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Terminal stages of complement hemolysis: Technical comment
Immunology Letters, 3 (1981) 51-55
© Elsevier/North-Holland Biomedical Press
TERMINAL STAGES OF COMPLEMENT HEMOLYSIS: TECHNICAL COMMENT George H. WlRTZ and Shirley S. WESTFALL Department of Biochemistry, School of Medicine, West Virginia University, Morgantown, WV 26506, U.S.A.
(Received 27 November 1980) (Accepted 3 December 1980)
1. Summary E,boun d (a_~)E,inserte d ~ In studying terminal lytic events of sensitized erythrocytes which have reacted with all 9 components of complement, we agree in general with the conclusions of Boyle and Borsos [1 ] that the cellular intermediate passes through the stages E *b°und , E *inserted, and E *d°°med. However, we differ with these workers on the influence of 3 variables on the terminal reactions. These variables are: time of hemolysis; temperature of E* preparation; and the use of EDTA to manipulate E*. 2. Introduction
After sensitized erythrocytes have reacted with all 9 components of the classical complement pathway, the intermediate E* is formed which can be maintained intact for a considerable period of time. Cell lysis will occur when E* has passed through several reaction stages leading to one or more lesions in the cell membrane. Frank et al. [2] first described the various stages of the terminal transformation. More recently, Boyle et al. [3-6] re-examined the terminal stages of immune hemolysis and have proposed a reaction sequence similar to that of Frank et al. [2] but which includes several significant new features. The sequence suggested by Boyle et al., can be summarized as follows: Abbreviations: E, sheep erythrocytes; EA, sheep erythrocytes
sensitized with anti-Forsmann antibo4y; EAC1-7, EA which has reacted with complement components C1 through C7; EAC1-8 and EAC1-91 analogous definitions; E*, EA which has reacted with all 9 complement components but has not yet lysed (synonym of EAC1-9); Hb, hemoglobin.
E,doomed .(C!~Eghost + Hb.
E *b°und is formed by the reaction of EAC1-8 with C9; its hemolytic activity is destroyed by the action of trypsin, presumably on C9. Reaction (a) is temperature-sensitive and occurs very readily at 37°C. At the E *inserted stage, the hemolytic activity is no longer vulnerable to trypsin, presumably because C9 (or some other reactant) has become inserted into the membrane. Reaction (b) was reported to be inhibitable by the metal ions, Cu 2÷ Zn 2÷, and UO2 ÷ [4]. At the E *d°°med stage, the lesion has been formed. The functional size of the lesion (or hole) is governed by the number of C9 molecules reacting with the EAC 1-8 site; Kolb and Miiller-Eberhard [7] concluded that up to 6 C9s can react per C8. The existence of lesions of various sizes can be demonstrated by the use of solutes of different sizes at their isotonic concentrations [6]. For example, the lysis of cells with large lesions (formed at high C9/C8 ratio) will be prevented by 25% albumin because this solute cannot move through the lesion into the cell; lysis of these cells is not prevented, however, by isotonic glucose solutions since this solute will pass through the lesion. On the other hand, isotonic glucose (but not isotonic NaC1) will prevent the lysis of E* with small lesions. While studying the mechanism of an inhibitor of complement lysis, we undertook the preparation of the various E* intermediates of Boyle et al., and although our experience was consistent with the E *b°und , E *inserted , and E *d°°med stages described by those workers, we differ on the influence of 3 variables on the E* intermediates.
51
3. Materials and methods With minor modifications (noted below), the following buffers were prepared according to Rapp and Borsos [8]: Veronal-buffered saline (VBS) contained 0.05% gelatin (rather than 0.1% used by Rapp and Borsos); Veronal-buffered sucrose (VB-sucrose); 0.09 M EDTA, 0.05% gelatin; the isotonic EDTA of Rapp and Borsos was 0.1 M EDTA and lacked gelatin. Partially purified guinea pig C1 was prepared according to Nelson [9]. Highly purified human C8 was prepared according to Hammer et al. [10] and had a concentration of 1.3 × 1012 effective molecules per ml. Partially purified human C9 was prepared according to Hammer et al. [10] and had a concentration of 7.5 X 1013 effective molecules per ml; the contamination with C8 was found to be less than 1 in 10,000 based on an assay of effective molecules. Guinea pig C8 and C9 were obtained from Cordis Corporation. IgM anti-Forsmann antibody was kindly provided by Ms. Thelma Gaither, National Institute of Allergy and Infectious Diseases, Bethesda, MD; to sensitize red cells, this antibody was used at 3 times its optimal concentration as defined by Mayer [11 ]. E, EA, EAC 1-7 and EAC 1-8 were prepared according to Boyle et al. [3]; we used guinea pig C1 while Boyle et al. used human C 1. The C 4 - 7 reagent, used to prepare EAC1-7, was obtained from Cordis Corporation. EAC1-9 (E*) was made by treating EAC1-8 with C9 for various times and temperatures as described in the individual experiments. In most of the experiments reported here, we used EAC1-8 carrying a limiting number of C8 sites; these were treated with an excess of C9 [5]. With each set of reagents, preliminary titrations were necessary to establish the dilutions required for the desired quantities. E* was also prepared by treating EA (1 × 109/ml) with an equal volume of 1/20 guinea pig serum, all in VBS. After 4 min at 37°C, the cells were washed with 0°C VB-sucrose. Centrifugation during cell preparation was carried out at 4°C. The extent of lysis of the red ceils was determined by measuring, after centrifugation, the absorbance of the supernatant solution at 412 nm. The number of effective lesions per cell (Z) was calculated, according to the one-hit hypothesis, by the method described by Rapp and Borsos [8]. Trypsin (2 times crystallized, 10,000 units/mg) 52
was obtained from Sigma Chemicals. It was dissolved in VBS without gelatin to yield a concentration of 0.5 mg/ml. Preliminary experiments showed this was approximately twice the concentration needed to optimally inactivate E *b°und : Soybean trypsin inhibitor (2 times crystallized) obtained from Sigma Chemicals was dissolved in VBS without gelatin to yield a concentration of 0.5 mg/ml. Raffinose obtained from Sigma Chemicals was dissolved in 0.005 M Veronal buffer (pH 7.4) to yield a 0.3 M solution. 4. Results 4.1. Time required for E* lysis When studying the reactions leading to the several E* intermediates, the final experimental manipulation is to lyse the resulting EAC1-9 cell so that the extent o f reaction can be calculated. Boyle et al. [3] concluded that lysis of E* is completed in 90 min. Although we agree that this is the case when guinea pig C8 and C9 are used, Fig. 1 shows that when using
d U
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t-- 0.8 (J d
0,4
0,6 I
~
1
0,2
0
50
60
I
90
I
I
I
I
I
I
120 150 180 210 240 270
TIME (minutes)
Fig. 1. Comparison of kinetics of lysis of EAC1-9 prepared at 0°C and at 37°C. To a button of 1 × 10s EAC1-8 ceils carrying a limiting number of human C8 was added 0.10 ml of C9 solution diluted to provide an excess of C9; the cells were suspended by vortexing. Sample A was incubated 4 min at 0°C and Sample B 1.5 rain at 37°C. The EAC1-8 plus C9 reaction was stopped by adding 3.0 ml of 0°C 0.09 M EDTA; the cells were washed once with 0.09 M EDTA. The E* were resuspended to a concentration of 1× 107 cells/ml in VBS. The lytic reaction was carried out at 37°C. At various times, 1.0 ml of cell suspension was removed and centrifuged and the absorbance of the supernatant measured at 412 nm.
using limiting quantities of C8 and excess C9. It is apparent that though the endpoint of lysis for each cell type is the same, the initial lytic rate of E* made at 37°C is significantly higher. When we attempted to demonstrate, by means of trypsin digestion, the insertion reaction we again found that temperature was a crucial parameter. In addition, the time course of insertion was different from that found by Boyle et al. [3]. E* were prepared using human C8 (limiting) and human C9 (excess).
human C8 and C9, lysis is far from complete at 90 min. 4.2. Temperature of the EACI -8 plus C9 reaction When we undertook the manipulation of the terminal reactions, we found that temperature had a profound influence on the qualitative and quantitative nature of the intermediates. Fig.1 illustrates kinetically the lysis at 37’C of E* made at 0°C and 37°C from human C8 and C9; these cells were prepared
0.8
8 ____---
8 _I_______---
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0.6 c 0.5
0.4 0.3 (f *d-C
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0. I
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9
0’
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I
I
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C9 BINDING CONDITIONS O” 4
min.
Ttypsin
B
O” 4
min.
Control
C
37O 1% min.
Trypsin
D
37O 1% min.
Control
I
I
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5
6
7
8
9
TIME
TREATMENT
A
I
IO
I
11
I
12
(minutes)
Fig. 2. Temperature of the EACI-8 plus C9 reaction and its influence on the achievement of trypsin invulnerability (insertion). To a button of 2 X 10’ EAC1-8 cells carrying a limiting number of human C8 sites was added 0.20 ml of human C9 solution to provide an excess of C9. The suspension was vortexed and incubated for the time and temperature shown on the table in Fig. 2. The reaction was stopped with 3 ml 0°C VBS and the cells washed with the same buffer. The cells were resuspended to a concentration of 1 X lOa cells/ml in 0°C VBS lacking gelatin and dispensed in O.lO-ml portions into a series of tubes and incubated at 37°C for times varying from 0 to 12 mm (insertion step) after which they were transferred to an ice bath. The control cells received 1.4 ml VBS. The treated cells received 0.10 ml trypsin and were held at 0°C for 15 min after which 0.10 ml soybean trypsin inhibitor and 1.2 ml VBS were added. All cell samples were allowed to lyse at 37°C for 90 min and the extent of lysis determined spectrophotometrically. Controls were included for EACI-8 fragility to trypsin and for the ability of soybean trypsin inhibitor to stop trypsin action on E*.
53
EAC1-8 cells were treated with C9 either at 0°C for 4 rain or 37°C for various times up to 2.5 rain. The insertion reaction on EAC 1-9 was permitted to occur for various times at 37°C after which the E* was subjected to trypsin at 0°C. After washing, the cells were allowed to lyse at 37°C. The results are shown in Fig. 2. Curves A and B show that EAC 1-9 prepared at 0°C are indeed vulnerable to trypsin. But unlike Boyle et al. [3], we were not able to attain complete insertion (or trypsin invulnerability) during the 12-rain incubation period; Boyle et al. [3] were able to show complete insertion in 3 rain. On the other hand, when the EAC1-8 plus C9 reaction was carried out at 37°C for 2 min, the resulting E* was invulnerable to trypsin; apparently C9 insertion was completed during the 2 rain that C9 was reacting with EAC1-8 (data not shown). By carrying out the EAC1-8 plus C9 reaction at 37°C for a shorter time ( 1 - 1 / 3 min), it was possible to demonstrate trypsin vulnerability and complete insertion (curves C and D). Even in this case, the time required for insertion (11 rain) was much longer than that found by Boyle et al. (3 min). Furthermore, only about half of the lytic sites were available to trypsin at zero time, suggesting that a significant amount of insertion had already taken place. 4.3. Action o f EDTA on E* Boyle et al. [5] have concluded that EDTA at its isotonic concentration will block osmotically the lysis of E *d°°m~. We have found that in addition, 0.09 M EDTA can block a reaction prior to the E *d°°med stage. This is shown in Fig. 3 which illustrates some of the characteristics of E* lysis. E* were prepared using guinea pig C8 and C9. When such cells were preincubated in 0.3 M raffinose (as an osmotic blocker) at 37°C for 110 min, the cells were converted to E *d°°med as shown by their rapid lysis in VBS (curve A). But if such cells were preincubated in 0.09 M EDTA at 37°C (curve C) or held in raffinose at 0°C as a control (curve B), the lysis in VBS was very much slower indicating that they were held at an earlier E* stage, probably E *inserted . In a separate experiment not shown here, we showed that cells assumed to be E *d°°med were in fact at that stage by the observation that they displayed the same lyric kinetics at 0°C as they did at 37°C [5]. 54
8O 70 03
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Fig. 3. Kinetics of E* lysis after preincubation in isotonic EDTA (37°C) or isotonic raffinose (0°C and 37°C). EACI-8 cells carrying a limiting n u m b e r of guinea pig C8 sites were prepared using Cordis C 8 . 0 . 5 ml of Cordis guinea pig C9 diluted 1/2.5 (to provide an excess of C9) was added to a b u t t o n of 5 × l0 s EAC1-8 and the cells were suspended by vortexing. The cells were incubated 10 min at 0°C and the reaction stopped by adding 3.0 ml of 0°C VB-sucrose; the cells were washed and resuspended in cold VB-sucrose, divided into several portions and centrifuged and drained. The cells were pretreated by suspending in either 0.3 M raffinose or 0.09 M EDTA and incubating at either 0°C or 37°C for 110 min. The E* were centrifuged, drained and resuspended to a concentration o f 1 X 107 cells per ml in VBS. The lytic reaction was carried out at 37°C. At various times, the supernatant from 1.0 ml of suspension was collected by centrifugation and the extent o f lysis measured spectrophotometrically.
5. Discussion
The terminal stages of immune hemolysis constitute a complex series of reactions taking place in a poorly defined milieu (the red cell membrane). Thus, it would not be surprising if small differences in the chemistry of the reactants or their milieu produced significant difference in the course of the tytic reaction. The general conclusions we have drawn from the work reported here is that each laboratory must carefully establish the conditions to unambiguously carry out the terminal reactions. Boyle et al. [3] found that the lysis of E* is complete in 90 rain. Fig. 1 shows that in our hands, E* made using human C8 and C9 requires considerably more than 90 rain to reach the end point. In carrying out the EAC 1-8 plus C9 reaction, Boyle et al. [3] state that at 0°C maximum C9 binding is achieved by 5 rain. Boyle et al. cite the work of Rommel and Mayer [12] and of Kolb and MOllerEberhard [7] in relation to the influence of temper-
ature on C9 binding; both of these groups emphasized that the EAC1-8 plus C9 reaction is relatively insensitive to temperature when com1~ared to enzyme-catalyzed reactions. Nevertheless, Figs. 1 and 2 demonstrate that temperature does have a profound influence on the nature of the E* produced. Fig. 1 shows that E* (human) made at 0°C will display significantly different kinetics of lysis from those made at 37°C. This could be accounted for by a greater multiplicity of C9 per C8 site being achieved at 37°C; the more rapid kinetics would be consistent with larger lesions being formed at 37°C. In an experiment not presented here, we found that as the temperature of C9 binding was raised from 0°C to 37°C, the fraction of lytic sites osmotically blocked by sucrose was halved; i.e. the average size of the lesions was greater when they were formed at the higher temperature. Fig. 2 shows that the temperature o f the EAC1-8 + C9 reaction has a profound effect on the nature of the intermediate as assessed by trypsin vulnerability. When Boyle et al. [3] made E* at 0°C using human C8 and C9, the resulting E *b°und was converted to E *inserted in 3 min at 37°C. Fig.2 shows that our E*, made in a similar way, had not completed the insertion reaction after 12 rain. If we carried out the EAC1-8 + C9 reaction at 37°C for 2 rain, none of the E* was trypsin-vulnerable (these data are not shown on the graph); apparently, the insertion reaction had taken place almost immediately. Thus, the thermal history of this system governs the ease with which insertion occurs: when C9 encounters EACI-8 at 37°C (2 min), the insertion is rapid; but if the temperature is 0°C when C9 first encounters EAC1-8, insertion is very slow even after the temperature is raised to 37°C. The implications this may have for membrane transformations and protein-membrane interactions are too speculative to discuss here. It is quite possible that the nature of this phenomenon will vary with the source of sheep erythrocytes. It should be noted that in the experiments of Boyle et al. [3], the C8 sites were in excess and the C9 was limiting, which is the reverse of the experiments reported here. We also did the experiment (not presented) using excess C8 and limiting C9; we again found that the insertion reaction was very slow. In experiments not presented here, we found that E* prepared with guinea pig C8 and C9 did not display trypsin vulnerability; presumably insertion was very rapid even at 0°C.
When we attempted to use 0.09 M EDTA as an osmotic blocker similar to 0.3 M raffinose or 25% albumin, we found that the action of EDTA was more complex (Fig. 3). EDTA apparently could act at a stage before E *d°°med ; when we used another sheep as a source of red cells, we obtained the same result. This is completely different from the findings of Boyle et al. [5] who concluded that isotonic EDTA acts only to prevent lysis of E *d°°med . We have no way of accounting for this disparity. It should be noted that in experiments done by Boyle et al. [5], E* prepared with excess C8 were used; we used limiting C8. In any case, it would seem advisable on general principles to avoid the use of concentrated EDTA for this purpose since in addition to having a certain Stoke's radius, this compound has another property of biological significance (i.e. the ability to chelate divalent cations). An acceptable alternative would be raffinose since results of Boyle et al. [6] indicate it has about the same functional size as EDTA.
Acknowledgements Supported by NIH Grant 2-RO1-AM 19716 and by a Biomedical Research Support Grant from the School of Medicine, West Virginia University.
References [ 1] Boyle, M. D. P. and Borsos, T. (1980) Mol. Immunol. 17,425-432. [2] Frank, M. M., Rapp, H. J. and Borsos, T. (1965) J. Immunol. 94,295. [3] Boyle, M. D. P., Langone, J. J. and Borsos, T. (1978) J. Immunol. 120, 1721. [4] Boyle, M. D. P., Langone, J. J. and Borsos, T. (1979) J. Immunol. 122, 1209. [5 ] Boyle, M. D. P. and Borsos, T. (1979) J. Immunol. 123, 71. [6] Boyle, M. D. P., Gee, A. P. and Borsos, T. (1979) J. Immunol. 123, 77. [7] Kolb, W. P. and Miiller-Eberhard, H. J. (1974) J. Immunol. 13,479. [8] Rapp, H. J. and Borsos, T. (1970) in: Molecular Basis of Complement Action, Appleton-Century-Crofts, New York. [9] Nelson, R. A., Jensen, J., Gigli, I. and Tamura, N. (1966) Immunochemistry 3, 111. [10] Hammer, C. H., Wirtz, G. H., Renfer, L., Gresham, H. D. and Tack, F. F. (1980) J. Biol. Chem. (in press). [ 11 ] Mayer, M. M. (1969) in: Experimental Immunochemistry (Kabat, E. A. ed.) p. 133. Charles C. Thomas, Springfield, IL [12] Rommel, F. A. and Mayer, M. M. (1973) J. Immunol. 110,637. 55
© Elsevier/North-Holland Biomedical Press
TERMINAL STAGES OF COMPLEMENT HEMOLYSIS: TECHNICAL COMMENT George H. WlRTZ and Shirley S. WESTFALL Department of Biochemistry, School of Medicine, West Virginia University, Morgantown, WV 26506, U.S.A.
(Received 27 November 1980) (Accepted 3 December 1980)
1. Summary E,boun d (a_~)E,inserte d ~ In studying terminal lytic events of sensitized erythrocytes which have reacted with all 9 components of complement, we agree in general with the conclusions of Boyle and Borsos [1 ] that the cellular intermediate passes through the stages E *b°und , E *inserted, and E *d°°med. However, we differ with these workers on the influence of 3 variables on the terminal reactions. These variables are: time of hemolysis; temperature of E* preparation; and the use of EDTA to manipulate E*. 2. Introduction
After sensitized erythrocytes have reacted with all 9 components of the classical complement pathway, the intermediate E* is formed which can be maintained intact for a considerable period of time. Cell lysis will occur when E* has passed through several reaction stages leading to one or more lesions in the cell membrane. Frank et al. [2] first described the various stages of the terminal transformation. More recently, Boyle et al. [3-6] re-examined the terminal stages of immune hemolysis and have proposed a reaction sequence similar to that of Frank et al. [2] but which includes several significant new features. The sequence suggested by Boyle et al., can be summarized as follows: Abbreviations: E, sheep erythrocytes; EA, sheep erythrocytes
sensitized with anti-Forsmann antibo4y; EAC1-7, EA which has reacted with complement components C1 through C7; EAC1-8 and EAC1-91 analogous definitions; E*, EA which has reacted with all 9 complement components but has not yet lysed (synonym of EAC1-9); Hb, hemoglobin.
E,doomed .(C!~Eghost + Hb.
E *b°und is formed by the reaction of EAC1-8 with C9; its hemolytic activity is destroyed by the action of trypsin, presumably on C9. Reaction (a) is temperature-sensitive and occurs very readily at 37°C. At the E *inserted stage, the hemolytic activity is no longer vulnerable to trypsin, presumably because C9 (or some other reactant) has become inserted into the membrane. Reaction (b) was reported to be inhibitable by the metal ions, Cu 2÷ Zn 2÷, and UO2 ÷ [4]. At the E *d°°med stage, the lesion has been formed. The functional size of the lesion (or hole) is governed by the number of C9 molecules reacting with the EAC 1-8 site; Kolb and Miiller-Eberhard [7] concluded that up to 6 C9s can react per C8. The existence of lesions of various sizes can be demonstrated by the use of solutes of different sizes at their isotonic concentrations [6]. For example, the lysis of cells with large lesions (formed at high C9/C8 ratio) will be prevented by 25% albumin because this solute cannot move through the lesion into the cell; lysis of these cells is not prevented, however, by isotonic glucose solutions since this solute will pass through the lesion. On the other hand, isotonic glucose (but not isotonic NaC1) will prevent the lysis of E* with small lesions. While studying the mechanism of an inhibitor of complement lysis, we undertook the preparation of the various E* intermediates of Boyle et al., and although our experience was consistent with the E *b°und , E *inserted , and E *d°°med stages described by those workers, we differ on the influence of 3 variables on the E* intermediates.
51
3. Materials and methods With minor modifications (noted below), the following buffers were prepared according to Rapp and Borsos [8]: Veronal-buffered saline (VBS) contained 0.05% gelatin (rather than 0.1% used by Rapp and Borsos); Veronal-buffered sucrose (VB-sucrose); 0.09 M EDTA, 0.05% gelatin; the isotonic EDTA of Rapp and Borsos was 0.1 M EDTA and lacked gelatin. Partially purified guinea pig C1 was prepared according to Nelson [9]. Highly purified human C8 was prepared according to Hammer et al. [10] and had a concentration of 1.3 × 1012 effective molecules per ml. Partially purified human C9 was prepared according to Hammer et al. [10] and had a concentration of 7.5 X 1013 effective molecules per ml; the contamination with C8 was found to be less than 1 in 10,000 based on an assay of effective molecules. Guinea pig C8 and C9 were obtained from Cordis Corporation. IgM anti-Forsmann antibody was kindly provided by Ms. Thelma Gaither, National Institute of Allergy and Infectious Diseases, Bethesda, MD; to sensitize red cells, this antibody was used at 3 times its optimal concentration as defined by Mayer [11 ]. E, EA, EAC 1-7 and EAC 1-8 were prepared according to Boyle et al. [3]; we used guinea pig C1 while Boyle et al. used human C 1. The C 4 - 7 reagent, used to prepare EAC1-7, was obtained from Cordis Corporation. EAC1-9 (E*) was made by treating EAC1-8 with C9 for various times and temperatures as described in the individual experiments. In most of the experiments reported here, we used EAC1-8 carrying a limiting number of C8 sites; these were treated with an excess of C9 [5]. With each set of reagents, preliminary titrations were necessary to establish the dilutions required for the desired quantities. E* was also prepared by treating EA (1 × 109/ml) with an equal volume of 1/20 guinea pig serum, all in VBS. After 4 min at 37°C, the cells were washed with 0°C VB-sucrose. Centrifugation during cell preparation was carried out at 4°C. The extent of lysis of the red ceils was determined by measuring, after centrifugation, the absorbance of the supernatant solution at 412 nm. The number of effective lesions per cell (Z) was calculated, according to the one-hit hypothesis, by the method described by Rapp and Borsos [8]. Trypsin (2 times crystallized, 10,000 units/mg) 52
was obtained from Sigma Chemicals. It was dissolved in VBS without gelatin to yield a concentration of 0.5 mg/ml. Preliminary experiments showed this was approximately twice the concentration needed to optimally inactivate E *b°und : Soybean trypsin inhibitor (2 times crystallized) obtained from Sigma Chemicals was dissolved in VBS without gelatin to yield a concentration of 0.5 mg/ml. Raffinose obtained from Sigma Chemicals was dissolved in 0.005 M Veronal buffer (pH 7.4) to yield a 0.3 M solution. 4. Results 4.1. Time required for E* lysis When studying the reactions leading to the several E* intermediates, the final experimental manipulation is to lyse the resulting EAC1-9 cell so that the extent o f reaction can be calculated. Boyle et al. [3] concluded that lysis of E* is completed in 90 min. Although we agree that this is the case when guinea pig C8 and C9 are used, Fig. 1 shows that when using
d U
1.2 1.0
t-- 0.8 (J d
0,4
0,6 I
~
1
0,2
0
50
60
I
90
I
I
I
I
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120 150 180 210 240 270
TIME (minutes)
Fig. 1. Comparison of kinetics of lysis of EAC1-9 prepared at 0°C and at 37°C. To a button of 1 × 10s EAC1-8 ceils carrying a limiting number of human C8 was added 0.10 ml of C9 solution diluted to provide an excess of C9; the cells were suspended by vortexing. Sample A was incubated 4 min at 0°C and Sample B 1.5 rain at 37°C. The EAC1-8 plus C9 reaction was stopped by adding 3.0 ml of 0°C 0.09 M EDTA; the cells were washed once with 0.09 M EDTA. The E* were resuspended to a concentration of 1× 107 cells/ml in VBS. The lytic reaction was carried out at 37°C. At various times, 1.0 ml of cell suspension was removed and centrifuged and the absorbance of the supernatant measured at 412 nm.
using limiting quantities of C8 and excess C9. It is apparent that though the endpoint of lysis for each cell type is the same, the initial lytic rate of E* made at 37°C is significantly higher. When we attempted to demonstrate, by means of trypsin digestion, the insertion reaction we again found that temperature was a crucial parameter. In addition, the time course of insertion was different from that found by Boyle et al. [3]. E* were prepared using human C8 (limiting) and human C9 (excess).
human C8 and C9, lysis is far from complete at 90 min. 4.2. Temperature of the EACI -8 plus C9 reaction When we undertook the manipulation of the terminal reactions, we found that temperature had a profound influence on the qualitative and quantitative nature of the intermediates. Fig.1 illustrates kinetically the lysis at 37’C of E* made at 0°C and 37°C from human C8 and C9; these cells were prepared
0.8
8 ____---
8 _I_______---
0.7
0.6 c 0.5
0.4 0.3 (f *d-C
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E’ SAMPLE
9
0’
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I
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Ttypsin
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Control
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Control
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5
6
7
8
9
TIME
TREATMENT
A
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(minutes)
Fig. 2. Temperature of the EACI-8 plus C9 reaction and its influence on the achievement of trypsin invulnerability (insertion). To a button of 2 X 10’ EAC1-8 cells carrying a limiting number of human C8 sites was added 0.20 ml of human C9 solution to provide an excess of C9. The suspension was vortexed and incubated for the time and temperature shown on the table in Fig. 2. The reaction was stopped with 3 ml 0°C VBS and the cells washed with the same buffer. The cells were resuspended to a concentration of 1 X lOa cells/ml in 0°C VBS lacking gelatin and dispensed in O.lO-ml portions into a series of tubes and incubated at 37°C for times varying from 0 to 12 mm (insertion step) after which they were transferred to an ice bath. The control cells received 1.4 ml VBS. The treated cells received 0.10 ml trypsin and were held at 0°C for 15 min after which 0.10 ml soybean trypsin inhibitor and 1.2 ml VBS were added. All cell samples were allowed to lyse at 37°C for 90 min and the extent of lysis determined spectrophotometrically. Controls were included for EACI-8 fragility to trypsin and for the ability of soybean trypsin inhibitor to stop trypsin action on E*.
53
EAC1-8 cells were treated with C9 either at 0°C for 4 rain or 37°C for various times up to 2.5 rain. The insertion reaction on EAC 1-9 was permitted to occur for various times at 37°C after which the E* was subjected to trypsin at 0°C. After washing, the cells were allowed to lyse at 37°C. The results are shown in Fig. 2. Curves A and B show that EAC 1-9 prepared at 0°C are indeed vulnerable to trypsin. But unlike Boyle et al. [3], we were not able to attain complete insertion (or trypsin invulnerability) during the 12-rain incubation period; Boyle et al. [3] were able to show complete insertion in 3 rain. On the other hand, when the EAC1-8 plus C9 reaction was carried out at 37°C for 2 min, the resulting E* was invulnerable to trypsin; apparently C9 insertion was completed during the 2 rain that C9 was reacting with EAC1-8 (data not shown). By carrying out the EAC1-8 plus C9 reaction at 37°C for a shorter time ( 1 - 1 / 3 min), it was possible to demonstrate trypsin vulnerability and complete insertion (curves C and D). Even in this case, the time required for insertion (11 rain) was much longer than that found by Boyle et al. (3 min). Furthermore, only about half of the lytic sites were available to trypsin at zero time, suggesting that a significant amount of insertion had already taken place. 4.3. Action o f EDTA on E* Boyle et al. [5] have concluded that EDTA at its isotonic concentration will block osmotically the lysis of E *d°°m~. We have found that in addition, 0.09 M EDTA can block a reaction prior to the E *d°°med stage. This is shown in Fig. 3 which illustrates some of the characteristics of E* lysis. E* were prepared using guinea pig C8 and C9. When such cells were preincubated in 0.3 M raffinose (as an osmotic blocker) at 37°C for 110 min, the cells were converted to E *d°°med as shown by their rapid lysis in VBS (curve A). But if such cells were preincubated in 0.09 M EDTA at 37°C (curve C) or held in raffinose at 0°C as a control (curve B), the lysis in VBS was very much slower indicating that they were held at an earlier E* stage, probably E *inserted . In a separate experiment not shown here, we showed that cells assumed to be E *d°°med were in fact at that stage by the observation that they displayed the same lyric kinetics at 0°C as they did at 37°C [5]. 54
8O 70 03
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Fig. 3. Kinetics of E* lysis after preincubation in isotonic EDTA (37°C) or isotonic raffinose (0°C and 37°C). EACI-8 cells carrying a limiting n u m b e r of guinea pig C8 sites were prepared using Cordis C 8 . 0 . 5 ml of Cordis guinea pig C9 diluted 1/2.5 (to provide an excess of C9) was added to a b u t t o n of 5 × l0 s EAC1-8 and the cells were suspended by vortexing. The cells were incubated 10 min at 0°C and the reaction stopped by adding 3.0 ml of 0°C VB-sucrose; the cells were washed and resuspended in cold VB-sucrose, divided into several portions and centrifuged and drained. The cells were pretreated by suspending in either 0.3 M raffinose or 0.09 M EDTA and incubating at either 0°C or 37°C for 110 min. The E* were centrifuged, drained and resuspended to a concentration o f 1 X 107 cells per ml in VBS. The lytic reaction was carried out at 37°C. At various times, the supernatant from 1.0 ml of suspension was collected by centrifugation and the extent o f lysis measured spectrophotometrically.
5. Discussion
The terminal stages of immune hemolysis constitute a complex series of reactions taking place in a poorly defined milieu (the red cell membrane). Thus, it would not be surprising if small differences in the chemistry of the reactants or their milieu produced significant difference in the course of the tytic reaction. The general conclusions we have drawn from the work reported here is that each laboratory must carefully establish the conditions to unambiguously carry out the terminal reactions. Boyle et al. [3] found that the lysis of E* is complete in 90 rain. Fig. 1 shows that in our hands, E* made using human C8 and C9 requires considerably more than 90 rain to reach the end point. In carrying out the EAC 1-8 plus C9 reaction, Boyle et al. [3] state that at 0°C maximum C9 binding is achieved by 5 rain. Boyle et al. cite the work of Rommel and Mayer [12] and of Kolb and MOllerEberhard [7] in relation to the influence of temper-
ature on C9 binding; both of these groups emphasized that the EAC1-8 plus C9 reaction is relatively insensitive to temperature when com1~ared to enzyme-catalyzed reactions. Nevertheless, Figs. 1 and 2 demonstrate that temperature does have a profound influence on the nature of the E* produced. Fig. 1 shows that E* (human) made at 0°C will display significantly different kinetics of lysis from those made at 37°C. This could be accounted for by a greater multiplicity of C9 per C8 site being achieved at 37°C; the more rapid kinetics would be consistent with larger lesions being formed at 37°C. In an experiment not presented here, we found that as the temperature of C9 binding was raised from 0°C to 37°C, the fraction of lytic sites osmotically blocked by sucrose was halved; i.e. the average size of the lesions was greater when they were formed at the higher temperature. Fig. 2 shows that the temperature o f the EAC1-8 + C9 reaction has a profound effect on the nature of the intermediate as assessed by trypsin vulnerability. When Boyle et al. [3] made E* at 0°C using human C8 and C9, the resulting E *b°und was converted to E *inserted in 3 min at 37°C. Fig.2 shows that our E*, made in a similar way, had not completed the insertion reaction after 12 rain. If we carried out the EAC1-8 + C9 reaction at 37°C for 2 rain, none of the E* was trypsin-vulnerable (these data are not shown on the graph); apparently, the insertion reaction had taken place almost immediately. Thus, the thermal history of this system governs the ease with which insertion occurs: when C9 encounters EACI-8 at 37°C (2 min), the insertion is rapid; but if the temperature is 0°C when C9 first encounters EAC1-8, insertion is very slow even after the temperature is raised to 37°C. The implications this may have for membrane transformations and protein-membrane interactions are too speculative to discuss here. It is quite possible that the nature of this phenomenon will vary with the source of sheep erythrocytes. It should be noted that in the experiments of Boyle et al. [3], the C8 sites were in excess and the C9 was limiting, which is the reverse of the experiments reported here. We also did the experiment (not presented) using excess C8 and limiting C9; we again found that the insertion reaction was very slow. In experiments not presented here, we found that E* prepared with guinea pig C8 and C9 did not display trypsin vulnerability; presumably insertion was very rapid even at 0°C.
When we attempted to use 0.09 M EDTA as an osmotic blocker similar to 0.3 M raffinose or 25% albumin, we found that the action of EDTA was more complex (Fig. 3). EDTA apparently could act at a stage before E *d°°med ; when we used another sheep as a source of red cells, we obtained the same result. This is completely different from the findings of Boyle et al. [5] who concluded that isotonic EDTA acts only to prevent lysis of E *d°°med . We have no way of accounting for this disparity. It should be noted that in experiments done by Boyle et al. [5], E* prepared with excess C8 were used; we used limiting C8. In any case, it would seem advisable on general principles to avoid the use of concentrated EDTA for this purpose since in addition to having a certain Stoke's radius, this compound has another property of biological significance (i.e. the ability to chelate divalent cations). An acceptable alternative would be raffinose since results of Boyle et al. [6] indicate it has about the same functional size as EDTA.
Acknowledgements Supported by NIH Grant 2-RO1-AM 19716 and by a Biomedical Research Support Grant from the School of Medicine, West Virginia University.
References [ 1] Boyle, M. D. P. and Borsos, T. (1980) Mol. Immunol. 17,425-432. [2] Frank, M. M., Rapp, H. J. and Borsos, T. (1965) J. Immunol. 94,295. [3] Boyle, M. D. P., Langone, J. J. and Borsos, T. (1978) J. Immunol. 120, 1721. [4] Boyle, M. D. P., Langone, J. J. and Borsos, T. (1979) J. Immunol. 122, 1209. [5 ] Boyle, M. D. P. and Borsos, T. (1979) J. Immunol. 123, 71. [6] Boyle, M. D. P., Gee, A. P. and Borsos, T. (1979) J. Immunol. 123, 77. [7] Kolb, W. P. and Miiller-Eberhard, H. J. (1974) J. Immunol. 13,479. [8] Rapp, H. J. and Borsos, T. (1970) in: Molecular Basis of Complement Action, Appleton-Century-Crofts, New York. [9] Nelson, R. A., Jensen, J., Gigli, I. and Tamura, N. (1966) Immunochemistry 3, 111. [10] Hammer, C. H., Wirtz, G. H., Renfer, L., Gresham, H. D. and Tack, F. F. (1980) J. Biol. Chem. (in press). [ 11 ] Mayer, M. M. (1969) in: Experimental Immunochemistry (Kabat, E. A. ed.) p. 133. Charles C. Thomas, Springfield, IL [12] Rommel, F. A. and Mayer, M. M. (1973) J. Immunol. 110,637. 55