Proc. Nati. Acad. Sci. USAVol. 73, No. 9, pp. 3268-3272, September 1976Immunology

Control of the amplification convertase of complement by the plasmaprotein j1lH

(amplification convertase inhibitor/C3b inactivator/properdin/C3 nephritic factor/alternative complement pathway)

JOHN M. WEILER, MOHAMED R. DAHA, K. FRANK AUSTEN*, AND DOUGLAS T. FEARON

Departments of Medicine, Harvard Medical School and Robert B. Brigham Hospital, Boston, Massachusetts 02120

Contributed by K. Frank Austen, June 9,1976

ABSTRACT An inhibitory activity for an erythrocyte in-termediate bearing the properdin (P)-stabilized amplificationC3 convertase, PC3bBb, was recognized in whole normal humanserum and separated from C3b inactivator by its distinct phys-icochemical and functional characteristics. The inhibitory ac-tivity was found to reside in a protein that was purified to ho-mogeneity and elicited a monospecific antibody in a rabbit. Thisprotein was identified as P11H and found to have a serum con-centration of 516 + 89 yg/ml (mean ±1 SD).-#1H produced a dose related, first-order loss of convertase

function and release of 125I-Bb from the P-stabilized interme-diate, indicating a mechanism of action by decay-dissociationof Bb from the complex, PC3bBb. PI1H exhibited only a limitedcapacity to accelerate decay of C3bBb sites stabilized with C3nephritic factor or to release 125I-Bb from such sites.

Amplification of C3 cleavage by C3bBb may well determinewhether initial complement activation by the classical or al-ternative activating sequence is beneficial or detrimental to thehost. Regulation of this amplifying function is now recognizedto occur at at least three steps: intrinsic decay which reflects theinherent lability of the C3bBb convertase; extrinsic decay-dis-sociation of Bb which is mediated by the effect 4f P1H; andinactivation of exposed C3b by C3b inactivator. The stabiliza-tion of C3bBb by activated properdin minimizes intrinsic decayand protects C3b in the bimolecular complex from C3b inacti-vator. P1H restores control of the system by decay-dissociationof the bimolecular complex, thereby exposing C3b to C3b in-activator whose irreversible action prevents regeneration of theconvertase at that site.

The complement system, which is comprised of at least 18plasma proteins, consists of four functional divisions: twopathways for activation, the classical and alternative (proper-din); a single amplification mechanism that is recruited by eachactivating pathway; and a final common effector pathway towhich the activating and amplifying sequences are directedand from which are derived the biologic activities of comple-ment (1, 2). Activation of the classical pathway by antigen-antibody complexes containing immunoglobulin of the ap-propriate class involves conversion of C1 from its precursor formto an active state, C1 (3), with subsequent cleavage of C4 (4)and C2 to form C4b2a (5), the classical C3 convertase that ini-tiates cleavage of C3. Activation of the alternative pathwayoccurs with certain microbial polysaccharides that interact withB, D, C3, and other proteins to achieve initial CS cleavage (6).The major cleavage fragment of CS, C3b, generated by eitheractivating sequence, then interacts with B and D (7) to form theamplification convertase, C3bBb (8, 9).

Abbreviations: P, activated properdin; PC3bBb, properdin-stabilizedamplification CS convertase; C3NeF, CS nephritic factor; CMbINA,C3b inactivator; DGVB++, half-isotonic Veronal-buffered saline, 0.1%gelatin, 0.5 mM magnesium, 0.15 mM calcium, and 2.5% dextrose;DGVB-EDTA, half-isotonic Veronal-buffered saline, 0.1% gelatin, 0.01M EDTA, and 2.5% dextrose; C-EDTA, rat serum diluted 1:20 inVeronal-buffered saline, 0.1% gelatin and 0.04 M EDTA.* To whom reprint requests should be addressed.

In the amplification step, C3b serves as a receptor for B (9)in a magnesium-dependent binding reaction'that partially re-veals the proteolytic site in B for C3 (10). D, a protease of theserine esterase class (11), cleaves bound B to release the Bafragment and to uncover fully the CS-cleaving site on Bb whichremains bound. The C3bBb complex is labile because of irre-versible decay-dissociation of Bb, but can be regenerated onthe residual C3b by the uptake of additional B and its cleavageby D (9). Activated properdin (P) binds to C3b (12-14), and CSnephritic factor (C3NeF), a serum protein found in some pa-tients with hypocomplementemic membranoproliferativeglomerulonephritis, binds to C3bBb (15); both proteins stabilizethe convertase and increase its half-life by as much as 10-fold,thereby profoundly augmenting amplification of CS cleavage(12, 16).

Control of the amplification pathway is regulated by theinherent lability of the C3bBb convertase (9) and by inactiva-tion of the residual C3b by C3b inactivator (C3bINA) (17, 18)which thereby prevents regeneration of the convertase (19).These controls are circumvented in the stabilized convertases,because intrinsic decay is retarded and C3bINA cannot inac-tivate C3b carrying Bb (15, 20). #l1H (21), a plasma protein of175,000 molecular weight, accelerates decay of C3bBb, evenwhen stabilized by P. and thus allows C3bINA to reassert itscontrol function. In contrast, amplification convertase stabilizedby C3NeF, a protein isolated from pathologic sera, is relativelyresistant to the reestablishment of controls by #31H.

MATERIALS AND METHODSSephadex G-200 (superfine), Sephadex G-200, Sephadex G-25,quaternary aminoethyl Sephadex A-50 (Pharmacia FineChemicals, Inc., Piscataway, N.J.), hydroxylapatite (HypatiteC, Clarkson Chemical Co., Inc., Williamsport, Pa.), Na125I(New England Nuclear, Cambridge, Mass.), polyethylene glycol20,000 (Carbowax), agarose (Fisher Scientific Co., Somerville,N.J.), UM10 Diaflo ultrafiltration membranes (Amicon Corp.,Lexington, Mass.), and insolubilized lactoperoxidase (Worth-ington Biochemical Corp., Freehold, N.J.) were obtained asindicated. Disc gel electrophoresis was performed in 5% poly-acrylamide gels according to instructions supplied by themanufacturer (Canalco, Inc., Rockville, Md.).Complement Components and Assays. Half-isotonic Ver-

onal-buffered saline at pH 7.5, containing 0.1% gelatin, 5 X10-4 M magnesium, and 1.5 X 10-4M calcium (GVB++), and2.5% dextrose (DGVB++), and half-isotonic Veronal-bufferedsaline containing 0.1% gelatin, 0.01 M EDTA, and 2.5% dex-trose (DGVB-EDTA) were used as diluents in hemolytic as-says.

Proteins B (7), D (12), P (22), and C3 (21, 23) were purifiedto homogeneity and quantitated as described. Sixty microgramsof B were trace labeled with 125I and insolubilized lactoperox-

3268

Proc. Nati. Acad. Sci. USA 73 (1976) 3269

idase (24) which yielded a specific activity of 50,000 cpm/,ugof protein. Guinea pig C1, guinea pig C2, (25), and C3NeF (16)were functionally purified and quantitated; C5, C6, C7, C8,and C9 were obtained from Cordis Corporation, Miami, Fla.Serum C3bINA eluted from quaternary aminoethyl Sephadexthat was equilibrated with 0.01 M Tris, 0.05 M NaCi at pH 7.5between 5.8 and 6.2 milliSiemens (mS) and was filtered onG-200 (superfine) equilibrated with Veronal-buffered salinemade hypertonic with additional 0.15 M NaCl and 0.002 MEDTA. Functional C3bINA (20) was concentrated to 60% ofplasma concentration and was free of B, D, C3, and P functionalactivities and fl1H on radial immunodiffusion.To assess serum or its fractions for their capacity to inhibit

the amplification convertase, an assay was developed using anerythrocyte-bound, P-stabilized convertase. Limited B, 100 ng,of D, and 3.0 ,ug of P/108 cells (EAC43), prepared with 80 ,gof C3/109 cells (EAC142) (9), were incubated at 30° for 30 minto generate EAC43PB. Dilutions of serum or its fractions inDGVB-EDTA were incubated with 1 X 107 EAC43PB in 0.2ml of DGVB-EDTA for 15 min at 300, and residual convertasesites were developed by addition of 0.3 ml of a 1:20 dilution ofrat serum in Veronal-buffered saline containing 0.1% gelatinand 0.04 M EDTA (C-EDTA), and further incubation for 60min at 370. The amount of B used to make the hemolytic in-termediate was selected to permit approximately 63% hemolysisin the absence of inhibitor. Inhibitory units/ml of serum or itsfraction were calculated as follows:

Inhibitory units/mi = dilution X ln (A - A)bAnon inh - Arbwhere, at A414 n, Ainh is the lysis of the inhibited cellular in-termediate, Anon inh is cell lysis of EAC43PB in the absence ofinhibitor, and Arb is the reagent blank consisting of EAC43 cellsinteracted with 1:20 dilution of C-EDTA.

RESULTSNormal human serum inhibited P-stabilized amplificationconvertase sites on EAC43PB in dose-response fashion with theinhibitory titer/ml of between 2000 and 3000. Although itseemed unlikely that this inhibitory activity of serum for astabilized convertase was due to C3bINA, the initial chroma-tographic isolation of the inhibitory activity utilized a step inwhich the elution characteristics of C3bINA were known.

Purification and Identification of Amplification Con-vertase Inhibitory Protein. Twenty-five milliliters of freshserum were diluted with ice-cold distilled water to 3.3 mS,adjusted to pH 7.5 with dilute HC1 and applied to a 2.5 X 40cm column containing quaternary aminoethyl Sephadexequilibrated with 0.01 M Tris, 0.05 M NaCl, 0.002 M EDTAat pH 7.5. The column was washed with 300 ml of startingbuffer, and eluted with a 2 liter linear gradient of 0.05-0.33 MNaCl. C3bINA eluted between 5.8 and 6.2 mS as assessed byirreversible inactivation of an EAC43 intermediate (20), whilea single peak of inhibitory activity for the EAC43PB interme-diate was obtained between 9.0 and 10.0 mS. The inhibitoryactivity for EAC43PB was concentrated to 18 ml by ultrafil-tration, adjusted to pH 5.5 with dilute HCl, and dialyzedovernight at 40 against 6000 ml of 0.1 M sodium acetate, 0.002M EDTA at pH 5.5. The resulting precipitate was washed threetimes in dialysis buffer, dissolved in Veronal-buffered salinecontaining additional 0.15 M NaCl and 0.002 M EDTA, andapplied to a 2.5 X 80 cm column containing Sephadex G-200(superfine) equilibrated with the same buffer. The inhibitoryactivity was in fractions containing the major protein peakwhich corresponded to an apparent molecular weight of

175,000. An 8 ml pool containing peak inhibitory activity wasdialyzed overnight against 1000 ml of phosphate buffer, 4 mS,at pH 7.9 and applied to a 1.5 X 11 cm column of hydroxyla-patite equilibrated with dialysis buffer. The column was washedwith 50 ml of buffer and eluted with a 400 ml linear phosphategradient to 18 mS. Fractions containing peak inhibitory activityeluted between 8.0 and 9.0 mS and were pooled and concen-trated in a dialysis bag against dry G-200. Approximately 5%of the concentrate containing 75 ,g of protein was applied toeach of two 7.5 cm alkaline disc gels; one disc gel was stainedand revealed a single band 2.5mm from the cathodal end whichcorresponded to the area from which function was eluted fromthe replicate gel.

In a separate experiment, eluates from alkaline disc gels werepooled, emulsified with Freund's complete adjuvant, and usedto immunize a single rabbit. Antiserum was obtained whichgave a single precipitin arc in the jB globulin region on immu-noelectrophoretic analysis of normal human serum in 1.5%agarose. On Ouchterlony analysis this antiserum and an anti-31H antiserum provided by Dr. U. R. Nilsson gave a line ofidentity between the protein which each recognized in wholehuman serum and the hydroxylapatite-purified inhibitoryprotein. Protein concentration of the purified #1H was deter-mined by Folin analysis with purified C3 as the referenceprotein. Purified #1H was used to standardize radial immu-nodiffusion plates prepared with monospecific anti-#IH at a1:30 dilution with 1.5% agarose in isotonic Veronal-bufferedsaline containing 0.1% gelatin and 0.01 M EDTA. Twentynormal sera, quantitated by radial immunodiffusion, containedan average of 516 ± 89MAg of i1lH/ml (mean +1 SD). The iso-lation procedure resulted in a 100-fold purification and anapproximately 10% yield.

Inhibition by #1H of Unstabilized and Stabilized Ampli-fication Convertases. Dose response effects of fl1H on P-sta-bilized amplification convertase were studied kinetically bymeasuring residual convertase sites. EAC43, 4.3 X 108 cells,were incubated with 3.5 ng of B, 13.7 Mg of P, and 0.43 Mg ofD in 8.6 ml of DGVB++ at 30' for 30 min to generateEAC43PB. EAC43, 6 X 107 cells, were incubated with 9.9 ngof B and 66 ng of D in 1.2 ml of DGVB++ at 300 for 30 min toform EAC43B. The intermediates were washed twice in ice-cold DGVB-EDTA, and 1 X 108 EAC43PB cells were resus-pended at 300 in 2 ml of DGVB-EDTA alone or containing 0.84Mug of 61H, 0.28 ,g of B1H, and 0.094 Mg of #1H, respectively;6 X 107 EAC43B cells were resuspended in 1.2 ml of bufferalone. Incubation was continued at 300, during which 0.2 mlsamples were removed from each reaction mixture at timedintervals and added to 0.3 ml of C-EDTA to develop residualconvertase sites per cell (Z) during a 60 min incubation at 370as assessed by lysis (Fig. 1). The half-life of the stabilized con-vertase in buffer alone was 55 min and diminished in a dose-related manner with increasing inputs of ,B1H. The #1H-mediated loss of convertase sites was first-order at each input,and the highest input reduced the half-life to that of the un-stabilized convertase.

In a comparable experiment, 0.48 ,ug of ,B1H per 108 cellsaccelerated the decay of the unstabilized convertase from a4-min to a 1.5-min half-life but failed to affect the 70-minhalf-life of the C3NeF-stabilized convertase. Three higherconcentrations of #I1H were used to assess further the apparentrefractoriness of the C3NeF-stabilized convertase to ,31H.EAC43, 4.3 X 108 cells, were incubated with 28.3 ng of B, 0.34units of C3NeF, and 0.43 Mug of D in 8.6 ml of DGVB++ at 300for 30 min to generate EAC43BNeF; 6 X 107 EAC43B cellswere prepared as in the experiment depicted in Fig. 1. The

Immunology: Weiler et al.

3270 Immunology: Weiler et al.

1.0

0.5z7

10 20 30MINUTES

40 50

FIG1. Decay ofEAC43PB alone (M - *), and in the presence of0.094 Mg of#1H (0 - - 0), 0.28 Mg of 61H (A - - A), or 0.84 MIg of f1H(O - - 0); decay of EAC43B alone (0 - 0).

intermediates were washed twice in ice-cold DGVBEDTA and1 X 108 EAC43BNeF cells were resuspended at 300 in 2 ml ofDGVB-EDTA alone or containing 21 Mg of j1H, 4.2 ,g of (31H,and 0.84 Mug of #1H, respectively; the EAC43B cells were re-

suspended in 1.2 ml of buffer alone. Incubation was continuedat 300 and 0.2 ml samples were removed from each reactionmixture at timed intervals, diluted with 3 ml of ice-coldDGVB-EDTA, sedimented, resuspended in 0.2 ml ofDGVB-EDTA and residual convertase sites developed as in theexperiment depicted in Fig. 1. The half-life of C3NeF-stabi-lized convertase site was 48 rnin in buffer alone and diminished

with the highest concentration of (31H to 20 min which was stillin excess of the 3-min half-life for the unstabilized convertase(Fig. 2).

Release of 125I-B by PJH. Since j#1H accelerated the func-tional decay of stabilized and unstabilized convertase, its ca-

pacity to release radiolabeled B from the two classes of stabilizedconvertase was determined. EAC43, 2.2 X 108 cells, 0.22 Mug ofD, and 3 gg of 125I-B in 4.2 ml of DGVB++ were incubated witheither 14 Mug of P or 0.09 units of C3NeF for 60 min at 300,washed three times in ice-cold DGVB++, and resuspended with2.2 ml of DGVB++. EAC43, 6.6 X 107 cells, interacted with 0.9Mug of 125I-B in 1.32 ml of DGVB++ were treated in an identical

2.0~~~~~~~K

0f.1 11I10 20 30 40 50 60

MINUTESFIG. 2. Decay ofEAC43BNeF alone (U *), and in the presence

of 0.84 gg of 1H ( ---0), 4.2 gg of AlH (A - - A), or 21 gg of,1H(O - - 0); decay of EAC43B alone (O ).

Table 1. Residual 12"I-Bb molecules per cell aftertreatment with 1H

Bb molecules/cellularintermediate

plH addition*(Mg) EAC43PB EAC43BNeF

None (unincubated) 1400 1800None 580 14500.02 2500.07 1100.21 500.2 14500.7 13502.1 1250

* Reactions were incubated at 300 for 30 mim except where noted.

fashion and served as a control for non-1D-dependent bindingof 125I-B. Replicate samples containing 5 X 107 of each cellularintermediate were assessed for bound l25I-Bb at time zero andthen incubated for 30 min at 300 with 0.5 ml of DGVB++ aloneor containing 0.21 jig of 01H, 0.07 Mg of g31H, and 0.023 gg of(31H, respectively, in the case of the EAC43PB intermediateand 2.1 Mg of #1H, 0.7 Mg of (31H, and 0.23 Mug of #1H, respec-

tively, for EAC43BNeF cells. The reaction mixtures were

centrifuged at 40, the intermediates washed three times withice-cold DGVB++, resuspended with 0.5 ml of DGVB++ andassessed for residual cell-bound 125I-Bb. The results are ex-

pressed as molecules of 125I-Bb per cell. No '25I-Bb was boundin the absence of D. 125I-Bb was released from both stabilizedintermediates during the incubation period in buffer alone, and(J1HI released additional 125I-Bb from both intermediates in adose-related manner (Table 1). Whereas 0.21 Mg of ft1H dis-sociated 530 of 580 P-stabilized 125I-Bb molecules per cell, 10times that amount dissociated only 200 of 1450 C3NeF-stabi-lized 125I-Bb molecules per cell.

Sequential Action of (ilH and C3bINA on the P-stabilizedIntermediate. The ability of (31H to accelerate decay of Bbfrom the P-stabilized intermediate suggested that this actionof this protein may allow C3bINA access to C3b. Fourteen re-

action mixtures containing 5 X 107 EAC43 cells bearing limitedamounts of C3b per cell, as a result of being prepared with 0.12Mg of C3 per 109 EAC142 cells, were each incubated in 0.5 mlof DGVB++ with 0.164 ,ug of B, 1.6 Mg of P, and 50 ng of D at300 for 30 min. The cells were washed three times and resus-

pended in 0.5 ml of DGVB-EDTA; a 0.1 ml portion of each wasremoved to assess the initial number of convertase sites. The 14cell suspensions were incubated with buffer alone or with thecontrol proteins for 15 min at 300, then collected by sedimen-tation, washed, and resuspended to 0.4 ml in buffer with or

without the control proteins as indicated in Table 2. After a

further incubation for 15 min at 300, the cells were sedimented,washed, and resuspended in 0.4 ml of DGVB++, and a 0.1 mlportion was assessed for residual convertase sites. The remainingcells were assessed for residual cell-bound C3b by their capacityto regenerate convertase sites by interaction with B, P, and Din their original concentrations for 30 min at 300. All samplescontained 1 X 107 cells in 0.1 ml of DGVB++ and were devel-oped with 0.3 ml of G-EDTA. As shown in Table 2, only 26%of the initial i-stabilized convertase sites remained after the twoincubation periods in buffer alone, and this loss was not in-creased by the presence of C3bINA during the first incubation.The presence of #1H during the first incubation did give a

dose-related further diminution of convertase sites. The cells

A\ 0\\\ \

\\ \ \\\\; \ \R\\b

Proc. Natl. Acad. Sci. USA 73 (1976)

Proc. Natl. Acad. Sci. USA 73 (1976) 3271

Table 2. Sequential action of ,B1H and C3bINA* on theP-stabilized convertase

Percent of initialSequence of treatment convertase sites

First Second Residual Restored

Buffer Buffer 26 100C3bINA Buffer 26 94fl1H (jug/ml)

1 Buffer 7 1000.5 Buffer 12 970.25 Buffer 23 95

,slH(pg/ml) +C3bINA

1 Buffer 14 200.5 Buffer 16. 360.25 Buffer 20 63

,B1H (jug/ml )1 C3bINA 7 140.5 C3bINA 9 360.25 C3bINA 18 57

f1H (yg/ml)C3bINA 1 8 99C3bINA 0.5 13 100C3bINA 0.25 16 100

* C3bINA was used at a final dilution of 1:100.

exposed to either C3bINA or to incremental doses of /B1Hduring the first incubation were comparable to buffer treatedcells in residual functional cell-bound C3b. The combinedpresence of C3bINA and (B1H during the first incubation re-duced the convertase sites in a manner comparable to jB1Halone, but in addition, yielded a #1H dose-related suppressionof convertase regeneration, presumably because of the actionof the C3bINA on the exposed C3b. The introduction of ,1Hduring the first incubation and C3bINA during the second in-cubation suppressed regeneration in a manner comparable withtheir simultaneous presence in the first incubation. In contrast,when the sequence of inhibitory proteins was reversed so thatC3bINA was present during the first incubation and #1H waspresent during the second, loss of convertase sites was not as-sociated with loss of cell-bound C3b function in that regener-ation of convertase was as complete as for buffer-treatedcells.

DISCUSSIONThe circumvention of the recognized control steps once thelabile amplification convertase C3bBb has been stabilized byproteins in normal or pathologic sera prompted a search foradditional control mechanisms. An inhibitory activity for anintermediate bearing the P-stabilized convertase was recognizedin whole normal human serum, and was separable fromC3bINA by its distinct physicochemical and functional char-acteristics. After chromatography of whole serum on quater-nary aminoethyl Sephadex, C3bINA eluted early with trans-ferrin and was inactive on the stabilized intermediate whileinhibitory activity for the stabilized intermediate eluted withalbumin. The latter was further purified by euglobulin pre-cipitation, gel filtration, and chromatography on hydroxyla-patite. The final product was present as a single stained bandon alkaline disc gel electrophoresis, and elution of a replicateunstained gel yielded inhibitory activity in the correspondingregion. Antisera raised against the material eluted from disc gels

recognized a single protein in the # region on immunoelectro-phoretic analysis of whole human serum. On Ouchterlonyanalysis this antiserum and an anti-#IH antiserum providedby Dr. U. R. Nilsson gave a line of identity between the proteinwhich each recognized in whole human serum and the hy-droxylapatite-purified protein. Quantitation of #I1H by radialimmunodiffusion indicated a serum concentration of 516 I 89,gg/mil (mean 4 1 SD), and, thus, this protein constitutes ap-proximately 1% of the serum protein concentration. The overallrecovery was approximately 10% with a purification of 100-foldas compared with starting serum. #I1H has independently beenrecognized to be antigenically identical with AcceleratorsC3bINA (26), a protein which augments C3b inactivation (27)by C3bINA and in addition suppresses formation of the P-sta-bilized amplification convertase.

Since the inhibitory action of 6i1H was directed against thefunctional integrity of the P-stabilized convertase, the timecourse of this effect was assessed at different #I1H concentrations(Fig. 1). Accelerated loss of convertase function by (#1H wasdose-related and first order which indicates an immediate andprogressive action on the convertase. The stabilizing action ofP was entirely reversible by ,6H. The capacity of (B1H to ac-celerate loss of convertase function was also apparent for theunstabilized amplification convertase and suggests an effecton Bb, possibly by accelerating its decay-dissociation from theC3b site. Indeed, the release of 125I-Bb (Table 1) from the P-stabilized convertase by j01H in a dose-related fashion indicatesthat the mechanism of its action is decay-dissociation of thebimolecular complex. Although such an action would reversestabilization, regeneration of convertase on cell-bound C3b withfresh B, P, and D (Table 2) could still occur. However, in thepresence of C3bINA, augmented decay-dissociation of theconvertase by fl1H would restore the irreversible controlfunction of C3bINA which cannot be expressed when C3b isprotected by Bb in the bimolecular complex. This point iscritically illustrated in Table 2. Cell-bound C3b permits re-generation of the convertase when the P-stabilized intermediateis treated with C3bINA before decay-dissociation of the con-vertase by #1H. In contrast, when the P-stabilized convertaseis decay-dissociated by fB1H and then treated with C3bINA,there is loss of residual functional C3b that is dose-related to theoriginal #1H input.

Dose-response studies (Fig. 2) indicated that the amount of(01H required to reduce the half-life of the C3NeF-stabilizedconvertase by 50% was 200-fold greater than that needed toachieve the same relative effect on the P-stabilized convertase.The resistance of the C3NeF-stabilized convertase to decay-dissociation was confirmed by the limited ability of 031H todissociate 25I-Bb from the C3NeF-stabilized convertase (Table1).

Control of the amplification pathway is critical to its regu-lated utilization and may well determine whether the initialactivation of the classical or alternative activating sequence isbeneficial or detrimental to the host. This regulation occurs atat least three levels (Fig. 3): intrinsic decay which reflects theinherent lability of the C3bBb convertase; extrinsic decay-dissociation of Bb which is mediated by the effect of #IH; andinactivation of exposed C3b by C3bINA thereby preventinginitial formation ahd regeneration of the amplification con-vertase. The stabilization of the amplification convertase by Fminimizes intrinsic decay and protects C3b in the bimolecularcomplex from C3bINA. #1H restores control by decay-disso-ciation of Bb which exposes C3b to the irreversible action ofC3bINA. Thus, it is the sequential action of #1H and C3bINAthat fully reverses the augmenting effect of F on the amplifi-

Immunology: Weiler et al.

Proc. Natl. Acad. Sci. USA 73 (1976)

GENERATION STABILIZATION CONTROL

INTRINSIC PX1H:ETRINSIC C3bINA: ESCAPEI IDECAY DECAY INACTMION

'C3b +BA- C3bBb 3b -C3c + C3d

PFC3bBb t 3 PC3c+ CMdI Ba I I '~~~~~~~BiB

C3bBb(C3NeF) C3bBb(C !eI

81 81IBi

FIG. 3. Schematic representation of three levels of control of the amplification convertase of the complement system.

cation convertase. It is noteworthy that C3NeF, which createsa stabilized convertase that is relatively resistant to #1H controLis found in individuals with persistently depressed serum levelsof C3 and often with renal disease such as hypocomplemen-temic membranoproliferative glomerulonephritis (28, 29).

This work was supported by Grants AI-07722, AM-05577, RR-05669,and AI-10356 from the National Institutes of Health. D.T.F. is apostdoctoral fellow of the Helen Hay Whitney Foundation, M.R.D.of the Netherlands Organization for the Advancement of Pure Re-search, and J.M.W. of Training Grant AI-00366 from the NationalInstitutes of Health.

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