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Heterogeneity in the size and stability of transmembrane channels produced by whole complement
CLINICAL
IMMUNOLOGY
AND
lMMUNOPATHOLOCY
20, 287-295 (1981)
Heterogeneity in the Size and Stability of Transmembrane Channels Produced by Whole Complement MICHAEL D. P. BOYLE,’ Laboratory
of Immunobiology,
ADRIAN P. GEE, AND TIBOR BORSOS
National Bethesda,
Cancer Institute, Maryland 20205
National
Institates
of Health,
The functional size of complement (C) lesions produced by treatment of EAIgM with human or guinea pig C was studied. In all cases, a distribution of stable C lesions differing in size could be distinguished. Differences between the distribution of differing-sized lesions were observed depending on whether EAIgM were lysed with human (Hu) or guinea pig (GP) C. This difference appeared to be related to the kinetics of transformation prepared with HuC lysed more slowly than those of E*wecursor to Eghosts; E*III.CUIGUI prepared with GPC. The significance of these findings to the wide range of estimates reported for the size of the C lesion is briefly discussed.
INTRODUCTION
In studies of the lysis of EACl-8 by C9, we have shown that the more C9 was bound to a given SACl-8 the larger the C2 lesion produced (l-3), although binding of a single C9 molecule is sufficient for lesion production (4-5). In these and other studies of the lytic effects of individual functionally pure components, experimental conditions were usually controlled so that the degree of lysis was governed solely by the C component being studied, i.e., the reaction was performed under pseudo-first-order conditions. By contrast, the activity of C in whole serum consists of the interaction of C components as well as regulating proteins and consequently the final reaction step with C9 may or may not behave as a pseudofirst-order reaction. Thus, it has not been established whether the heterogeneity in the size of C channels was a consequence of the sequential addition of purified components in an artificial system in which EACl-8 were treated with varying concentrations of C9, or, if such variations in the size of the C lesion would occur when whole C was used. The purpose of the present study was to examine a variety of experimental conditions of lysis of EA by whole C to determine whether heterogeneity of C lesions could be detected. MATERIALS
AND METHODS
Cells. Sheep red blood cells (E) were collected and washed as described in (6). Buffers. Isotonic veronal-buffered saline (VBS), pH 7.4, containing 0.1% gelatin I Present address: Dr. Michael D. P. Boyle, Department of Immunology and Medical Microbiology, College of Medicine, Box J-266, JHMHC, University of Florida, Gainesville, FL 32610. p Abbreviations used: E, sheep erythrocytes; VBS-gel, isotonic Veronal-buffered saline containing 0.1% gelatin, 0.001 M Mg”’ and O.OOO15M Cd L+; VBS-EDTA, isotonic Veronal-buffered saline containing 0.1% gelatin and disodium ethylenediaminetetraacetate; E*, sheep erythrocytes with at least one cell bound antigen-antibody complex and all the necessary complement components for killing to occur: Hu or hu, human: C, complement: BSA, bovine serum albumin. 287 OO90-1229/81/O90287-O9$01.00/0 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
288
BOYLE,
GEE,
AND
BORSOS
(Mann Research Labs., N.Y.), 0.001 M Mg’+, and 0.0015 A4 Ca” (VBS-gel); sucrose buffer, Al. = 0.065 (VBS-sucrose); 0. I M EDTA buffer, were prepared as described by Rapp and Borsos (6). Glucose monohydrate was obtained from Baker Chemical Company. Philadelphia. Pennsylvania, sucrose (ultracentrifuge grade) was obtained from Schwarz-Mann. Orangeburg, New York, and raffinose pentahydrate was obtained from Vega Chemicals. Tucson. Arizona. All sugars were used as 0.3 M solutions in water. Albumin. Bovine serum albumin (Pathocyte 5) was obtained from Miles Laboratories. Elkhart, Indiana, and was used in all experiments at a concentration of 25%. Anrihody. Anti-Forssman antiserum was raised in New Zealand albino rabbits according to the procedures given in (7). The IgM and IgG fractions were separated on DEAE as described by Boyle and Langone (8). Complement. Fresh guinea pig serum (pooled) (JEM Research Inc., Kensington. Md.) or human serum were used as complement sources. Both were absorbed with packed E and stored in aliquots at -30°C. ComplPmrnt components. Functionally purified complement components were obtained from the Cordis Corporation, Miami. Florida. Indicator cells. EAIgM and EAIgG were prepared by incubating E at 109/ml with an equal volume of a dilution of either the IgM or IgG fraction of rabbit anti-Forssman antiserum which produced 100% lysis of the cells within 15 min upon addition of excess C. Hrmoglohin &termination. Relative hemoglobin levels were determined on a Micromedic MS2 spectrophotometer by measuring the absorbance at a wavelength of 412 or 541 nm. RESULTS
During the interaction of EA with C, cells may be in one of the following states: pre-E*, E* or Eghost (see Fig. 1). Eghost are cells that have released their hemoglobin, E* are cells that will release their hemoglobin in the absence of any further interaction with fluid-phase C, while pre-E* are cells at any stage between EA and EACl-8. The proportion of cells in each of these states can be quantified by appropriate experimental procedures. For example, cells that have not reached the E* precursor stage will not lyse during a subsequent incubation in VBS-gel unless fluid-phase C is added (see Fig. 1). Cells in any of the E* states will lyse in the presence of VBS-gel but not in the presence of 25% albumin, and cells in the Eghost state have already released their hemoglobin. Consequently. by measuring the proportion of cells from which hemoglobin was released during the initial exposure to fluid-phase C, or during subsequent incubation in VBS-gel or 25% albumin, in the absence of fluid-phase C, it is possible to determine the percentage of cells that(i) have not formed E*, (ii) that have formed E* but not lysed (i.e., in the E* transformation sequence), and (iii) have already lysed (Eghost). In the first series of experiments the kinetics of production of E*preeursorand the subsequent transformation of these cells to Eghost was followed. These studies were performed using 1.5 x lo* E sensitized with excess IgM anti-Forssman
SIZE
VARIATION
AND
STABILITY
E* E*precursor tsmpePa~e dependent ’ inserted
EA + C-----b I
OF
289
C LESIONS
inhibitable by ainc or ’ uranyz salts
blocked by 25x nlbwlrin~
E*dLIOWd
E gtmst +
HB
I I
(EAC1-g--=*
(EACI-9boun*)
I
REQ”mES FLUIDPHASE c
I
(sAC1-9*o~me*)
)
DOES NOT REQ”IKE FL”ID-PHASE
c
I
FIG.
1. Reaction
sequence
for the transformation
of pre-E*
to Eghost.
antibody and incubated for varying times with 1 ml of a l/200 dilution of GPC or HuC diluted 11100. At the appropriate times the reactions were stopped by adding 5 ml ice-cold 0.1 M EDTA and the cells centrifuged. The degree of lysis occurring at the time of sampling was determined by measuring the amount of hemoglobin in the supernatant fluid (Eghost) and the degree of E* formation was quantified by measuring the amount of lysis in VBS-gel or 25% BSA following a further 60-min incubation of the cells at 37°C. The results obtained with EAIgM and GPC indicate that E*PreCUm,,rwas formed, rapidly followed by the appearance of Eghosts (Fig. 2A). The maximum percentage of cells that could be isolated in the E* state was 54%. In the experiments using HuC as the C source, a number of differences were observed (Fig. 2B). While the formation of E* Prec,,rSOr from EAIgM was rapid in the presence of HuC, unlike the equivalent situation with GPC, the transformation of E* to Eghost was much slower and it was possible to isolate >90% of the cells in the E* transformation sequence (Fig. 2B). Similar E* intermediates with an endpoint lysis potential of >80% could never be achieved using GPC because of Eghost formation.
EAlgM
+ GPC
EAlgM
100
E Ghost
-2 GM CiI 2
q E Ghost
E”
c b
P60 Y 540 8 g20
+ HuC
r
bC a
a
EX A 0 K:KI 0
4
8
12
160 4 TIME (min.1
8 8
12
16
20
24
FIG. 2. Kinetics of E* formation and lysis using GPC or HuC. (A) E* prepared from GPC: (B) E* prepared from EAIgM and HuC. No lysis of E* occurred in the continuing 25% albumin. The size of C lesions on the E* at various stages (a, b, and c) was examined See text for precise experimental details.
EAIgM and presence of (See Fig. 3).
290
BOYLE,
GEE.
AND
BORSOS
A similar pattern of hemolysis was obtained with EAlgG and GPC although some minor differences as compared to EAIgM were apparent, For example. the rate of E”’ transformation to Eghosts appeared slower than for the equivalent intermediates prepared with IgM and consequently the maximum percentage of cells that could be isolated in the E* states was greater (approximately 80%). Cells in any of the E’% states could be prevented from lysing by incubation in 25% BSA (data not shown). At the times indicated in Figs. 2A and B. E:;: were obtained and their ability to transform to Eghosts in the presence of isotonic solutes of differing Stokes’ radii was compared. The solutes chosen were NaCl with a Stokes’ radius of 1.4 A. glucose with a Stokes’ radius of 3.6 A. raffinose with a Stokes’ radius of 5.6 A, and albumin with a Stokes’ radius of 36 A. The degree of E*: transformation to Eghost in each solution was measured following incubation for 1 hr at 37°C. The results for E* prepared from EAIgM and GPC are shown in Fig. 3 (top) and for EAIgM and HuC are shown in Fig. 3 (bottom), and they indicate that a distribution of different-sized C lesions could be identified. A comparison of the results in Figs. 3A and B indicate differences between the distribution of different-sized C lesions produced by GPC and HuC. The distinction between the smallest-sized lesion blocked by glucose (Stokes’ radius 3.6 A), but not NaCl (Stokes’ radius 1.4 A) and the next-sized lesion EhlgM
+ GPC
EAlgM
+ HuC
n
FIG. 3. Transformation of ES prepared with GPC or HuC were taken at the times marked a, b, or c in Fig. 2 and their ability to lyse in isotonic buffers of differing Stokes‘ radii was tested. Hemolysis was determined following incubation in the solutes for I.5 hr at 37°C. See text for precise experimental details.
SIZE
VARIATION
AND
STABILITY
OF
C LESIONS
291
blocked by raffinose (Stokes’ radius 5.6 A), but not by glucose, was not as marked for the same endpoint lysis when the E*s were generated with HuC rather than with GPC. This was most pronounced at later time points when the distinction between the various sized channels on E*s produced by HuC almost disappeared (Fig. 3). In these cases more than 80% of the cells were in the E* states in the absence of Eghost formation. We have also demonstrated that lysis of E* pRc,,rsorgoes through a series of reactions before lysis results (1). The rate of the E* transformation reaction for cell population with equivalent potential end point lysis has been shown to be influenced by the species of C8 in the C attack complex (9). When HuC8 was present the rate of achieving maximal lysis was much slower than equivalent E* intermediates containing GPCS (9). These findings could account for the major difference between the rate of transformation of E*,,reCUrS,,I. observed in Fig. 2A and B. In addition, since E* intermediates containing HuC8 required longer to transform to Eghost than those containing GPC8, additional opportunity to react with more C9 would exist and consequently the production of larger-sized lesions would be favored. Such an explanation might account for the differing-sized distribution profiles obtained with EAIgM and GPC and EAIgM and HuC (Fig. 3) for E* preparations with equivalent potential endpoint lysis. If such a difference was responsible for the experimental observations, then it should be possible, by changing the conditions of the reaction between EAIgM and GPC, to change the size of the lesions produced. In the next series of experiments, we tested the effect of inhibiting E* transformation on the size distribution of C lesions produced during the interaction of EAIgM with GPC. The inhibitor chosen was ZnSO, which we have previously shown reversibly inhibited the action of inserted C9 prior to the formation of the stable C lesion (10). In these experiments E* were prepared by incubation of an equal volume (1.5 x lO”/ml) of EAIgM and a 1:200 dilution of GPC for 3.5 min at 37°C. The cells were washed free of unbound C and resuspended in VBS-gel containing lop3 M ZnSO, at a concentration of 1.5 x 108/ml. Three equal samples were taken. The first was incubated for a further 10 min at 37°C with an equal volume of buffer: the second sample was incubated for 10 min at 37°C with a 1:2000 dilution of GPC; the third sample was incubated with 100 CH50 units of functionally purifjed GPC9 for 10 min at 37°C. The samples were then pelleted and the supernatant discarded: each pellet was resuspended in 200 ~1 of ice-cold 0.1 M EDTA and 10 ~1 was added to tubes containing 1 ml of the solutes of differing Stokes’ radii. The tubes were incubated for 1.5 hr at 37°C and the quantity of hemoglobin release under these conditions measured. The results in Fig. 4 indicate that addition of GPC9 or the 1:2000 dilution of GPC did not increase the extent of lysis of the preformed E* incubated in NaCl, i.e., under these experimental conditions, no additional complement lesions were formed. However, the results presented in Fig. 4 demonstrate the addition of purified GPC9 or a 1:2000 dilution of GPC allowed the smaller-sized lesions to be converted to the larger-sized lesions. The results of the experiment presented in Fig. 4 would suggest that the varioussized lesions were stable and consequently it should be possible by reducing the
292
BOYLE,
0
GEE,
AND
BORSOS
’ ’ ’ ’ ’ ’ ’ ’
E”+Buffer
C]
40 Xl m 10 01
u; e P 3 5’
FIG. 4. Expansion of C lesion of E” by interaction with fluid-phase GPC or functionaily purified GPC9. For these experiments E* were prepared by incubation of EAIgM with a I:200 dilution of GPC for 3.5 min at 37°C. The cells were washed free of GPC to provide cells in the E* state and maintained in that state by addition of IO-” M ZnSO, to the reaction mixture. To one aliquot of these cells was added GPC9 (panel A); to a second a 112000 dilution of GPC (panel B): and to the third buffer (panel C) and the mixtures incubated for 10 min at 37°C. The cells were then pelleted and the supematant discarded: each pellet was resuspended in 200 liters of ice-cold 0. I M EDTA and IO-liter aliquots were added to tubes containing 1 ml of solutes of differing Stokes’ radii. The degree of lysis of the cells was measured following 1.5 hr incubation at 37°C.
size of the blocking sugar to express sequentially the various-sized lesions in a red cell membrane. This was tested in the next series of experiments. E* were prepared by incubation 5 x 10Hof EA suspended in 2 ml of 25% BSA with 0.5 ml of GPC for 3 min at 37°C. The unbound GPC was then removed by washing two times with 5 ml of 25% BSA. The production of E* in albumin was chosen since this prevents any Eghost formation during the interaction of EA with GPC (11). The E* prepared in this way were then suspended in 5 ml 25% BSA and four l-ml aliquots were prepared and the cells pelleted. The cell peliets were resuspended in 5 ml of either NaCl, 0.3 M glucose, 0.3 M raftinose, or 25% albumin and incubated at 37°C for 3 hr. At this time the degree of lysis was measured (see large blocks on Fig. 5). The unlysed cell pellets following incubation with solute (i.e.. NaCl.
SIZE VARIATION 1~
b
n Albumin
AND STABILITY kl
293
OF C LESIONS
Raffincoe H Glucose 0 NaCl
t
1
NaCl
FIG. 5. Stability of differing-sized lesions on E*. E* were prepared by reacting EAIgM and GPC in 25% albumin. These cells were first incubated for 3 hr at 37°C in the solutes shown on the x-axis and the degree of lysis measured. Aliquots of the unlysed cells were transferred to solutes of differing Stokes’ radii and reincubated for 3 hr at 37°C before determining the degree of lysis. The solutes used in the second incubation are shown in the key at the top of the figure. For precise experimental detail see text.
glucose, raffinose, or albumin) were resuspended in 0.5 ml 25% BSA and O.l-ml samples were added to 1 ml of either NaCl, 0.3 M glucose, 0.3 M raflinose, or 25% albumin. These samples were then incubated for a further 3 hr at 37°C before the extent of lysis was measured (see small blocks on Fig. 5). The results in Fig. 5 indicate that 25% albumin was capable of blocking cell lysis. However, when the albumin block was replaced by NaCl, glucose. or raffnose, a profile of lysis was obtained that was similar (within the limits of experimental error) to that observed in the original buffers when no blocking by albumin took place. Similar results were observed with the cells first incubated in raffinose or glucose following transfer to glucose and NaCl or NaCl, respectively. These results suggest that differing-sized functional C lesions in red cell membranes are produced and that once these structures have been formed, they are stable for at least 3 hr. DISCUSSION
The experiments reported in this paper have demonstrated that the functional size of the C lesion produced by serum C is not uniform. These studies have revealed that a variety of differing-sized stable lesions can be produced in red cell membranes and that the size of the lesion can be increased by addition of purified C9.,A similar series of observations had been made in a system that used EACl-8 and functionally purified C9 (2, 3). These experiments have been criticized for using ratios of CS to C9 that could not be found in whole serum. However, the results of the present study indicate that similar results can be obtained with whole serum depending on the experimental conditions used to prepare E*. In particular, the critical factor in determining the distribution of lesion sizes would appear to be the balance between the rate of E*PreCUTSOI. formation (binding of all the necessary components for cell lysis to occur) and transformation of E*precur. SWto Eghosts (the reactions required to generate a functional C lesion by the bound components). A comparison of the lytic reaction of EAIgM and GPC (Fig.
294
BOYLE,
GEE,
AND
BORSOS
3A) and EAIgM and HuC (Fig. 2B) demonstrate a considerable difference in the maximum percentage of cells that can be isolated in an E* state prior to detectable cell lysis. Previous studies of the terminal stages of immune hemolysis have indicated kinetic differences in the lysis of E*s containing HuC8 and GPC8 (9). The differences between C8 species appear to occur both in the insertion reaction and on the subsequent reactions required to generate a C channel (9). Cells having HuC8 in the E* have been found to lyse more slowly (9) and consequently. during E* transformation additional interaction with fluid-phase C can occur. This would lead to the formation of new lesions, as well as expanding existing lesions by binding of further C9 molecules to an already potentially lytic but not saturated SACl-89 complex. By contrast, during the equivalent period with GPC. the cells would lyse and any tendency to form the larger-sized lesions would not be measured under the experimental conditions being used. If this explanation was valid. it should be possible by use of inhibitors of E” transformation to change the distribution of hole sizes resulting from the interaction of EAIgM and GPC. The results in Fig. 4 indicate that this was the case and that inhibiting E* transformation resulted in the production of larger-sized lesions provided fluid-phase C9 was still present in the reaction mixture. While the osmotic blockers of varying size will distinguish different populations of C lesions, they do not appear to influence these lesions in any way once they have been formed. In the experiments reported in Fig. 5 it was possible to change progressively from the largest blockers to the smallest one (NaCl Stokes’ radius 1.8 A) without any substantial loss in the total potential lysis of the E* population under study. These results would suggest that all of the differing-sized lesions once formed were stable for at least 3 hr at 37°C. The results presented in this paper indicate that the size of the C lesion formed can be influenced by the method of preparation of the E”:, in particular, the source of C. It would appear that if fluid-phase C (in particular, C9) is still present after the earliest formation of E*, it will continue to bind and influence the properties of the C lesion during the transformation reaction. It is not clear from these experiments whether C9 can continue to bind and change the properties of C lesions on already lysed cell membranes (Eghost). These findings would suggest that in studying the size of the C lesion or the immunochemical composition of C complexes isolation from ghost membranes attempts should be made to differentiate between events occurring during the initial formation of the potential lesion (E* I)pccu~SoJ and the collection of the Eghost material used for analysis. Until experimental approaches are standardized to define the size distribution of lesions being studied, the reasons for the wide range of estimates for the size of the functional C lesion (12- 17) and wide range of molecular weights and proposed molecular composition of the isolated C attack complex (18-20) measured by different techniques are unlikely to be resolved. REFERENCES I. Boyle, 7. Boyle. 3. DeLisi.
M. D. P.. and Borsos. T., M. D. P.. Gee. A. P.. and C., Boyle, M. D. P.. and
Mo(. Immurwl. 17. 425. Borsos. T., J. Immunol. Borsos, T.. J. Immrtnol.
1980.
123, 77. 1979. 125,
2055.
1980.
SIZE VARIATION
AND STABILITY
OF C LESIONS
295
4. Boyle, M. D. P., Langone, J. J., and Borsos, T., J. Zmmunol. 120, 1721, 1978. 5. Rommel. F. A.. and Mayer, M. M., J. Immunol. 110, 637, 1973. pp. 75-79, Appleton6. Rapp, H. J., and Borsos, T., “Molecular Basis of Complementation,” Century-Crofts, New York, 1970. 7. Kabat, E., and Mayer, M. M. “Kabat and Mayer’s Experimental Immunochemistry.” pp. 871-877. Thomas, Springfield, Ill., 1961. 8. Boyle. M. D. P., and Langone, J. J. J. Zmmunol. Methods 32, 51, 1980. 9. Okada, M.. Boyle, M. D. P., and Borsos. T., Biochem. Biophys. Res. Commun. 94, 406, 1980. 10. Boyle, M. D. P., Langone, J. J., and Borsos, T., J. Zmmunol. 122, 1209, 1979. 11. Boyle. M. D. P., and Borsos, T., J. Zmmunol. 123, 71, 1979. 12. Humphrey, J. H.. and Dourmashkin, R. R., Advan. Zmmunol. 11, 75, 1969. 13. Sears, D. A., Weed, R. I., and Swisher, S. N. J. C/in. Invest. 43, 975, 1964. 14. Michaels, D. W., Abramovitz, A. S.. Hammer, C. H., and Mayer, M. M. J. Zmmunol. 120, 1785, 1978. 15. Giavedoni, E. B., Chow, Y. M., and Dalmasso, A. P., J. Zmmunol. 122, 240, 1979. 16. Sims, P. J., and Lauf, P. K.. Proc. Nut. Acad. Sri. USA 75, 5669, 1978. 17. Ramm, L. E.. and Mayer, M. M. J. Zmmunol. 124, 2281, 1980. 18. Kolb, W. P., and Mtiller-Eberhard, H. J. J. Exp. Med. 141, 724, 1975. 19. Bhakdi, S., Ey. P., and Bhakdi-Lehnen, B.. Biochim. Biophys. Acta 419, 445, 1976. 20. Biesecker, G., Podack, E. R., Halverson, C. A., and Miiller-Eberhard, H. J., J. Exp. Med. 149, 448, 1979. Received December 15. 1980; accepted March 27, 1981.
IMMUNOLOGY
AND
lMMUNOPATHOLOCY
20, 287-295 (1981)
Heterogeneity in the Size and Stability of Transmembrane Channels Produced by Whole Complement MICHAEL D. P. BOYLE,’ Laboratory
of Immunobiology,
ADRIAN P. GEE, AND TIBOR BORSOS
National Bethesda,
Cancer Institute, Maryland 20205
National
Institates
of Health,
The functional size of complement (C) lesions produced by treatment of EAIgM with human or guinea pig C was studied. In all cases, a distribution of stable C lesions differing in size could be distinguished. Differences between the distribution of differing-sized lesions were observed depending on whether EAIgM were lysed with human (Hu) or guinea pig (GP) C. This difference appeared to be related to the kinetics of transformation prepared with HuC lysed more slowly than those of E*wecursor to Eghosts; E*III.CUIGUI prepared with GPC. The significance of these findings to the wide range of estimates reported for the size of the C lesion is briefly discussed.
INTRODUCTION
In studies of the lysis of EACl-8 by C9, we have shown that the more C9 was bound to a given SACl-8 the larger the C2 lesion produced (l-3), although binding of a single C9 molecule is sufficient for lesion production (4-5). In these and other studies of the lytic effects of individual functionally pure components, experimental conditions were usually controlled so that the degree of lysis was governed solely by the C component being studied, i.e., the reaction was performed under pseudo-first-order conditions. By contrast, the activity of C in whole serum consists of the interaction of C components as well as regulating proteins and consequently the final reaction step with C9 may or may not behave as a pseudofirst-order reaction. Thus, it has not been established whether the heterogeneity in the size of C channels was a consequence of the sequential addition of purified components in an artificial system in which EACl-8 were treated with varying concentrations of C9, or, if such variations in the size of the C lesion would occur when whole C was used. The purpose of the present study was to examine a variety of experimental conditions of lysis of EA by whole C to determine whether heterogeneity of C lesions could be detected. MATERIALS
AND METHODS
Cells. Sheep red blood cells (E) were collected and washed as described in (6). Buffers. Isotonic veronal-buffered saline (VBS), pH 7.4, containing 0.1% gelatin I Present address: Dr. Michael D. P. Boyle, Department of Immunology and Medical Microbiology, College of Medicine, Box J-266, JHMHC, University of Florida, Gainesville, FL 32610. p Abbreviations used: E, sheep erythrocytes; VBS-gel, isotonic Veronal-buffered saline containing 0.1% gelatin, 0.001 M Mg”’ and O.OOO15M Cd L+; VBS-EDTA, isotonic Veronal-buffered saline containing 0.1% gelatin and disodium ethylenediaminetetraacetate; E*, sheep erythrocytes with at least one cell bound antigen-antibody complex and all the necessary complement components for killing to occur: Hu or hu, human: C, complement: BSA, bovine serum albumin. 287 OO90-1229/81/O90287-O9$01.00/0 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
288
BOYLE,
GEE,
AND
BORSOS
(Mann Research Labs., N.Y.), 0.001 M Mg’+, and 0.0015 A4 Ca” (VBS-gel); sucrose buffer, Al. = 0.065 (VBS-sucrose); 0. I M EDTA buffer, were prepared as described by Rapp and Borsos (6). Glucose monohydrate was obtained from Baker Chemical Company. Philadelphia. Pennsylvania, sucrose (ultracentrifuge grade) was obtained from Schwarz-Mann. Orangeburg, New York, and raffinose pentahydrate was obtained from Vega Chemicals. Tucson. Arizona. All sugars were used as 0.3 M solutions in water. Albumin. Bovine serum albumin (Pathocyte 5) was obtained from Miles Laboratories. Elkhart, Indiana, and was used in all experiments at a concentration of 25%. Anrihody. Anti-Forssman antiserum was raised in New Zealand albino rabbits according to the procedures given in (7). The IgM and IgG fractions were separated on DEAE as described by Boyle and Langone (8). Complement. Fresh guinea pig serum (pooled) (JEM Research Inc., Kensington. Md.) or human serum were used as complement sources. Both were absorbed with packed E and stored in aliquots at -30°C. ComplPmrnt components. Functionally purified complement components were obtained from the Cordis Corporation, Miami. Florida. Indicator cells. EAIgM and EAIgG were prepared by incubating E at 109/ml with an equal volume of a dilution of either the IgM or IgG fraction of rabbit anti-Forssman antiserum which produced 100% lysis of the cells within 15 min upon addition of excess C. Hrmoglohin &termination. Relative hemoglobin levels were determined on a Micromedic MS2 spectrophotometer by measuring the absorbance at a wavelength of 412 or 541 nm. RESULTS
During the interaction of EA with C, cells may be in one of the following states: pre-E*, E* or Eghost (see Fig. 1). Eghost are cells that have released their hemoglobin, E* are cells that will release their hemoglobin in the absence of any further interaction with fluid-phase C, while pre-E* are cells at any stage between EA and EACl-8. The proportion of cells in each of these states can be quantified by appropriate experimental procedures. For example, cells that have not reached the E* precursor stage will not lyse during a subsequent incubation in VBS-gel unless fluid-phase C is added (see Fig. 1). Cells in any of the E* states will lyse in the presence of VBS-gel but not in the presence of 25% albumin, and cells in the Eghost state have already released their hemoglobin. Consequently. by measuring the proportion of cells from which hemoglobin was released during the initial exposure to fluid-phase C, or during subsequent incubation in VBS-gel or 25% albumin, in the absence of fluid-phase C, it is possible to determine the percentage of cells that(i) have not formed E*, (ii) that have formed E* but not lysed (i.e., in the E* transformation sequence), and (iii) have already lysed (Eghost). In the first series of experiments the kinetics of production of E*preeursorand the subsequent transformation of these cells to Eghost was followed. These studies were performed using 1.5 x lo* E sensitized with excess IgM anti-Forssman
SIZE
VARIATION
AND
STABILITY
E* E*precursor tsmpePa~e dependent ’ inserted
EA + C-----b I
OF
289
C LESIONS
inhibitable by ainc or ’ uranyz salts
blocked by 25x nlbwlrin~
E*dLIOWd
E gtmst +
HB
I I
(EAC1-g--=*
(EACI-9boun*)
I
REQ”mES FLUIDPHASE c
I
(sAC1-9*o~me*)
)
DOES NOT REQ”IKE FL”ID-PHASE
c
I
FIG.
1. Reaction
sequence
for the transformation
of pre-E*
to Eghost.
antibody and incubated for varying times with 1 ml of a l/200 dilution of GPC or HuC diluted 11100. At the appropriate times the reactions were stopped by adding 5 ml ice-cold 0.1 M EDTA and the cells centrifuged. The degree of lysis occurring at the time of sampling was determined by measuring the amount of hemoglobin in the supernatant fluid (Eghost) and the degree of E* formation was quantified by measuring the amount of lysis in VBS-gel or 25% BSA following a further 60-min incubation of the cells at 37°C. The results obtained with EAIgM and GPC indicate that E*PreCUm,,rwas formed, rapidly followed by the appearance of Eghosts (Fig. 2A). The maximum percentage of cells that could be isolated in the E* state was 54%. In the experiments using HuC as the C source, a number of differences were observed (Fig. 2B). While the formation of E* Prec,,rSOr from EAIgM was rapid in the presence of HuC, unlike the equivalent situation with GPC, the transformation of E* to Eghost was much slower and it was possible to isolate >90% of the cells in the E* transformation sequence (Fig. 2B). Similar E* intermediates with an endpoint lysis potential of >80% could never be achieved using GPC because of Eghost formation.
EAlgM
+ GPC
EAlgM
100
E Ghost
-2 GM CiI 2
q E Ghost
E”
c b
P60 Y 540 8 g20
+ HuC
r
bC a
a
EX A 0 K:KI 0
4
8
12
160 4 TIME (min.1
8 8
12
16
20
24
FIG. 2. Kinetics of E* formation and lysis using GPC or HuC. (A) E* prepared from GPC: (B) E* prepared from EAIgM and HuC. No lysis of E* occurred in the continuing 25% albumin. The size of C lesions on the E* at various stages (a, b, and c) was examined See text for precise experimental details.
EAIgM and presence of (See Fig. 3).
290
BOYLE,
GEE.
AND
BORSOS
A similar pattern of hemolysis was obtained with EAlgG and GPC although some minor differences as compared to EAIgM were apparent, For example. the rate of E”’ transformation to Eghosts appeared slower than for the equivalent intermediates prepared with IgM and consequently the maximum percentage of cells that could be isolated in the E* states was greater (approximately 80%). Cells in any of the E’% states could be prevented from lysing by incubation in 25% BSA (data not shown). At the times indicated in Figs. 2A and B. E:;: were obtained and their ability to transform to Eghosts in the presence of isotonic solutes of differing Stokes’ radii was compared. The solutes chosen were NaCl with a Stokes’ radius of 1.4 A. glucose with a Stokes’ radius of 3.6 A. raffinose with a Stokes’ radius of 5.6 A, and albumin with a Stokes’ radius of 36 A. The degree of E*: transformation to Eghost in each solution was measured following incubation for 1 hr at 37°C. The results for E* prepared from EAIgM and GPC are shown in Fig. 3 (top) and for EAIgM and HuC are shown in Fig. 3 (bottom), and they indicate that a distribution of different-sized C lesions could be identified. A comparison of the results in Figs. 3A and B indicate differences between the distribution of different-sized C lesions produced by GPC and HuC. The distinction between the smallest-sized lesion blocked by glucose (Stokes’ radius 3.6 A), but not NaCl (Stokes’ radius 1.4 A) and the next-sized lesion EhlgM
+ GPC
EAlgM
+ HuC
n
FIG. 3. Transformation of ES prepared with GPC or HuC were taken at the times marked a, b, or c in Fig. 2 and their ability to lyse in isotonic buffers of differing Stokes‘ radii was tested. Hemolysis was determined following incubation in the solutes for I.5 hr at 37°C. See text for precise experimental details.
SIZE
VARIATION
AND
STABILITY
OF
C LESIONS
291
blocked by raffinose (Stokes’ radius 5.6 A), but not by glucose, was not as marked for the same endpoint lysis when the E*s were generated with HuC rather than with GPC. This was most pronounced at later time points when the distinction between the various sized channels on E*s produced by HuC almost disappeared (Fig. 3). In these cases more than 80% of the cells were in the E* states in the absence of Eghost formation. We have also demonstrated that lysis of E* pRc,,rsorgoes through a series of reactions before lysis results (1). The rate of the E* transformation reaction for cell population with equivalent potential end point lysis has been shown to be influenced by the species of C8 in the C attack complex (9). When HuC8 was present the rate of achieving maximal lysis was much slower than equivalent E* intermediates containing GPCS (9). These findings could account for the major difference between the rate of transformation of E*,,reCUrS,,I. observed in Fig. 2A and B. In addition, since E* intermediates containing HuC8 required longer to transform to Eghost than those containing GPC8, additional opportunity to react with more C9 would exist and consequently the production of larger-sized lesions would be favored. Such an explanation might account for the differing-sized distribution profiles obtained with EAIgM and GPC and EAIgM and HuC (Fig. 3) for E* preparations with equivalent potential endpoint lysis. If such a difference was responsible for the experimental observations, then it should be possible, by changing the conditions of the reaction between EAIgM and GPC, to change the size of the lesions produced. In the next series of experiments, we tested the effect of inhibiting E* transformation on the size distribution of C lesions produced during the interaction of EAIgM with GPC. The inhibitor chosen was ZnSO, which we have previously shown reversibly inhibited the action of inserted C9 prior to the formation of the stable C lesion (10). In these experiments E* were prepared by incubation of an equal volume (1.5 x lO”/ml) of EAIgM and a 1:200 dilution of GPC for 3.5 min at 37°C. The cells were washed free of unbound C and resuspended in VBS-gel containing lop3 M ZnSO, at a concentration of 1.5 x 108/ml. Three equal samples were taken. The first was incubated for a further 10 min at 37°C with an equal volume of buffer: the second sample was incubated for 10 min at 37°C with a 1:2000 dilution of GPC; the third sample was incubated with 100 CH50 units of functionally purifjed GPC9 for 10 min at 37°C. The samples were then pelleted and the supernatant discarded: each pellet was resuspended in 200 ~1 of ice-cold 0.1 M EDTA and 10 ~1 was added to tubes containing 1 ml of the solutes of differing Stokes’ radii. The tubes were incubated for 1.5 hr at 37°C and the quantity of hemoglobin release under these conditions measured. The results in Fig. 4 indicate that addition of GPC9 or the 1:2000 dilution of GPC did not increase the extent of lysis of the preformed E* incubated in NaCl, i.e., under these experimental conditions, no additional complement lesions were formed. However, the results presented in Fig. 4 demonstrate the addition of purified GPC9 or a 1:2000 dilution of GPC allowed the smaller-sized lesions to be converted to the larger-sized lesions. The results of the experiment presented in Fig. 4 would suggest that the varioussized lesions were stable and consequently it should be possible by reducing the
292
BOYLE,
0
GEE,
AND
BORSOS
’ ’ ’ ’ ’ ’ ’ ’
E”+Buffer
C]
40 Xl m 10 01
u; e P 3 5’
FIG. 4. Expansion of C lesion of E” by interaction with fluid-phase GPC or functionaily purified GPC9. For these experiments E* were prepared by incubation of EAIgM with a I:200 dilution of GPC for 3.5 min at 37°C. The cells were washed free of GPC to provide cells in the E* state and maintained in that state by addition of IO-” M ZnSO, to the reaction mixture. To one aliquot of these cells was added GPC9 (panel A); to a second a 112000 dilution of GPC (panel B): and to the third buffer (panel C) and the mixtures incubated for 10 min at 37°C. The cells were then pelleted and the supematant discarded: each pellet was resuspended in 200 liters of ice-cold 0. I M EDTA and IO-liter aliquots were added to tubes containing 1 ml of solutes of differing Stokes’ radii. The degree of lysis of the cells was measured following 1.5 hr incubation at 37°C.
size of the blocking sugar to express sequentially the various-sized lesions in a red cell membrane. This was tested in the next series of experiments. E* were prepared by incubation 5 x 10Hof EA suspended in 2 ml of 25% BSA with 0.5 ml of GPC for 3 min at 37°C. The unbound GPC was then removed by washing two times with 5 ml of 25% BSA. The production of E* in albumin was chosen since this prevents any Eghost formation during the interaction of EA with GPC (11). The E* prepared in this way were then suspended in 5 ml 25% BSA and four l-ml aliquots were prepared and the cells pelleted. The cell peliets were resuspended in 5 ml of either NaCl, 0.3 M glucose, 0.3 M raftinose, or 25% albumin and incubated at 37°C for 3 hr. At this time the degree of lysis was measured (see large blocks on Fig. 5). The unlysed cell pellets following incubation with solute (i.e.. NaCl.
SIZE VARIATION 1~
b
n Albumin
AND STABILITY kl
293
OF C LESIONS
Raffincoe H Glucose 0 NaCl
t
1
NaCl
FIG. 5. Stability of differing-sized lesions on E*. E* were prepared by reacting EAIgM and GPC in 25% albumin. These cells were first incubated for 3 hr at 37°C in the solutes shown on the x-axis and the degree of lysis measured. Aliquots of the unlysed cells were transferred to solutes of differing Stokes’ radii and reincubated for 3 hr at 37°C before determining the degree of lysis. The solutes used in the second incubation are shown in the key at the top of the figure. For precise experimental detail see text.
glucose, raffinose, or albumin) were resuspended in 0.5 ml 25% BSA and O.l-ml samples were added to 1 ml of either NaCl, 0.3 M glucose, 0.3 M raflinose, or 25% albumin. These samples were then incubated for a further 3 hr at 37°C before the extent of lysis was measured (see small blocks on Fig. 5). The results in Fig. 5 indicate that 25% albumin was capable of blocking cell lysis. However, when the albumin block was replaced by NaCl, glucose. or raffnose, a profile of lysis was obtained that was similar (within the limits of experimental error) to that observed in the original buffers when no blocking by albumin took place. Similar results were observed with the cells first incubated in raffinose or glucose following transfer to glucose and NaCl or NaCl, respectively. These results suggest that differing-sized functional C lesions in red cell membranes are produced and that once these structures have been formed, they are stable for at least 3 hr. DISCUSSION
The experiments reported in this paper have demonstrated that the functional size of the C lesion produced by serum C is not uniform. These studies have revealed that a variety of differing-sized stable lesions can be produced in red cell membranes and that the size of the lesion can be increased by addition of purified C9.,A similar series of observations had been made in a system that used EACl-8 and functionally purified C9 (2, 3). These experiments have been criticized for using ratios of CS to C9 that could not be found in whole serum. However, the results of the present study indicate that similar results can be obtained with whole serum depending on the experimental conditions used to prepare E*. In particular, the critical factor in determining the distribution of lesion sizes would appear to be the balance between the rate of E*PreCUTSOI. formation (binding of all the necessary components for cell lysis to occur) and transformation of E*precur. SWto Eghosts (the reactions required to generate a functional C lesion by the bound components). A comparison of the lytic reaction of EAIgM and GPC (Fig.
294
BOYLE,
GEE,
AND
BORSOS
3A) and EAIgM and HuC (Fig. 2B) demonstrate a considerable difference in the maximum percentage of cells that can be isolated in an E* state prior to detectable cell lysis. Previous studies of the terminal stages of immune hemolysis have indicated kinetic differences in the lysis of E*s containing HuC8 and GPC8 (9). The differences between C8 species appear to occur both in the insertion reaction and on the subsequent reactions required to generate a C channel (9). Cells having HuC8 in the E* have been found to lyse more slowly (9) and consequently. during E* transformation additional interaction with fluid-phase C can occur. This would lead to the formation of new lesions, as well as expanding existing lesions by binding of further C9 molecules to an already potentially lytic but not saturated SACl-89 complex. By contrast, during the equivalent period with GPC. the cells would lyse and any tendency to form the larger-sized lesions would not be measured under the experimental conditions being used. If this explanation was valid. it should be possible by use of inhibitors of E” transformation to change the distribution of hole sizes resulting from the interaction of EAIgM and GPC. The results in Fig. 4 indicate that this was the case and that inhibiting E* transformation resulted in the production of larger-sized lesions provided fluid-phase C9 was still present in the reaction mixture. While the osmotic blockers of varying size will distinguish different populations of C lesions, they do not appear to influence these lesions in any way once they have been formed. In the experiments reported in Fig. 5 it was possible to change progressively from the largest blockers to the smallest one (NaCl Stokes’ radius 1.8 A) without any substantial loss in the total potential lysis of the E* population under study. These results would suggest that all of the differing-sized lesions once formed were stable for at least 3 hr at 37°C. The results presented in this paper indicate that the size of the C lesion formed can be influenced by the method of preparation of the E”:, in particular, the source of C. It would appear that if fluid-phase C (in particular, C9) is still present after the earliest formation of E*, it will continue to bind and influence the properties of the C lesion during the transformation reaction. It is not clear from these experiments whether C9 can continue to bind and change the properties of C lesions on already lysed cell membranes (Eghost). These findings would suggest that in studying the size of the C lesion or the immunochemical composition of C complexes isolation from ghost membranes attempts should be made to differentiate between events occurring during the initial formation of the potential lesion (E* I)pccu~SoJ and the collection of the Eghost material used for analysis. Until experimental approaches are standardized to define the size distribution of lesions being studied, the reasons for the wide range of estimates for the size of the functional C lesion (12- 17) and wide range of molecular weights and proposed molecular composition of the isolated C attack complex (18-20) measured by different techniques are unlikely to be resolved. REFERENCES I. Boyle, 7. Boyle. 3. DeLisi.
M. D. P.. and Borsos. T., M. D. P.. Gee. A. P.. and C., Boyle, M. D. P.. and
Mo(. Immurwl. 17. 425. Borsos. T., J. Immunol. Borsos, T.. J. Immrtnol.
1980.
123, 77. 1979. 125,
2055.
1980.
SIZE VARIATION
AND STABILITY
OF C LESIONS
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4. Boyle, M. D. P., Langone, J. J., and Borsos, T., J. Zmmunol. 120, 1721, 1978. 5. Rommel. F. A.. and Mayer, M. M., J. Immunol. 110, 637, 1973. pp. 75-79, Appleton6. Rapp, H. J., and Borsos, T., “Molecular Basis of Complementation,” Century-Crofts, New York, 1970. 7. Kabat, E., and Mayer, M. M. “Kabat and Mayer’s Experimental Immunochemistry.” pp. 871-877. Thomas, Springfield, Ill., 1961. 8. Boyle. M. D. P., and Langone, J. J. J. Zmmunol. Methods 32, 51, 1980. 9. Okada, M.. Boyle, M. D. P., and Borsos. T., Biochem. Biophys. Res. Commun. 94, 406, 1980. 10. Boyle, M. D. P., Langone, J. J., and Borsos, T., J. Zmmunol. 122, 1209, 1979. 11. Boyle. M. D. P., and Borsos, T., J. Zmmunol. 123, 71, 1979. 12. Humphrey, J. H.. and Dourmashkin, R. R., Advan. Zmmunol. 11, 75, 1969. 13. Sears, D. A., Weed, R. I., and Swisher, S. N. J. C/in. Invest. 43, 975, 1964. 14. Michaels, D. W., Abramovitz, A. S.. Hammer, C. H., and Mayer, M. M. J. Zmmunol. 120, 1785, 1978. 15. Giavedoni, E. B., Chow, Y. M., and Dalmasso, A. P., J. Zmmunol. 122, 240, 1979. 16. Sims, P. J., and Lauf, P. K.. Proc. Nut. Acad. Sri. USA 75, 5669, 1978. 17. Ramm, L. E.. and Mayer, M. M. J. Zmmunol. 124, 2281, 1980. 18. Kolb, W. P., and Mtiller-Eberhard, H. J. J. Exp. Med. 141, 724, 1975. 19. Bhakdi, S., Ey. P., and Bhakdi-Lehnen, B.. Biochim. Biophys. Acta 419, 445, 1976. 20. Biesecker, G., Podack, E. R., Halverson, C. A., and Miiller-Eberhard, H. J., J. Exp. Med. 149, 448, 1979. Received December 15. 1980; accepted March 27, 1981.