activation of the pathway to proceed (Nydegger, Fearon & Austen, 1978). If haemoglobin is released into the circulation in substantial amounts, it seems likely that many of the biological consequences of complement activation would follow. This mechanism might be of importance in the genesis of shock and coagulation abnormalities associated with acute intravascular haemolysis. Chronic, low grade intravascular haemolysis occurs in sickle cell anaemia (Neely et al., 1969; Naumann et al., 1971), and haemoglobin released from ruptured sickled cells in capillary beds could cause local
144
W.A. Wilson & E. Jean Thomas
consumption of alternative pathway factors. This could account for reduced serum alternative pathway activity seen in some patients (Wilson et al., 1976; Auderset et al., 1977); reduced alternative pathway activity has been linked to defective bacterial opsonization, especially of pneumococci and salmonella organisms (Johnston, Newman & Struth, 1973; Hand & King, 1978) which have a marked predilection for the infection of patients with sickle cell anaemia (Eeckles, Gatti & Renoirte, 1967). The extent to which these mechanisms are of importance in vivo may depend on the binding of haemoglobin to carrier proteins in the blood, or the rate of its removal by the reticuloendothelial system. Further investigation of these aspects is needed. We thank Dr J.G.P. Sissons who prepared the pure factor B and C3 used here, and Drs E. Morrison and M. Wilson for help with fractionation. This work was supported by the Wellcome Trust, to whom we are grateful.
REFERENCES AUDERSET, M.J., CASALI, P. & LAMBERT, P.H. (1977) Decreased factor B activity in B thalassaemia major. Path. Biol. 25, 391 (abstract). BRYANT, R.E. & JENKINS, D.E., JR. (1968) Calcium requirements for complement dependent haemolytic reactions. I Immunol. 101, 664. EECKLES, R., GATTI, R. & RENOIRTE, A.M. (1967) Abnormal distribution of haemoglobin genotypes in negro children with severe bacterial infections. Nature (Lond.), 216, 382. FEARON, D.T. (1978) Regulation by membrane sialic acid of ,flH dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway. Proc. Nat. Acad. Sci. (Wash.), 75, 1971. FEARON, D.T. & AuSTEN, K.F. (1977) Activation of the alternative complement pathway by circumvention of the regulatory action of endogenous proteins. J. exp. Med. 146, 22. GOTZE, 0. & MULLER-EBERHARD, H.J. (1976) The alternative pathway of complement activation. Adv. Immunol. 23, 1. HAND, W.L. & KING, N.L. (1978) Serum opsonification of salmonella in sickle cell anaemia. Amer. J. Med. 64, 388. HUGH-JONES, N.C., GARDNER, B. & ROWLANDS, J. (1977) Activation of human complement by glutaraldehydetreated red cells. Nature (Lond.), 270, 613. JOHNSTON, R.B., NEWMAN, S.K. & STRUTH, A.G. (1973) An abnormality of the alternative pathway of complement activation in sickle cell disease. N. Engl. _7. Med. 288, 803. LACHMANN, P.J., HOBART, M.J. & ASTON, W.P. (1973) Complement technology. Handbook of Experimental Immunology (ed. by D.M. Weir) Chap. 5, p. 1. Blackwell, Oxford.
LAURELL, C.B. (1965) Antigen-antibody crossed electro-
phoresis. Analyt. Biochem. 10, 358. MARTIN, A., LACHMANN, P.J., HALBWACHS, L. & HOBART, M.J. (1976) Haemolytic diffusion plate assays for factors B and D of the alternative pathway of complement
activation. Immunochemistry, 13, 317. NAUMANN, H.N., DIGGS, L.W., BARRERAs, L. & WILLIAMS, B.J. (1971) Plasma haemoglobin and haemoglobin fractions in sickle cell crisis. Amer. ]. clin. Path. 56, 137. NEELY, C.L., WAJIMA, T., KRAUS, A.P., DIGGS, L.W. & BARRERAS, L. (1969) Lactic acid dehydrogenase activity and plasma haemoglobin elevations in sickle cell disease. Amer. ]. clin. Path. 52, 167. NYDEGGER, U.E., FEARON, D.T. & AUSTEN, F.K. (1978) The modulation of the alternative pathway of complement in C2 deficient human serum by changes in concentration of component and control proteins. ]. Immunol. 120, 1404. PLATTS-MILLS, T.E. & ISHIZAKA, K. (1974) Activation of the alternate pathway of human complement by rabbit cells. 5. Immunol. 113, 348. POSKITT, T.R., FORTWENGLER, H.P. & LUNSKIS, B.J. (1973) Activation of the alternative complement pathway by autologous red cell stroma. ]. exp. Med. 138, 715. WILSON, W.A., HUGHES, G.R.V. & LACHMANN, P.J. (1976) Deficiency of factor B of the complement system in sickle cell anaemia. Brit. Med. ]. i, 367. WILSON, W.A., THOMAS, E.J. & SISsoNS, J.G.P. (1979) Complement activation in asymptomatic patients with sickle cell anaemia. Clin. exp. Immunol. 36, 130.
Activation of the alternative pathway of human complement by haemoglobin W. A. WILS ON & E. JEA N THOMA S Department of Medicine, University of the West Indies, Kingston 7, Jamaica
(Accepted for publication 26 October 1978)
SUMMARY
Haemoglobin solutions (concentration> 1 5 mg/ml), prepared from lysates of erythrocytes from a normal subject and from a patient with sickle cell anaemia, caused factor B and C3 cleavage and loss of haemolytic activity of factor B when incubated with fresh autologous serum. Under the same experimental conditions, preparations of erythrocyte stroma or of buffy coat lysates did not produce factor B and C3 cleavage. This reaction required Mg' + but not Clq or C4, indicating that the alternative complement pathway was activated.
INTRODUCTION The recruitment of complement and its biological sequelae occurs by two pathways-the classical and the alternative (Gotze & Muller-Eberhard, 1976). A large number of substances is known to activate the alternative pathway independently of the classical route, including aggregated IgA and IgG under special circumstances. Polysaccharides, e.g. from pneumococci or gram-negative organisms, and also inulin, zymosan, rabbit erythrocytes and certain polyanionic substances, e.g. dextran sulphate, can also activate the alternative pathway. Of these, perhaps only the first three could be said to have much in vivo immunological significance in man. The possibility that red cell constituents might produce activation of the complement system in man was suggested by the association of haemolytic disorders, specifically sickle cell anaemia and ,3 thalassaemia major, with evidence of hypercatabolism of complement proteins (Wilson, Hughes & Lachmann, 1976; Auderset, Casali & Lambert, 1977; Wilson, Thomas & Sissons, 1979). For this reason we investigated the complement-activating properties of various components of human blood and of haemoglobin and erythrocyte stroma in particular. MATERIALS AND METHODS In order to test their ability to activate complement in autologous human serum, haemoglobin solutions and erythrocyte stroma were prepared as follows. Blood was anti-coagulated with acid citrate dextrose, centrifuged at 500 g, the plasma discarded, and the buffy coat (with upper 5% of erythrocytes) removed. The remaining erythrocytes were washed thrice in 20 volumes of phosphate buffered saline (PBS, 0*15 M pH 7-4) and the upper 5% of cells discarded after each wash. The packed erythrocytes were lysed in 50 volumes of hypotonic PBS (0-02 M pH 7.4) at 4°C for 1 hr, and centrifuged for 1 hr at 25,000 g. The supernatant haemoglobin was pressure filtered through an Amicon XM 100 membrane. The haemoglobin containing filtrate was concentrated and filtered through a Sephadex G200 column in PBS; haemoglobin was the only component found. It was re-concentrated to 50 mg/ml and used in the incubation experiments described below. The residue from the centrifugation of the haemolysate (erythrocyte stroma) was diluted to a concentration equivalent (with respect to whole blood) to that present in the haemoglobin solution, using a dilution factor calculated by the formula: Hb concentration in whole blood (mg/ml) x 100 percentage haematocrit x 50 (mg/ml) The haemoglobin concentration in erythrocyte stroma prepared in this way was approximately 2.0 mg/ml. The buffy coat removed initially was also washed thrice in 20 volumes of isotonic PBS, incubated for 1 hr at 4°C in hypotonic PBS, Correspondence: Dr W. A. Wilson, Department of Medicine, University of the West Indies, Kingston 7, Jamaica. 0099-9104/79/0040-0140$02.00 (© 1979 Blackwell Scientific Publications
140
Complement activation by haemoglobin
141
centrifuged at 25,000 g for 1 hr, and the supernatant re-concentrated by the same factor as described above for the haemolysate. The anti-coagulant and all the buffers or solutions used had sodium azide 2-5 mm added to prevent bacterial contamination. Purified C3 and factor B, and partly purified factor D0 for use in the incubation experiments described below were obtained from human blood as described previously (Lachmann, Hobart & Aston, 1973; Martin et al., 1976). Purified factor B and semi-purified factor D were tested for functional activity in haemolytic plate assays (Martin et al., 1976). Factors B and ID, and C3 contained no Clq or C4 as detected by the Ouchterlony assay, and the haemolytic plate assay for C4 (Lachmann et al., 1973). Incubation experiments. (a) Solutions of haemoglobin of varying concentration, or suspensions of erythrocyte stroma of concentration equivalent (with respect to whole blood) to the haemoglobin solutions (see above) were incubated with fresh autologous serum at 370C for 30 min in glass tubes, then EDTA 0-01 M was added to stop the reaction. Control tubes contained serum and PBS alone. C3 conversion to C3c was measured by crossed immunoelectrophoresis (Laurell, 1965). Percentage conversion of C3 to C3c in test and control mixtures was calculated as follows: area under C3c precipitin arc x 100 area under C3c precipitin arc+area under C3 precipitin arc
Mean percentage C3 conversion in control tubes was 23% (s.d. 5%, n = 4) and was subtracted from the percentage C3 conversion in the test mixtures to obtain the net percentage C3 conversion given in Fig. 1 and Table 1. Results of these experiments are shown in Fig. 1. 50 40-
0 C
c30-
20 10
O
0
-I
4 8 12 16 Haemoglobin concentration (mg/ml)
I
20
FIG. 1. Net immunochemical C3 conversion (calculated as stated in the Materials and Methods section) in autologous human serum produced by normal (o 0) haemoglobin, and by equivo) and sickle ( alent concentrations (see the Materials and Methods section) of normal erythrocyte stroma (oL a) and erythrocyte stroma from a patient with sickle cell anaemia (U U).
(b) In order to determine requirements for Ca+ + and Mg+ +, and whether C4 was consumed in the reaction mixtures, the following procedure was adopted, the results of which are shown in Table 1. Ethyleneglycoltetraacetic acid (EGTA) 0 01 M, pH 7-2, which chelates both Ca+ + and Mg+ +, or EGTA 0 01 M pH 7-2 containing Mg 0-0035 M (MgEGTA) were included in some incubation mixtures. Immediately after incubation at 37°C for 30 min, factor B in the incubation mixtures was measured by haemolytic plate assays (Martin et al., 1976), and C4 by haemolytic assay using C4-deficient guinea-pig serum (Lachmann et al., 1973). Standard curves for the haemolytic assays were produced by using dilutions of pooled normal human serum, and loss of haemolytic activity was expressed as a percentage of the haemolytic activity present in the control tubes (serum and PBS alone). EDTA (0-01 M) was added to the incubation mixture to stop the reaction (after removal of samples for factor B and C4 haemolytic assay), and the percentage immunochemical conversion of C3 to C3c and of factor B to Bb was measured by crossed immunoelectrophoresis (see above). Mean factor B conversion in the control tubes was 9% ks.d. 3%, n = 4) and was subtracted from the percentage conversion in each test mixture to give the percentage immunochemical conversion given in Table 1. (c) In order to determine requirements for Clq and C4 in haemoglobin-induced C3 conversion, incubation experiments were carried out using purified C3 and factor B, and partly purified factor 1D. Factor B and ID were diluted to haemolytic concentrations of 100% of pooled normal serum, and C3 was diluted to a concentration of 1-2 mg/ml. Equal volumes of the three proteins and a four-fold concentrated complement fixation diluent (CFD, Oxoid) were mixed, and haemoglobin AA at a final concentration of 5*0 mg/ml or 10 mg/ml was added. After incubation at 37°C for 15 min, C3 conversion was assessed by crossed immunoelectrophoresis.
142
W.A. Wilson & E. Jean Thomas TABLE 1. Effect of haemoglobin, erythrocyte stroma and buffy coat lysate on C3 and factor B in fresh autologous serum Percentage immunochemical conversion
Incubation mixture
C3
Factor B
HS+PBS-+ MgEGTA HS + PBS A- inulin HS + PBS + MgEGTA+ inulin HS - IgG HS +MgEGTA+ IgG
2 42 34 41 7 44 0 39 40 0 35 3 2 3
0 20 25 0 0 21 0 24 23 0 20 0 0 0
HS+HbAA HS +EGTA+ HbAA HSI-MgEGTA+HbAA HS+HbSS
HS+EGTA+ HbSS HS + MgEGTA 4 HbSS HS+stroma AA HS4-stroma SS HS+buffy coat lysate
Percentage loss of haemolytic activity C4
Factor B
-
-
7
43
-
-
51
15
-
-
0
30
-
-
-
7
27
-
--
5 6 0
0 3 4
Requirements for Ca+ + and Mg+ +, and for Ca+ + were assessed by using EGTA and MgEGTA, respectively. Loss of C4 and factor B haemolytic activity was measured simultaneously. HS = autologous serum, PBS = phosphate buffered saline 0 015 M pH 7.4, IgG = human heat-aggregated IgG at a final concentration of 1 0 mg/ml. Inulin = inulin suspension (sonicated) at a final concentration of 100 pg/ml. HbAA = normal haemoglobin at a final concentration of 10 mg/ml, HbSS = sickle haemoglobin at a final concentration of 10 mg/ml. Percentage immunochemical conversion and percentage loss of haemolytic activity were calculated as stated in the Materials and Methods section.
RESULTS Fig. 1 shows that haemoglobin in solution produced C3 conversion and that the percentage of C3 conversion increased with the increasing haemoglobin concentration. Haemoglobin which had not been subjected to Sephadex G200 or pressure filtration gave similar results, as did preparations of haemoglobin from subjects with homozygous sickle cell (HbSS) disease (Fig. 1). As shown in Table 1, haemo-
C3 +
B:0+:D + CFU
C31:C\ ..
.
HB(B)
+
C3 I
C3
B+
D+CFD+HBA)
C3+B+D+CFD C3
C3
C3c .
..
,
:
F .Tl"..
FIG. 2. Immunochemical C3 conversion (assessed by crossed immunoelectrophoresis) by haemoglobin AA in incubation mixtures containing purified C3 and factor B, and semi-purified factor ID, and in the absence of Clq and C4. HB(A) = Normal adult haemoglobin at a final concentration of 50 mg/ml. HB(s) = Normal adult haemoglobin at a final concentration of 10 mg/ml.
Complement activation by haemoglobin
143
globin produced C3 and factor B cleavage and loss of haemolytic activity of factor B. EGTA 0-01 M pH 7-2, which chelates both Ca' + and Mg' +, thereby blocking both complement pathways, inhibited C3 and factor B cleavage, but EGTA 0 01 M pH 7-2 containing Mg"+ 00035 M (MgEGTA) (Bryant & Jenkins, 1968; Platts-Mills & Ishizaka, 1974) did not. Aggregated IgG produced C3 conversion which was blocked by Mg EGTA, whereas the conversion produced by inulin was not; MgEGTA, under these conditions, therefore inhibited the classical pathway, but provided enough Mg' + for alternative pathway activation to proceed. Table 1 also shows that C4 consumption did not occur when C3 and factor B activation were produced by haemoglobin or inulin, but did so when it was produced by aggregated IgG. Experiments using haemoglobin solutions and purified factors B, C3, and 17 showed (Fig. 2) that C3 conversion was produced by haemoglobin in the absence of antigenically detectable Clq or C4 and of functionally detectable C4.
DISCUSSION In these experiments, haemoglobin, whether normal (HbAA) or sickle (HbSS), produced C3 and factor B conversion in autologous serum; this reaction was blocked by EGTA, but permitted by MgEGTA which in our experiments was shown to selectively block classical pathway activity. This conversion did not consume C4 and occurred in its absence, as well as in the absence of Clq. These features indicate that haemoglobin in solution activates the alternative pathway and that the activity was present in haemoglobin solutions of a concentration of 1-5 mg/ml or greater. Compared with inulin, haemoglobin is a weak alternative pathway activator. It seems unlikely that activation was due to a trace contaminant present in the haemoglobin, as no complement-activating properties were found in our preparations of erythrocyte stroma (Fig. 1), nor in our lysates of the buffy coat (Table 1), thus excluding a possible contribution by platelet and leucocyte components which may have been present in the haemolysate. The precautions taken against contamination with endotoxin and the method of treating the haemolysate are further arguments against the presence of a non-haemoglobin activator. The absence of factor B splitting activity in our preparations of erythrocyte stroma is in contrast with a previous report (Poskitt, Fortwengler & Lunskis, 1973), in which it is unclear by which pathway factor B was split and special precautions were apparently not taken against contamination with endotoxin. It should also be noted that our preparations of haemoglobin and erythrocyte stroma were fresh (less than 5 days old), as it is possible that storage may alter the properties of stroma. Glutaraldehyde-treated erythrocytes, for example, have been shown to activate the classical complement pathway (Hugh-Jones, Gardner & Rowlands, 1977), and loss of sialic acid from sheep red cell membranes converts these membranes to an activator of the alternative pathway (Fearon, 1978). This study does not indicate how haemoglobin activates the alternative pathway. C3 conversion by this pathway occurs mainly by a positive feed-back cycle (Gotze & Muller-Eberhard, 1976) in which factor B binds to the major cleavage fragment of C3, C3b, and is then split by factor 17 to give the C3 converting enzyme C3bBb. Properdin delays the intrinsic decay of this otheru-ise labile enzyme, which is also inactivated by the plasma protein ,B1H and by C3b inactivator. The initial C3b needed for this cycle may be generated by the classical pathway or independently of Cl, C4 and C2 by the alternative pathway. There is still some uncertainty about the way in which the alternative pathway is initiated independently of the classical pathway (Fearon & Austen, 1977). Haemoglobin may conceivably act either by initiating the alternative pathway proper, or by potentiating the feed-back cycle. It may also possibly prevent the decay of C3b or C3bBb, or interact with ,B1H or C3b INA, as the concentration of these inhibitors may also be of importance in allowing activation of the pathway to proceed (Nydegger, Fearon & Austen, 1978). If haemoglobin is released into the circulation in substantial amounts, it seems likely that many of the biological consequences of complement activation would follow. This mechanism might be of importance in the genesis of shock and coagulation abnormalities associated with acute intravascular haemolysis. Chronic, low grade intravascular haemolysis occurs in sickle cell anaemia (Neely et al., 1969; Naumann et al., 1971), and haemoglobin released from ruptured sickled cells in capillary beds could cause local
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consumption of alternative pathway factors. This could account for reduced serum alternative pathway activity seen in some patients (Wilson et al., 1976; Auderset et al., 1977); reduced alternative pathway activity has been linked to defective bacterial opsonization, especially of pneumococci and salmonella organisms (Johnston, Newman & Struth, 1973; Hand & King, 1978) which have a marked predilection for the infection of patients with sickle cell anaemia (Eeckles, Gatti & Renoirte, 1967). The extent to which these mechanisms are of importance in vivo may depend on the binding of haemoglobin to carrier proteins in the blood, or the rate of its removal by the reticuloendothelial system. Further investigation of these aspects is needed. We thank Dr J.G.P. Sissons who prepared the pure factor B and C3 used here, and Drs E. Morrison and M. Wilson for help with fractionation. This work was supported by the Wellcome Trust, to whom we are grateful.
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