studies on the inhibition of c56-initiated lysis (reactive lysis). iii

Immunology, 1975, 28, 133.

Studies on the Inhibition of C56-initiated Lysis (ReactiveLysis)

III. CHARACTERIZATION OF THE INHIBITORY ACTIVITYC567-INH AND ITS MODE OF ACTION*

B. MCLEOD, PATRICIA BAKER AND H. GEWURZ

Department of Immunology, Rush Medical College, and Department of Microbiology,University of Illinois Medical Center, Chicago, Illinois, U.S.A.

(Received 20th March 1974; acceptedfor publication 22nd May 1974)Summary. An activity in serum which inhibits reactive lysis has recently beenshown to do so by preventing the attachment of C567 complexes to cells, and hencehas been designated C567-INH. This report describes certain physicochemicalcharacteristics ofthe inhibitory activity. It behaves as a heat-stable pseudoglobulin,soluble in 20 per cent Na2SO4, and having al mobility on Pevikon block electro-phoresis. It is excluded from CM cellulose at pH 6-0, RSC = 0 007 M, is retainedby an XM-100 membrane and is heterogenous on Sephadex G-200, eluting in atleast two peaks. The combined active materials from the Sephadex column elutefrom DE-52 in at least four peaks. The mechanism of action of material fromeach of these four peaks is shown to involve prevention of attachment of C567complexes to membranes, and this is shown to involve an effect on C567 complexesin solution rather than an effect on the membrane. A less dramatic effect on thelysis of EC567 by limited quantities of C8 and C9 can be demonstrated. Haemo-lytic studies using cell-bound C567 suggest that the interaction of C567-INHwith C567 involves a loose reversible association. It is therefore postulated that C567-INH inhibits reactive lysis primarily by reversibly associating with the nascentC567 complex in solution, increasing its bulk and decreasing its diffusion capacityso that it is unable to reach a cell membrane before its haemolytic potential decays.

INTRODUCTION

One mechanism by which complement (C) can bring about lysis of unsensitized cellsis now well delineated. Thompson and Lachmann, through their studies of reactive lysis,showed that activated complexes of the fifth and sixth components, in the presence of C7,could alter a red cell (E) membrane, so that the cell could be lysed by C8 and C9(Thompson and Lachmann, 1970; Lachmann and Thompson, 1970). G6tze and Muller-Eberhard (1970) showed that activation ofpurified C5, C6 and C7 in mixtures ofEAC1423

* Presented in part to the American Society for Experimental Pathology, April 14, 1974.Correspondence: Professor H. Gewurz, Department of Immunology, Rush Medical College, 1725 West Harrison

Street, Chicago, Illinois 60612, U.S.A.

133

B. McLeod, Patricia Baker and H. Gewurz

and sheep red blood cells (E) could render the unsensitized cells susceptible to lysis byC8 and C9. On the strength of their haemolytic data, both groups postulated that theactivated trimolecular complex, C567 (Nilsson and Muller-Eberhard, 1967), was formedin solution and had the ability to attach to red cell membranes independently of antibodyand early complement components. Subsequent investigation has supported this conceptof a trimolecular functional unit with firm physical data, and has indicated that afterenzymatic activation of C5, assembly of the complex proceeds nonenzymatically, by aprocess of absorption (Kolb, Haxby, Arroyave and Muller-Eberhard, 1972, 1973;Arroyave and Muller-Eberhard, 1973).We have recently described an activity in normal serum which inhibits reactive lysis

(McLeod, Baker and Gewurz, 1973a, 1974a). The inhibitory activity was shown to bequite selective for reactive lysis in that it had little effect on the classical complement-mediated lysis of antibody-coated cells. A precise, sensitive means of quantification wasdeveloped. We report here the initial results ofour effort to isolate this inhibitory principlefrom serum, along with the results ofstudies ofthe mechanism ofaction ofpartially purifiedinhibitory preparations. We initially chose the symbol 'INH-RL' (for 'inhibitor of reactivelysis') to refer to this activity. Subsequent studies have indicated that the site of inhibitionis at the C567 trimolecular complex and have suggested that the symbol 'C567-INH'is more appropriate (McLeod, Baker and Gewurz, 1974b). Both designations have beenfound useful in this communication.

MATERIALS AND METHODSBuffersVBS refers to isotonic veronal-buffered saline, pH 7-3 (Mayer, 1961). GVB refers to

VBS containing 0.1 per cent gelatin. GVB-EDTA refers to the above buffer made 0O01 Min EDTA.

Sheep red blood cellsThis refers to sheep red blood cells prepared as described (McLeod, Baker and Gewurz,

1974a) and washed into GVB-EDTA before use.

Normal human serum (NHS)Blood from healthy laboratory personnel was drawn into acid-citrate-dextrose anti-

coagulant. Plasma was separated and allowed to clot after replenishment of metal ions.After removal of coagulum, the serum was stored in aliquots at - 700 until use.

Guinea-pig complement (GPC)Pooled fresh frozen whole guinea-pig serum was obtained from Texas Biological,

Incorporated, and maintained at - 700. It was thawed immediately prior to use.

Complement reagentsC56 was prepared and purified as described (McLeod et al., 1974a) by DE-52 chroma-

134

C56-initiated Lysis Inhibition, III. 135

tography of the euglobulin fraction obtained from a zymosan-activated reactor serum.C56 prepared in this way is by definition free of C7. It was also free of detectable C8 andC9 activity at the concentrations employed.Human C7, C8 and C9 were obtained from Cordis Laboratories.

Pevikon block electrophoresisPevikon was washed twice in barbital buffer, pH 8.6 (Beckman B-2 buffer), and then

suspended in the same buffer in a 20 x 40 x 0-6 cm plastic tray. The sample was appliedin a volume of 5 ml to a 1 cm strip in the center of the block. Electrophoresis was carriedout in the cold for 12 hours at 300 volts. The block was cut into 1-cm strips, and each stripwas eluted in 5 nil of VBS for 1 hour in the cold. The eluate from each strip was assayedfor OD280 and for INH-RL.

DEAE chromatographyThis was performed in a 50 x 2-5 cm glass column. DE-52 (Whatman) resin was sus-

pended in 0 05 M Tris, pH 4-5, degassed, and washed into 0 5 M Tris, pH 10-5. The columnwas then poured and equilibrated with 0-05 M Tris, pH 8-5 (starting buffer) until the pHand ionic strength of the effluent were equal to that of the starting buffer. Sample wasapplied in starting buffer, and eluted at 80 cc/hour. After the appearance of an excludedpeak, a 2000-ml linear salt gradient from 0 to 0 3 M NaCl in the Tris Buffer was developed.12f5-ml fractions were collected and assayed for OD280 and for INH-RL.

CM cellulose chromatographyCM-32 (Whatman) was washed twice in 0 5 M NaCl, once in 90 per cent ethanol, and

once in 0-1 M NaOH. It was then washed four times in 0-01 M acetate buffer, pH 6-0,and poured in a 2-5 x 30 cm glass column. The column was washed extensively with 0-01M phosphate buffer, 0-001 M NaN3, pH 6-0 (starting buffer) before application of a 20per cent Na2SO4 supernatant of normal human serum, which had been dialysed intostarting buffer. The column was run at 80 cc/hour, and after collection of the excludedpeak, a 2000-ml linear salt gradient was developed from 0-007 to 0 5 M NaCl in the phos-phate buffer. Twenty-millilitre fractions were collected and analysed for OD280 and forINH-RL.

Gel filtrationA 5 x 104 cm Sephadex G-200 (Pharmacia) column was equilibrated with 0*l M

Tris buffer, 0-5 M NaCl, 0-01 M EDTA, and 0-001 M NaN3, pH 7-5. The sample wasapplied in a volume of 20 ml and eluted at 35 ml/hour with the same buffer. 17 5-mlfractions were collected and assayed for OD280 and INH-RL. The column was standard-ized with NHS. IgM and IgG were determined by radial immunodiffusion with commer-cial kits and the third peak of OD280 was taken as the peak of albumin elution.


UltrafiltrationThis was performed in 10-, 50- or 450-ml stirred chambers (Amicon) under pressure

from N2 gas. When passage through an XM-100 membrane was used to effect proteinseparation, low pressures (<10 psi) and low protein concentrations (<0 1 mg/ml) weremaintained.

Reactive lysisThis procedure was performed in solution as described (McLeod et al., 1974a) using

an amount ofC56 just sufficient to give 80-90 per cent lysis of 5 x 106 E in a 04-ml reactionvolume containing GPC at a final dilution of 1: 80 in GVB-EDTA.

Inhibitory activity (IJNH-RL)Inhibitory activity of sample was assayed quantitatively in solution as described (Mc-

Leod et al., 1974a). Appropriate dilutions of unknown samples were included in the 04-ml reaction volume described above for reactive lysis, and the extent of reactive lysiswas determined in the mixture containing the unknown sample. Control tubes containingeither no added inhibitor or serial dilutions of a standard inhibitory material were runsimultaneously. For each unknown, a value for 'percentage P' representing [(number ofcells protected) /(number of cells at risk of reactive lysis)] x 100 was determined and wascompared to the dose-response curve of the standard material assayed simultaneously.The standard gave 60-80 per cent P at full strength and was arbitrarily assigned a con-centration of 100 u/ml.

EC567This cellular intermediate was usually prepared in a 04-ml reaction volume containing

5 x 10 E, 10 u of Cordis C7 and an amount of C56 just sufficient to give 80 per centformation of EC567. After a 15-minute incubation at 370, the cells were separated bycentrifugation and washed twice in 0 5 ml of cold GVB-EDTA. In some instances, largernumber of cells or more potent preparations of complement reagents were used in thestandard reaction volume.

Lysis ofEC567This was usually accomplished by a 15-minute incubation in 0 4 ml of dilute GPC-

EDTA to supply C8 and C9. Indeed, susceptibility of a cell to lysis in a 1: 80 dilution ofGPC-EDTA was a suitable criterion to establish that a cell had reached the EC567state. Some experiments utilized more dilute GPC-EDTA or purified C8 and C9 inlonger incubations to study the lysis of EC567 by limited amounts of C8 and C9.

EAC1-7This cellular intermediate was obtained from Cordis Laboratories. Cells were washed

into GVB-EDTA and lysed in the same manner as EC567.

136

C56-initiated Lysis Inhibition, III.

RESULTS

CHARACTERIZATION

SolubilityEarlier experiments (McLeod et al., 1974a) showed that most if not all of the INH-

RL in a serum sample remained in the supernatant (pseudoglobulin) when euglobulinswere precipitated by dialysis against distilled H20 at pH 6-0. It was also shown to belargely soluble in 20 per cent Na2SO4 and 17 5 per cent (NH4) 2SO4. Because we feltthat C7 might interfere in our assay, and because it is known to precipitate in 20 per centNa2SO4, we used the supernatant remaining after 20 per cent Na2SO4 precipitationof NHS as a source of INH-RL for further studies of its properties.

Trichloroacetic acid (TCA) precipitationA 20 per cent Na2SO4 supernatant of NHS was dialysed in VBS and mixed with a

equal volume of 10 per cent TCA. The resulting precipitate was removed by centrifugationand the supernatant dialysed into GVB-EDTA. No INH-RL was found in the supernatant.

Heat stabilityA 20 per cent Na2SO4 supernatant ofNHS was dialysed into VBS and heated in a water

bath at 560 for varying intervals. Each sample was then assayed for INH-RL. No loss ofactivity was observed after a 1-hour incubation. By 2 hours there was a 30 per cent dropin activity; at 4 hours there was a 50 per cent drop. More purified preparations describedbelow showed similar heat stability except for the material referred to as Pool 1, whichshowed 50 per cent loss of activity at 1 hour and total loss at 4 hours.

ElectrophoresisThe results of Pevikon block electrophoresis, performed on a 20 per cent Na2SO4

supernatant of NHS, are shown in Fig. 1. INH-RL migrated as a single peak with almobility in this system.Thus INH-RL behaves as a heat-stable pseudoglobulin with al electrophoretic mobil-

ity. It is a protein by the criterion of TCA precipitability (Table 1).

SEPARATION

UltrafiltrationExhaustive ultrafiltration of a 20 per cent Na2SO4 supernatant on an XM-100 mem-

brane showed that INH-RL was largely retained on the membrane while albumin, asexpected, was largely filtered. Since reduction in the albumin content of our sample wasexpected to increase the resolving power of other chromatographic steps, we oftenemployed extensive ultrafiltration of Na2SO4 supernatants at low pressures and lowprotein concentrations on an XM-100 membrane as a preliminary to further purificationprocedures.

137

138 B. McLeod, Patricia Baker and H. Gewurz

0: 07E ,/ '' 1 X

20 X4- 30

0-16-6 -4 -2 0 2 4 6 8 10 12 14 16 18

Dist--a from origin (cm)

FIG. 1. Pevikon block electrophoresis of INH-RL. The supernatant remaining after treatment ofNHS with 20 per cent Na2SO4 was applied in a 1-cm strip to Pevikon suspended in barbital buffer,pH 8-6, in a 40 x 20 x 0-6-cm plastic tray. Electrophoresis was carried out at 300 V for 12 hours at 4°.The block was cut into 1 cm strips which were eluted in 5 ml cold VBS and assayed for OD280 (0 0)and INH-RL units (0 --- o). INH-RL migrated as a single peak with al mobility.

TABLE 1

CHARACTERISTICS OF C567-INH (INH-RL)

(1) Soluble in distilled H20, pH 6.0 (pseudoglobulin).(2) Soluble in 20 per cent Na2SO4 and 17-5 per cent (NH4)2SO4.(3) Precipitated by TCA.(4) Stable at 560 for 60 minutes.(2) Alpha-l mobility on Pevikon block electrophoresis.(6) Retained (predominantly) on XM-100 membrane.(7) Retentate eluted from Sephadex G-200 in two peaks ( > 360,000 and 180,000 Daltons).(8) Excluded from CM-cellulose at pH 6-0, RSC = 0-007 M.(9) Eluted from DE-52, pH 8-5, in four peaks: RSC = 0 09 M; 0-14 M; 0-17 M; 0-21 M.

CM-cellulose chromatographyA 20 per cent Na2SO4 supernatant of NHS was subjected to extensive ultrafiltration

into starting buffer and applied to a CM-32 column as described. Essentially all of theC567-INH activity eluted in the excluded peak. A small peak of activity (> 0.01 per centof total) eluted late in the salt gradient, but was not studied further. Because this procedureeffected little separation, it was not used in the ultimate purification scheme.

Gel filtrationA 20 per cent Na2SO4 supernatant of NHS was subjected to extensive ultrafiltration

on an XM-100 membrane. It was then applied to a Sephadex G-200 column and elutedas described. The results of the analysis of fractions are shown in Fig. 2. Two peaks of


Q - 240

II ~~~~~-220

0200

3-6 80-IS3-2 - -160 La

IgM Ig S

2-8 - l-0KO

2-4 - ~~~~~1204220V 100

16~~~~~~~ 61-2-

o08 - 400~~~~~

2 4 6 8 10 12 14 16 18 20 22 24IgM IgG HSAVolume (ml x 100)

FIG. 2. Sephadex G-200 Chromatography of partially purified INH-RL, which had been concentratedon an XM-100 membrane. 0D280 (. *.INH-RL units (0 --- a).

activity were seen, one emerging with the void volume and one in the region of the IgGmarker.

DEAE-cellulose chromatographyAt the time the Sephadex G-200 column was done, the need for an internal standard in

the assay procedure for INH-RL had not been recognized and the existence of two dis-crete peaks of activity was not obvious. Therefore, except for small amounts held back forre-assay, the active fractions comprising both peaks were pooled, concentrated by ultra-filtration, dialysed overnight in the cold into 005 M Tris, pH 8-5, and applied to a DE-52column as described. Four peaks of activity were eluted in the linear NaCl gradient atRSC = 009 M, 014 M, 017 M and 021 M respectively (Fig. 3). The active fractions com-prising each peak were pooled separately and concentrated by ultrafiltration. They arereferred to as Pools 1, 2, 3 and 4 in accordance with the order in which they emerged fromthe column. The specific activities of the pools based on INH-RL activity and OD280were 17, 125, 91 and 25 units/mg ofprotein, respectively, compared with about 6 units/mgof protein in normal serum.

Thus, our scheme used for purification of INH-RL involved precipitation in 20 percent Na2SO4 at neutral pH, ultrafiltration on an XM-100 membrane, and chromato-graphy on Sephadex G-200 and DE-52. We observed two discrete peaks on gel filtrationwhich resolved into four peaks on an anion exchange resin. Analysis of the four pools

139


_-- * _ ~~~015 C)0-15 (1)

005

¼1 130

0~~~~~~~~~~~~~~~~~~~90.8 0I K 9

2 4 6 8 10 12 14167....... 1820I 22 ........... 24 26 28 30

2it 4 6ri, 8- 1tRC=0 12, 014,6018 20d022 24 26280 30 32N- nt

(o- --).

by immunoelectrophoresis and polyacrylamide gel electrophoresis revealed that each washeterogeneous.

MECHANISM OF ACTION

Site of action in the complement cascadeIn a previous publication, we analysed the site of action of crude preparations of JNH-

RL by considering that reactive lysis of E by C56 in dilute guinea-pig serum occurred inthree steps.

(1) C56+ C7---C567;_formation of C567 complexes.(2) E+C567---+EC567; attachment of C567 complexes to cells.(3) EC567+C8, C9--+E*; lysis of EC567.

Amounts of INH-RL which could completely block the formation of EC567 by C56 andC7 did not prevent the lysis of EC567 in comparable systems. This amount was judgednot to prevent formation of C567 complexes since: (1) inhibition of EC567 formationcould be not overcome by either excess C56 or C7 alone, but only by an excess of both,suggesting that the effect was on the product of C56 and C7, i.e. C567; (2) consumption ofC8 and C9 from solution by mixtures of C56 and C7 was not reduced by an amount ofinhibitor which would prevent EC567 formation by the same amounts of C56+C7.It was concluded that the paramount effect of INH-RL is to prevent attachment of C567complexes to E, and that it may properly be called the C567 inhibitor or C567-INH(McLeod et al., 1974b) .

140

C 56-initiated Lysis Inhibition, III.

The site of action of each of the pools from the DE-52 column was analysed in the sameway, with identical results. Thus all materials in serum which inhibit reactive lysis appearto do so by means of C567-INH activity.

Further studies of the mechanism of action of C567-INH were performed and aredescribed below. Each experiment was initially performed using C567-INH derived froma 20 per cent Na2SO4 supernatant of NHS which had been extensively ultrafiltered on anXM-100 membrane. The behavior of each of the four purified pools was investigatedafter optimum dosages and conditions for the experiment had been determined. Exceptwhere otherwise stated, the behaviour of C567-INH from all sources was found to beidentical.

Localization of the C567-INH effectA number of experiments were performed to determine whether the C567-INH effect

involved an interaction with the cell membrane, increasing its resistance to C567 attack,or an effect on the C567 complex itself in solution, preventing it from reaching andattaching to the cell membrane.

(1) Preincubation ofE with C567-IN1H. E were exposed to C567-INH for 15 minutes at370, then separated by centrifugation and removal of the supernatant. C56 and C7 wereadded to the cells in the procedure described in the methods section to form EC567.Cells processed in this way were found to be susceptible to lysis in dilute GPC-EDTA(i.e. to have formed EC567) even if pretreated with an amount of C567-INH ten timesthat which would prevent EC567 formation when presented along with C56 and C7.Conversely, there was no measurable loss of C567-INH from the supernatant when asolution containing it was preincubated with excess E and analysed for residual activity.Thus there appeared to be no firm attachment of C567-INH to red cell membranes.

(2) Manipulation of cell number. We postulated that if C567-INH worked via an effecton the cell membrane involving a loose, transient association, then the magnitude of itseffect should be related to the amount of C567-INH present per cell (i.e. per amount ofmembrane), and it should be possible to 'dilute out' the effect of a limited amount of C567-INH with larger numbers of cells. To test this postulate, the prevention of EC567 forma-tion by C567-INH was measured in reaction mixtures containing constant amounts ofC56 and C7 and variable numbers of cells. For each cell concentration, the number ofEC567 formed by C56 and C7 alone was determined. The deviation from this numberproduced by a given amount of C567-INH was taken to be the number of cells protectedby that amount of C567-INH.

Fig. 4a shows the results obtained with C567-INH present in a Na2SO4 supernatantafter ultrafiltration. For every amount of C567-INH in the dose-response curve, thenumber of cells protected increased as the number of cells presented increased. The slopesof these lines, which represent 'cells protected per unit C567-INH' over a wide range ofC567-INH concentrations, are plotted in Fig. 4b as a function ofnumber of cells presented.

141


(a) Iz

.P,D 0-8 0-it

08 10 20 A 20 0556 10 0

I)~~~~~~~~~X6

o~~~~~~~~~~~t 0h6 A Un 0o430i6c

cel wer wahd inuae t3°wt :8 P- Afo 5mntsadcnrfgdi h

o 0-4 -t 0h4e0 0.

02 ~~~~~~~~50.19

0.5 .0 2-0 4-0 5 10 15 20Nukmber Slope

edep of C567-INH of cells Numbr of celIs present (xlOpresent

FIG. 4. Effect of membrane concentration on efficiency of C567-INH. (a) Formation of EC567 usingthe same amounts of C56 and C7 in all experiments. A dose-response curve of inhibition of EC567formation was developed from0b5 to4m0u of C567-INH using each of four cell concentrations. Thecells were washed, incubated at 370 with i:80 GPC-EDTA for 15 minutes and centrifuged in thecold. The supernatant lysate was removed and the cells remaining were lysed in i0 ml of distilledwater. A value representing 0D412 of the cells lysed in each tube was obtained by comparison with acell blank. The difference between experimental tubes and a control tube to which no C567-INH wasadded was taken asoD412of the cells protected C567-INH. (b) The slope of each dose-response curvein (a) is plotted as a function of the number of cells present. (0) Experimental. -- )Predicted ifC567-INH acts on the membrane.

This plot clearly depicts an increasing efficiency of C567-INH with higher cell numbers,which is the opposite ofthe behaviour predicted for a membrane-related effect. An identicalpattern of behaviour was observed with more purified C567-INH from each of the fourpools derived from the DE-52 column. This data excludes a membrane related effect forC567-INH. It is readily explained by: (1) an increased binding efficiency of C567 at

higher cell concentrations, which is known to occur; (2) an inhibitory effect of C567-INH on C567 in solution.

Optimum reaction conditionsLittle variation in the effectiveness of C567-INH preparations in preventing EC567

formation was seen when the pH of the reaction mixture was varied between 6-0 and8-O, when ionic strength was varied from 0-O4 to 0-l5, or when the reaction temperaturewas varied from 140 to 370.

Effect ofC567-INH on lysis of EC567 by C8 and C9An important effect of C567-INH on lysis of EC567 was not demonstrable in the sys-

tems where we found that C567-INH would inhibit reactive lysis. However, before

142


discarding the possibility that a small effect of this type could occur, we examined the lysisof EC567 in very small amounts of C8 and C9. The lysis of EC567, made by the standardtechnique, in serial dilutions of guinea-pig serum was studied as shown in Fig. 5. Optimal

100 _

so _

80-

204

4 16 64 00 400 000 4000 16,000 64,000 256,000

Dilution of GPC-EDTA

FIG. 5. Effect of serum dilution on lysis of EC567. 5 x 106 EC567 were incubated for 30 minutes at 370with a wide range ofdilutions ofGPC-EDTA and the percentage lysis was determined. Efficiency of lysis,relatively low in whole serum, increases as the serum is diluted.

lysis of 5 x 106 cells after a 30-minute incubation in a 04-ml reaction volume first occurredat a dilution of 1: 80. Lower dilutions were equally effective in a 30-minute incubation,although other studies showed that the rate of lysis decreased as the serum was made morepilute. At dilutions of 1: 8000 or 1: 16,000, the extent of lysis began to decrease. Therefore,a dilution of 1 :12,000 was taken to contain a limiting amount of C8 and/or C9 in a30-minute incubation.The effect of graded doses of C567-INH on the lysis of 5 x 106 EC567 was studied in

04 ml of 1 :12,000 GPC-EDTA. The doses used (5-100 u) were relatively large, sinceonly 2 u of C567-INH would have sufficed to prevent formation of 80 per cent of theseEC567. Fig. 6 shows that large doses of C567-INH decrease the lysis of EC567 in thisexperiment. The results indicate that this is due to a reduction in the rate of lysis of EC567.For example, with 25 units of C567-INH, 30 minutes are required to achieve the sameextent of lysis (30 per cent) obtained at 15 minutes with 5 units.

(1) Localization of the effect of C567-IVH. When the lysis of EC567 was performed bysequential incubations with C8 and then C9, addition of C567-INH from any of the poolswas inhibitory at both steps, with almost equal potency at each step. Thus formationof EC5678 from EC567 was inhibited. Formation of EC56789 (E*) from EC5678 was alsoinhibited.

(2) Effect of C567 site density and C8, C9 concentration. EC567 judged to have varyingK

143


0

U40 asA 25

v U ~~~~~~50

A lao20-

15 30 Doseof

Tirre (rNrutes) C567 - INH(units)

FIG. 6. Effect ofC567-INH on lysis ofEC567 by limited quantities ofC8 and C9. EC567 were generatedby the standard procedure described in the text. 5 x 106 cells, of which 85 per cent were EC567, wereincubated for 15 minutes or 30 minutes at 370 in 0-4 ml of a dilution of GPC-EDTA chosen from Fig.5 to give 50-60 per cent lysis at 30 minutes. The effect of C567-INH in this system was studied atvarious doses. The results show that C567-INH exhibits dose-dependent suppression of this haemolysis.(e) All samples were at a 1: 80 dilution of GPC-EDTA.

numbers of C567 sites were prepared by incubating the same number of cells (107)with increasing concentrations of the reagents (C56 and C7) used to generate C567complexes. After two washings, the lysis of these cells by a 30-minute incubation in smallor in large amounts of purified C8 and C9 was studied, with or without the addition of10 units of C567-INH. The results are shown in Fig. 7. Over 80 per cent of cells in allpreparations had EC567 sites as judged by their ability to lyse in large quantities of C8and C9. When incubated for 60 minutes with small amounts of C8 and C9, however, thecells expected to have fewer C567 sites lysed poorly, and their lysis was totally suppressedby 10 u of C567-INH. Cells expected to have many C567 sites lysed respectably (60 percent) in small amounts ofC8 and C9, and their lysis was reduced but not totally suppressedby 10 u of C567-INH. Thus the EC567 expected to have higher C567 site densities areless sensitive to inhibition.The effect of the concentration of C8 and C9 was studied systematically and had the

expected influence on C567-INH. By increasing the concentration of C8 and C9, it waspossible to overcome the inhibition of EC567 lysis by C567-INH.

(3) Preincubation of EC567 with C567-IJVH. EC567 having increasing numbers of C567sites were incubated for 30 minutes at 370 with C567-INH and then separated by centri-fugation in an attempt to absorb C567-INH onto the cells. Cells having the lowest C567site density showed no difference from untreated controls in their lysis by limited amountsof C8 and C9. The supernatant which had been exposed to cells having the highest C567site density showed no difference from an untreated control in ability to inhibit EC567

144


It 60 U

14020

2 4 8

Relative atiities Of C56 and C7 used to prepare EC567

FIG. 7. Effect of C567 site density on inhibition of lysis of EC567 by C567-INH. EC567 were preparedas described in the text by incubating 5 x 106 E with C56 and C7 in amounts sufficient to give 80 percent formation of EC567. They were also prepared from 5 x 106 E and solutions of C56 and C7 whichwere 2, 4 and 8 times as concentrated as the standard mixtures. Such cells werejudged to have increasingnumbers of C567 sites per cell. After two washings in GVB-EDTA, EC567 were lysed by a 30 minuteincubation with either 150 u of C8 and 150 u of C9, or 1500 u of C8 and 1500 u of C9, with or withoutthe presence of 10 u of C567-INH. The results show that EC567 lyse more readily in large than in smallamounts of C8 and C9, and that inhibition of lysis of EC567 by C567-INH can be overcome byadding more C567 sites to the cell. 1500 u ofC8 and C9 alone (0 *) or with C567-INH (o o).150 u of C8 and C9 alone (-U ) or with C567-INH (n ri).

formation by fresh E, C56 and C7. Appreciable absorption of C567-INH to EC567 wasnot observed.

(4) Lysis ofEC567 in whole serum. It can be seen from Fig. 5 that lysis of EC567 proceededless well (25 per cent in 30 minutes) in whole guinea-pig serum than in dilute serum. Thesame pattern was seen with human serum. Substantial lysis (>50 per cent) could beobtained in whole human serum if the EC567 were prepared in an excess of C56 and C7and hence judged to have high C567 site density.

(5) Efect of C567-IJVH on lysis of EAC1-7. EAC1-7 were exposed to small quantities ofC8 and C9 in the presence of large quantities of each pool of C567-INH. No inhibition oftheir lysis was seen with any of the pools of C567-INH. In fact, large amounts of Pools2 and 4 seemed to potentiate lysis of EAC1-7. This difference in behavior of the sensitizedintermediate and EC567 might suggest that the C567 trimolecular unit is displayeddifferently at the two types of sites; however, it may simply be due to an inequality inthe number of C567 sites.

DISCUSSION

The ability of a C567 complex to attach to unsensitized cells represents one avenue by

145


which complement might escape the direction of antibody or foreign activating substancesand cause random membrane damage. The factors which influence attachment of C567complexes to cell membranes therefore constitute an important feature of the controlof complement function. Without such control, the production of C567 complexes insolution during complement activation for any reason could lead to extensive damageto host tissue.One factor restricting C567 attachment is the localization of complement activation;

however, this is not sufficient to contain it in all cases. The very occurrence of reactivelysis suggests that many alternate pathway activators lead to the formation of C56 andC567, which can escape from the ties of activation and are intrinsically capable of in-discriminate cell damage at a distance. It has also been shown that primary pathwayactivation (Gotze and Muller-Eberhard, 1970) and C5 activation by trypsin or plasmin(Arroyave and Muller-Eberhard, 1973) can lead to occurrence of the same intermediatesin solutions of purified complement components.

Another controlling factor is the evanescence of the capacity of a C567 complex tobind to a membrane. All investigators have found that the survival of the binding siteof C567 is brief and that the complex rapidly decays to an entity (which we abbreviateC567d) which, although haemolytically dead, remains capable ofinactivating C8 and C9.The decay of the ability of a mixture of C56 and C7 to form EC567 when added to Ehas been shown by several investigators to have a half-life of less than 30 seconds at tem-peratures between 300 and 370 (Lachmann and Thompson, 1970; Goldman, Ruddy andAusten, 1972). We have obtained similar results for a similar experiment. This figure,however, does not discriminate between the rate of formation of C567 from C56 and C7,and the rate of decay of C567 to C567d. Gotze and Muller-Eberhard (1970), studied thehalf-time of the latter reaction, and calculated the half-life for the membrane-bindingcapacity of the C567 complex to be less than 0 1 seconds. This is clearly a potent controlmechanism, but the same experiments on which its measurement is based showed that themobility of a haemolytically active C567 complex is appreciable relative to the averageintercellular distance at physiological cell densities. The brief period of reactivity istherefore not sufficient hindrance to prevent substantial membrane damage when C567is released in solution.

This report offers an initial description of the physicochemical nature of a newlyappreciated inhibitory activity which is involved in the control of C567, and presentsdata from haemolytic experiments which probe its mechanism of its action. The solubilityand electrophoretic characteristics of the activity are typical of an a-globulin and, togetherwith its chromatographic behaviour, suggest that negative charge is a characteristic of theinhibitory principle. Further attempts to characterize the activity, however, have allindicated that it is heterogeneous. Separations based on molecular sieving and ion-exchange techniques are shown here to yield multiple peaks of activity. Ultracentrifuga-tion has also shown heterogeneity. We cannot entirely exclude the possibility that partial

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decomposition of a homogeneous material, or its dissociations into subunits, might accountfor the apparent heterogeneity we have observed. It seems likely, however, that severalsubstances having negative charge and high molecular weight, account for the inhibitoryactivity in normal serum.We have presented evidence (McLeod et al., 1974b) that the primary effect of C567-

INH is to prevent formation of EC567 by preventing attachment of C567 complexes,implying that prevention of lysis of EC567 is not an important biological effect of C567-INH. In the studies described here, we have shown that the latter effect also can occur.Since the dose of C567-INH needed to interfere with lysis of EC567 in this reagent ismuch larger than that needed to prevent formation of EC567, and since EC567 mustform in order to lyse, the primary effect of C567-INH in preventing membrane damageprobably is to prevent C567 complexes formed in solution from attaching to membranes.The effect of C567-INH on C567 in solution has been shown to be one which allows

continued consumption of C8 and C9 by C567, although at high concentrations of C567-INH this reaction can also be impeded. Reduction in the rate of lysis of EC567 broughtabout by C567-INH could be overcome either by excess C567 (in the form of more C567sites per call) or by excess C8 and C9. This indicates that the reduction in rate is due toan interaction between C567-INH and cell-bound C567, which interferes with haemolyticaccess of C8 and C9. Since the interaction does not abolish haemolysis but only slows it,and since C567-INH is not absorbed from solution by EC567, the interaction betweenthem probably does not involve a strong bond between C567 and C567-INH and seemsto be readily reversible. Further, since the C567 is membrane bound in this instance, theinteraction between it and C567-INH need not involve its membrane-binding site.

This data, along with the estimate of the half-life of a membrane-binding site describedabove, make it possible to suggest a working model for the interactions of C567 andC567-INH which accounts for the haemolytic data. We propose that when a C567 complexis generated in a solution containing C567-INH an association occurs, bringing about anincrease in average particle size, a decrease in the effective diffusion coefficient of C567,and hence a further reduction in the already limited distance C567 can travel in solutionbefore its membrane-binding site decays. This would bring about a reduction in the rateof successful binding by C567 regardless of whether or not the C567 membrane-bindingsites were made inaccessible by C567-INH. The C567d complex left in solution after decayofthe binding site would still be able to inactivate C8 and C9, although it might be expectedto encounter these molecules more slowly. Cell-bound C567 would also be capable ofloose,reversible associations with C567-INH, causing reduced access ofC8 and C9. In this view itis not any property of the (C567)-(C567-INH) interaction per se, that makes attachment ofC567 to cells the crucial step in the sequence. Rather it is the instability of the membrane-binding site on C567 which makes this step so sensitive to inhibition. Such a brief periodof reactivity can be a control mechanism in itself, but in the case of C567 at physiologicalcell densities it is insufficient to prevent membrane damage unless it is reinforced by the

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effect of C567-INH. Since our model depicts only a loose, readily reversible association,it might be expected that extensive intermolecular congruity would not be required for theinhibitory interaction. This might account for the indications that several serum proteinscan serve as C567-INH. The functionally important property they share may well benegative charge since heparin and other polyanions have also been found to inhibitreactive lysis (McLeod et al., 1973a).The mechanism of action we propose is quite different from that of other well studied

complement inhibitors. These are either enzymes which alter a component so as to in-activate it (Cl -esterase inhibitor and C3-inactivator), or haemolytically inactive com-ponent complexes which compete with active complexes for cell sites or for more distalcomponents (Koethe, Austen and Gigli, 1973). We are intrigued by certain similaritiesbetween the activity described here, and the chemotactic factor inactivator (CFI)described by Berenberg and Ward (1973). Both are soluble at high ionic strength, bothare heterogeneous on further purification (although not identically so), and neither formsa strong bond with the factor it inhibits. CFI, however, was heat-labile and was thoughtby its investigators to act by irreversibly altering the bacterial factor.

It seems unlikely that C567-INH interferes with membrane damage mediated by theclassical complement pathway at sites identified by antibody. An inhibitory effect ofC567-INH on the lysis of EA or EAC142 is hard to demonstrate, and occurs only atphysiologically unrealistic ratios of C567-INH to distal complement components (Mc-Leod et al., 1974a). Also, C567-INH obviously does not prevent vigorous complement-induced lysis of sensitized cells in whole serum.

It seems likely that substances which activate complement by the alternate pathwayregularly produce fluid phase C56 and C567. Thus the effect of C567-INH should beparticularly important in the control of complement activation by this means, and mayaccount for the low efficiency of lysis which is seen with alternate pathway activation(May, Green and Frank, 1973). Work in progress at this laboratory indicates that whenC567-INH activity is blocked, passive haemolysis of bystander erythrocytes during alter-nate pathway activation by cobra venom factor is greatly enhanced (McLeod et al.,1973b).In summary, this report has indicated that C567-INH, the activity in serum which

inhibits reactive lysis, is localized in the a-globulin fraction and appears to be heterogen-eous by several criteria. Its mechanism of action has been shown to involve an effect onC567 complexes in solution that impedes their access to cells during the brief period inwhich they are capable of attaching to membranes. It is proposed that this is due to areversible interaction between C567-INH and C567 which increases the bulk and hencedecreases the effective mobility of C567. C567-INH probably has an important role inpreventing damage to host tissue during alternate pathway activation.

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C56-initiated Lysis Inhibition, III. 149

ACKNOWLEDGMENTS

This research was supported by grants from the National Institutes of Health (number5 ROl AI 10784-02), the American Heart Association, the Leukemia Research Founda-tion and the Fay-Hunter Trust. H.G. is the ThomasJ. Coogan, Senior, Professor and Chair-man of Immunology.

REFERENCES

ARROYAVE, C. M. and MULLER-EBERHARD, H. J.(1973). 'Interactions between human C5, C6 andC7 and their functional significance in complementdependent cytolysis.'_7. Immunol., 111, 536.

BERENBERG, J. L. and WARD, P. A. (1973). 'Chemo-tactic factor inactivator in normal human serum.'J. clin. Invest., 52, 1200.

GOLDMAN, J. N., RUDDY, S. and AUSTEN, K. F. (1972).'Reaction mechanism ofnascent 0567 (reactive lysis).I. Reaction characteristics for the production ofE0567 and lysis by C8 and C9.'J. Immunol., 109,353.

GOTZE, 0. and MULLER-EBERHARD, H. J. (1970).'Lysis of erythrocytes by complement in the absenceof antibody.'_J. exp. Med., 132, 898.

KOETHE, S. M., AUSTEN, K. F. and GIGLI, I. (1973).'Blocking of the hemolytic expression of the classicalcomplement sequence by products of complementactivation via the alternate pathway. Materialsresponsible for the blocking phenomenon andtheir proposed mechanism of action.' J. Immunol.,110, 390.

KOLB, W. P., HAXBY, J. A., ARROYAVE, C. M. andMULLER-EBERHARD, H. J. (1972). 'Molecularanalysis of the membrane attack mechanism ofcomplement.' J. exp. Med., 135, 549.

KOLB, W. P., HAXBY, J. A., ARROYAVE, C. M. andMULLER-EBERHARD, H. J. (1973). 'The membraneattack mechanism of complement: reversibleinteractions among the five native componentsin free solution.'_J. exp. Med., 138, 428.

LACHMANN, P. J. and THOMPSON, R. A. (1970). 'Re-active lysis: the complement mediated lysis ofunsensitized cells. II. The characterization of

activated reactor as C56 and the participation of C8and C9.'_J. exp. Med., 131, 643.

MAYER, M. M. (1961). 'Complement and complementfixation.' Experimental Immunochemistry (ed. by E. A.Kabat and M. M. Mayer), p. 133. Thomas, Spring-field.

MAY, J. E., GREEN, I. and FRANK, M. M. (1972).'The alternate complement pathway in cell damage:antibody mediated cytolysis of erythrocytes andnucleated cells.' 7. Immunol., 109, 595.

McLEOD, B. C., BAKER, P. and GEWURZ, H. (1973a).'Studies of an inhibitor of reactive lysis.' Fed. Proc.,32, 873.

McLEOD, B. C., BAKER, P. and GEWURZ, H. (1973b).'Regulation of membrane damage by the alternatecomplement pathway.' Clin. Res., 21, 873.

McLEOD, B. C., BAKER, P. and GEWURZ, H. (1974a).'Studies on the inhibition of C56 initiated lysis. I.Description of the phenomenon and method ofassay.' Immunology, 26, 1145.

McLEOD, B. C., BAKER, P. and GEWURZ, H. (1974b).'Studies on the inhibition of C56-initiated lysis. II.C567-INH-an inhibitor of the C567 trimolecularcomplex of complement.' Int. Arch. Allergy, 47, 643.

NILSSON, U. and MULLER-EBERHARD, H. J. (1967).'Studies on the mode of action of the fifth, sixth,and seventh components of human complement inimmune hemolysis.' Immunology, 13, 101.

THOMPSON, R. A. and LACHMANN, P. J. (1970).'Reactive lysis: the complement mediated lysisof unsensitized cells. I. The characterization ofthe indicator factor and its identification as C7.'3. exp. Med., 131, 629.

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