The Inhibitory Effect of Factor J on the Alternative Complement Pathway*

Vol. 269, No. 42, Issue of October 21, pp. 26017-26024, 1994

THEJOURNAL OF B I O ~ I C CHEMISTRY AL 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

The Inhibitory Effect of Factor J on the Alternative Complement Pathway* (Received forpublication, March 3, 1994, and in revised form, August 5 , 1994) Carolina Gonzalez-Rubio8,Miguel A. Jimhez-ClaveroO, Gumersindo Fontan,and Margarita L6pez-Trascasal From the Unidad de Inmunologla, Hospital la Paz, Paseo de la Castellana261,28046 Madrid, Spain Factor J (FJ) is a cationic glycoprotein with inhibitory activity in C1, the first component of the classical complement pathway. Thisstudy demonstrates that F J is able to regulate the activity of the alternativecomplement pathway.FJ inhibits the generation of fluid-phase and cell-bound alternative pathway C3 convertase, C3b,Bb (C3-cleaving enzyme). Thus,FJ interferes with the generation of alternative pathway C3 convertase when sheep erythrocytes bearing antibody and activated C3 and C4 (EAC4b,3b) are incubated with the individual complement components, factors B, D, and P. FJ accelerates the decay of C3 convertase with a time course similar to thatof factor H, and when both regulators are present together, the decay of enzyme activity is faster thanwhen they are added separately. Furthermore, F J is able to inhibit the cleavage of C3 byfactor B in a fluid-phase assay. FJ prevents the initiation of alternative pathway activation in Kmorestabilized systems’’ with well known activators of alternative pathwayC3 convertase such as C3 nephritic factor (an autoantibody against alternative pathway C3 convertase), cobra venom factor, and rabbit erythrocytes. In these systems, F J has no effect on C3 convertase stabilized by rabbit erythrocytes or cobra venom factor. In contrast, F J promotes the dissociation of C3 convertase stabilized by C3 nephritic factor, but with much lower efficiency than in preventing initiation. Direct interaction of FJ with individual components of C3 convertase was shown by a solid-phase binding assay using plates coated with C3, C3b, B, Bb, or FJ. FJ inhibitory activity in the alternative pathway can be modulated by polyanions like heparin. FJ-mediated inhibition in the alternative complement pathwaycan be modifiedby surface interactions, as occurs during alternative pathway C3 convertase activation. Thus, whenF J is adsorbed by and eluted from hydroxylapatite and reverse-phase columns, its inhibitory effect on morestabilized systems is lost. This loss of inhibitory activity is fully reversed when FJ is rechromatographed on heparin-Sepharose or Sepharose columns. Taking into account these data, F J may be included in the group of highly charged molecules that inhibit the activation of classical and alternative complement pathways (Le. eosinophil major basic protein, protamine, and heparin).

* This work was supported in part by Grant SAL 90/0122 from the Comisidn Interministerial de Ciencia y Tecnologia, Ministerio de Educacidn y Ciencia and Grant FISS 9M132 from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Ministerio de Sanidad. The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a grant from the Spanish Ministry of Education and Science. 5 Recipient of a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social. 1 To whom correspondence should be addressed. Fax: 341-729-11-79

and 341-729-22-80.

The complement system regulates the clearance or lysis of foreign cells, particles, macromolecules, and tissue debris. It is composed of a series of proteins, both membrane-bound and is actisoluble, that interact with each other when the system vated. Activation of the alternative pathway(AP)’ can be both antibody-dependent and antibody-independent. Antibody-independent activation can be triggered by a whole spectrum of substances located on the surfaces of bacteria, fungi, viruses, and tumorcells (1).AP is consistently activated at a low level in the blood and makes a small amount of metastable C3b (2). Metastable C3b has a reactive intramolecular thioester and can form a covalent linkage withan acceptor group on a target. C3b bound to the target surface reacts factors with Band D and becomes C3convertase (C3b,Bb). TheC3convertasethus formed generates multiple metastable C3b molecules. Whether this C3b is inactivated or initiates amplification of C3 convertase usually dependson the affinity of bound C3b for the regulator factor H (3, 41,although other mechanisms have been described (5).The fast spontaneousdecay of C3b,Bb is accelerated by H, which displaces the Bb subunit, whereas activators by binding to it. In turn, like properdin(P) stabilize the enzyme C3b-H affinitydepends on the interactionof these two proteins with neutral and anionic polysaccharides on the surface (3, 4, 6-9). Heparin interacts with factor H, and this modifies its activity (10). Many diverse effects of heparin on the classical and alternative pathwaysof complement activation have been reported (reviewed in Ref. 11).Recently, the interactionof heparin with variouscomplement components has been analyzed. Of the 22 proteins examined, 13 bound heparin and9 did not (12). Other polyionic substances have the ability to regulate a polycation, regulates complement activation.Protamine, fluid-phase and cell-bound C3 convertases aswell as the activity of preformed C3 convertase (13). Eosinophil major basic protein is a highly charged polycation released by mast cell degranulation and has theability to regulate theclassical and alternative pathways of complement activation (14). Factor J (FJ)is a novel complement regulator with inhibitory activity in theclassical pathway (CP)that specifically inhibits C1 activity. Functional andlor antigenic analysis indicates that FJ is distinct from other known inhibitors of C1, namely the C1 a n d C l qinhibitors (15). FJ was discovered in urine andis also present in serum (16, 17). Structural studiesshowed that FJ is a cationicglycoprotein with a content of sugar of 228% anda PI of 29.6 (16, 18).Our main purpose was to examine the ability of FJ, like otherpolycations, to regulate the alternative amplification pathway of C3 convertase in cellular intermediates in the presence of physiological activators of AP such as P or with (NEF), potent known activators such as C3 nephritic factor cobra venom factor (CVF’), or rabbit erythrocytes (1, 2). Morel The abbreviations used are: A P , alternative pathway; CP, classical pathway; FJ, factor J; NEF, C3 nephritic factor; CVF,cobravenom factor; C7DS, C7-deficient serum; GPS, guinea pig serum; FITC,fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis.

26017

26018

Inhibitory Effect

of FJ on Alternative Complement Pathway

over, the possible modulation of FJ activity by heparin and the Modulation of Inhibitory Activity by Heparin-In the process of FJ purification, heparin-Sepharose affinity chromatographywas used ( E ) , inhibitory activity of FJ in fluid phase were also examined. MATERIALSANDMETHODS Buffers VBS is Veronal-bufferedsaline, pH 7.4. GVBis VBS (ionic strength of 0.147) containing 0.1% gelatin, 0.5 mM MgCI,, and 0.15 mM CaC1,. DGVB consists of 1 part GVB and 1 part D5W,which is 5% (w/v) aqueous dextrose containing 0.5 mM MgCI, and 0.15 mM CaC1,. DGVB/Mg is DGVB containing 10 mM MgCI,. GVBlEGTA is VBS containing 0.1% gelatin, 10 m~ EGTA, and 2.4 mM MgCl,. GVBEDTA is GVB without added Ca" and Mg2' and with the addition of a stockbuffered 0.086 M EDTA solution to give a final EDTA concentration of 40 mM. Complement Reagents Factor J was purified following the original scheme (15) with slight modifications (17). Rabbit polyclonal anti-factor J antisera wereobtained as described previously(17). Human C 1 was isolated by euglobulin precipitation (19); guinea pig C2 (20), human C3 (21), B (22), and D (23) were purified as described. Sheep erythrocytes, sensitized with rabbit hemolysin and bearing human C4b (EAC4) (Diamedix Corp., Miami, FL), were converted to EAC1,4b by the addition of human C1 to EAC4 to yield, after washes, -400 EAC1,4b sitedcell. EAC4b,3b was prepared as described (24).Briefly, an excess of guinea pig C2 and C3 (5 pg1108 cells) was added to the EACl4 cells in DGVB, and the mixture was incubated at 20 "C for 20 min. To obtain adsorbed C7-deficient serum (C?DS), serum from a patient without C7 (25) was incubated with rabbit erythrocytes in the absence of metal ions to remove agglutinating antibodies. Packed rabbit erythrocytes (1ml) wasincubated for 1 h a t 0 "C with 1ml of C7DS. Purified P and H (Quidel,San Diego, CA), anti-C3 (Dako, Glostrup, Denmark), anti-B (Dako), cobra venom factor (Diamedix),rat serum (Calbiochem),and guinea pig serum (Pel-Freez W I ) were purchased from the indicated Biologicals,BrownDeer, manufacturers. Inhibitory Activity in the Alternative Pathway Hemolytic Assay without Properdin"EAC4b,3b cells (1 x 10') were incubated for 5 min at 30 "C by shaking with 1.5pg of B, 15 ng of D, and various concentrations of F J (0.075-5 pglml)in 200 pl of DGVB/Mg.The cells werethen washed with GVBEDTA, and each pellet was incubated for 60 min at 37 "C with 200 p1 of DGVB/Mg and 300 pl of guinea pig serum (GPS) diluted 1:20 in GVBlEDTA (GPSEDTA). Next, 2 ml of saline was added to each tube, except to the control tube of 100%lysis, which received 2 ml of water. The tubes were shaken and centrifuged, and lysis of red cells was determined by measuring the absorbance of the supernatant at 414 nm. Reagent blank tubes contained cellular intermediates and complement without factor D. Noninhibited tubes contained cellular intermediates and complement, but not FJ. The percentage of inhibition was calculated as follows: 100 - ((% of lysis in presence of FJ/% of lysis in absence of FJ) x 100). Hemolytic Assay with Properdin-The conditions regarding the assay without P were the same as those described under "Hemolytic Assay without Properdin," except for an incubation time of 30 min a t 30 "C. Cellular intermediates containing P (EAC3b,Bb,P) weregenerated by adding 3 ng of P/lO' EAC4b,3b cells. The inhibitory activity of FJ was tested during or after the formation of C3 convertase by two different methods. ( a ) To assay the possible interference of FJ with some individual convertase components, cellular intermediates B, D, and P were simultaneously incubated with FJ; and (b) to analyze the ability of FJ to dissociate the stabilized complex, FJ was added for 10 min at 30 "C after the formation of EAC3b,Bb,P. Finally, all the tubes were washed with GVBEDTA and subsequently incubated with GPSEDTA. Effect of Factor J on the Decay of C3 Conuertase-EAC4b,3b cells (107/tube)were incubated with B (1pg) and D(15 ng) for 2 min (t,& at 30 "C in DGVB/Mg. Then, FJ (0,0.44, 1.1, or 2.2 pg/ml) in 225 pl of DGVB/Mg was added, and the incubation was continued for different times ( 5 , 10, 20, and 60 min) at 30 "C. The decay of C3 convertase stabilized by P was also investigated. For this purpose, EAC3b,Bb,P cells were prepared as described above. The intermediates (10' cells/ tube) were washed twicein GVBlEDTAand resuspended at 30 "Cin 200 p1 of DGVB/Mg alone orcontaining 2.2 pg/ml FJ, 0.022 pg/mlfactor H, or a mixture of both inhibitors. A tube was used for each reaction time (0,5,10, and 30 mid. Inboth cases, 300 p1 of GPSiEDTA was added to develop residual convertase sites for 60 min at 37 "C. The reaction mixture was diluted with 2 ml of saline and centrifuged, and the absorbance of the supernatant at 414 nm was measured.

but the subsequent observation that FJ interacts with Sepharose matrix alone ledus to verifythe specificity of the FJ-heparin interaction in two different systems: (a)with FJ immobilized ona nitrocellulose sheet and probed with biotinylated heparin (for this purpose, 10 mg of heparin was labeled with 750 pg of d-biotin-N-hydroxysuccinimideester (Boehringer Mannheim) as described (26)) and ( b )by affinity chromatography on Affi-Prep-heparingel. To do this, 30 mgof heparin (Analema) was coupled to 2 ml of AfK-Prep gel (Bio-Rad) according to the manufacturer's instructions. Previously, we verified that FJ does not interact with this matrix. FJ and heparin were examined together to determine their combined effects on the ability of cellular intermediates to be lysed. For this experiment, reaction tubes contained EAC4b,3b (100 pl) a t 108/ml;B, D, and P in DGVB/Mg (see "Hemolytic Assay with Properdin"); and FJ alone (2.2 and 4.4 pg/ml) or with heparin at several different concentrations (0.55-4.44 pg/ml), or heparin alone at various concentrations, or neither FJ nor heparin in a total volume of 225 pl. The tubes were incubated in the usual manner a t 30 "C for 30 min, after which 300 pl of GPSEDTA wasadded, and the reaction was continued as described above.The percentage of inhibition was compared in the presence and absence of FJ. Hemolytic Assays with "More Stabilized Systems" Inhibition of the Alternative Pathway in Rabbit Cells-One-hundred p1 of rabbit erythrocytes adjusted to 5 x 107/ml was incubated with several concentrations of FJ (0.05-3.3 pglml) and with 20 pl of normal human serum in GVB/EGTA in a totalvolume of 300 pl. After a 30-min incubation at 37 "C, the reaction was stopped by the addition of GVBi EDTA (2 ml). Tubes were centrifuged, and the absorbance at 414 nm was read. The percentage of inhibition was calculated for each concentration of FJ. Inhibition of C3 Convertase Stabilized by C3 Nephritic Factor"c3 convertase was measured in the presence of serum from a patient with NEF antibody as described (27). Briefly, 40 p1 of normal human serum, 40 pl of NEF serum, and 10 p1 of GVBiEGTA containing several final concentrations of FJ (0.0374.8pglml) were incubated with 120 p1 of sheep erythrocytes at 5 x 10' celldml for 10 min at 30 "C in the presence of GVBEGTA. The followingcontrolswere introduced: (a) reagent blank tubes containing all the reagents except NEF-positiveserum and (b) positive controltubes containing all the reagents, but not FJ. After washing the sheep erythrocytes three times with GVB/EDTA, 200pl of 1:lO diluted rat serum in GVBEDTA wasadded and incubated for 1 h at 37 "C. Next, 2 ml of GVBEDTA was added to each tube except the 100% lysis tube, which received 2 ml of water. The tubes were shaken and centrifuged, and lysis was determined by measuring the absorbance of the supernatant at 414 nm. After the first incubation and washing the cells, the effect of FJ on C3 convertase stabilized by NEF was tested by adding FJ for 10 min at 30 "C. After that, the cells were washed, and the reaction was completed with 200 p1of diluted rat serum. Inhibition of C3 ConvertuseStabilized by Cobra Venom Factor"C3 convertases stabilized by CVF were measured in various red cells (28, 29). Rabbit, sheep, or guinea pig erythrocytes (lo7celldtube) was incubated for 20 min at 37 "C with guinea pig serum (20 pl) and CVF (900 ng for rabbit or guinea pig erythrocytes and 400 ng for sheep erythrocytes) in GVBEGTA, and different concentrations of FJ (0.09-3 pglml) were added to a final volume of 215 pl. After that, 1 ml of cold VBS was added, the tubes were centrifuged, and the absorbance of the supernatants at414 nm was measured. To examine the FJ effect on preformed C3 convertase, guinea pig erythrocytes ( I O 7 celldtube) were incubated for 30 min at 37 "C with CVF and 15 pl of C7DS. After washing, cells were incubated with two concentrations of FJ that induced effectiveinhibitions when added simultaneously with CVF. A second incubation for 10 min in 200 pl of GVBEGTA was performed. After that, 300 pl of 1:15 diluted GPS in GVBEDTA was added, and the incubation was continued for 30 min at 37 "C. Two ml of cold VBS was added to stop the reaction. Effect of FJ on the Initiation of the Alternative C3 Convertuse in Rabbit Erythrocytes"C3 and B were labeled with fluorescein isothiocyanate (FITC) (30), and afluoresceirdprotein ratio of >2 was obtained. With the aim of analyzing the effect of FJ on C3 and B deposition, the experiments were carried out as described (2). Rabbit erythrocytes (100 pl, 5 x 107/ml)were incubated in GVBEGTAfor 15 min at 37 "C with 40 pl of C7DS, -7.5 pgof FITC-labeled C3 or 3.5 pgof FITC-labeled B, and 7.5 pgof C3. In both cases, FJ was addedsimultaneously to the mixture at several concentrations (O.OOP3.12 pg/ml) in atotal volume of 160 p1 for tubes containing FITC-labeled C3 or 175 pl for tubes containing

26019

Inhibitory Effect of FJ on Alternative Complement Pathway FITC-labeledB. Afterthe incubation, the cells werewashed with GVB/ EDTA, and the pellet was resuspended in 400 pl of buffer. These experiments were alsocarried out by introducing FJ 10 min later than the generation of C3 convertase in the presence of the same amounts of C7DS and FITC-labeled C3 or B. Negative fluorescence controls were included in both assays: rabbit erythrocytes were incubated with FITClabeled C3 or B inthe absence of C7DS. Fluorescencewas analyzed with a FACScan instrument (Becton Dickinson, Mountain View, CA)with the acquisition of 3000 individual cells. Fluid-phase and Solid-phase Assays Fluid-phase Proteolytic Assaysofc3 in the PresenceofFJ-The timeand dose-dependent appearance of the a' chain was followed after conversion of C3 to C3b in the presence of B (31). C3 (substrate), purified from C3 (H,O) (21),was incubated at 37 "C with B (enzyme)in VBS and in the presence of 5 m~ MgCl,. In each experiment, the enzyme/ substrate molar ratio was 1:1(5 pg of C3 and 2.5 pg of B were used). The reaction was carried out intwo different ways: (a)at different times (0, 1,2, and 5 h) and in thepresence or absence of a fixed concentration of F J (33.3 pg/ml) or (b) in the presence of different concentrations of FJ (0.11-33.3 pg/ml) for 2 h. FJ was added before B was included in the reaction mixture. To stop the reaction, 15 p1 of each tube was combined with 15 p1 of a 2-fold concentrated sample buffer for SDS-PAGEunder reducing conditions and loadedon 8% acrylamide gels with a low percentage of cross-linker (acrylamidehisacrylamide ratio of 1:0.006) (32). Dry gels were subjected to densitometry (Scan v1.20, Molecular Dynamics, Inc.). Solid-phase Ligand Binding Assays-Wellsof microtiter plates (Costar Corp.) were coated overnight with C3 (0.5 pg/ml (0.26 pmol/ well), 100 pl/well),B (0.24 pg/ml(O.26 pmol/well),100 pUwelI), or purified F J (0.15 pg/ml, 100 pVwell) at 4 "C; back-coated with skimmed milk in phosphate-bufferedsaline (200 pl of a 5% solution);and incubated for 60 min at 37 "C. The plates were washed with phosphate-buffered salinefieen 20, and FJ atseveral concentrations ranging from 0.001 to 1pg/ml and eitherC3 or B at doses ranging from 0.04to 28.8 pmol/well (plates coated with FJ) were added and incubated for 30 min at 37 "C. After washing, to develop the added component, primary polyclonal antibodies were added anti-FJ, anti-C3, or anti-B. The plates were incubated for another 30 min at 37 "C, followed by washing and the addition of a specific secondary peroxidase-conjugated antibody. The plates were incubated as described above and washed, and the substrate 1,2-phenylenediamine(Merck) was used for enzymatic reaction. Reactions were developed at room temperature for 10-30 min, and the absorbance at 492 nm was read in an Anthos Reader 2001. Furthermore, we tested the ability of FJ to bind C3b and Bb with the scheme used forC3 and B.C3 was digested with 1%(w/w) trypsin (Sigma) for 2 min at 37 "C, and the reaction was stopped with 3% soybean trypsin inhibitor (Sigma). The C3b fragment was purified as described (21) with a Mono-Q high pressure liquid chromatography column (Pharmacia Biotech Inc.). Bb was obtained by digestion of B in the presence of C3b and D. The B/C3b/D weightratio was 1:0.05:0.0025 (33). The Bb fragment was purified with the Mono-Qcolumn as described (31). Evaluation ofthe Possible Inhibitory Effect of FJ on D-Cleavage of B inthe presence of C3b and Dwas analyzed by incubation of B/C3bD in VBS with 4 mM MgCI, at a weight ratio of 1:0.05:0.0025. Afterincubation for 3 h at 37 "C, the reaction was stopped with EDTA at a final concentration of 10 mM. The eventual inhibition induced by FJ was analyzed by incubating the same mixture in the presence of FJ at 0.5-50 pg/ml in a total volume of 40 p1. The Bb and Ba fragments were analyzed by 10% SDS-PAGE under reducing conditions (34). RESULTS Effect o f F J o n the Generationa n d Decay of C3 Convertases of the Alternative Complement Pathway on EAC4b,3b Cells-Fig. 1 shows that FJ inhibited the generationof EAC3b,Bb convertase in a dose-dependent manner.As shown in Fig. 2, this effect was also observed in C3 convertase stabilized byP in the same FJ concentration range. FJ inhibition inthe alternative pathway was compared withthat observed in the classical pathway. Inhibitory activity in C P w a s tested as described (15). Both inhibitions were in the same range if FJ was previously incubated with cellularintermediates: 50% inhibition was observed at -0.1 pg/ml FJ. In contrast, the amount of FJ necessary for inducing 50% lysis was -10 times higher in A p than in CP

1

0 10"

10'

Factor J (pg/ml) FIG.1. Effect of factor J on alternative C3 convertase generation. EAC4b,3b cellular intermediates (lo7)were incubated with factor B (1.5 pg) and factor D (15 ng) and several concentrations of FJ in DGVB/Mg for 5 min at 30 "C and then washed immediately with cold GVBIEDTA, and the incubation was continued for 60 minat 37 "C with diluted GPSLCDTA. The incubation was stopped by the addition of 2 ml of saline, mixing, and centrifugation. The released hemoglobinwas determined spectrophotometricallyin the supernatant at 414 nm. The percentage of inhibition was calculated as follows: 100 - ((% of lysis in presence of FJ/% of lysis in absence of FJ) x 100).Data areexpressed as the means * S.E. of five determinations.

0 10.2

10-

loo

10'

Factor J (pg/ml) FIG.2. Comparison of the inhibition induced by factor J in EAC1,4b and EAC4b,3b intermediates before and during convertase generation and the effectof FJ on the terminal complex. Factor J was preincubated for 10 min a t 30 "C(before)with EAC1,4b (W) or EAC4b,3b(x) and then washed and eitherincubated simultaneously with the same intermediates and C2 for10 min (during) (CP (0)) or with factors B, D, and P for 30 min (AP (0)). Finally, after washing with GVBIEDTA, cells were resuspended in 200 pl of DGVBMg, and 300 pl of GPSIEDTA was added, followed by incubation for 60 min at 37 "C. The effect on terminal components (in the AP assay) was analyzed by adding FJ at the same time as GPSIEDTA, as shown (01,FJ did not inhibit at any dose, and thus, the closed circles are on the horizontal axis. The percentage of inhibition was determined as described in the legend of Fig. 1.

when FJ was included during the generation of C3 convertase (Fig. 2). Furthermore, the extentof inhibition was nearly the same whether FJ was added during or after the formation of EAC3b,Bb,P (data not shown), and FJ did not act on the terminal components when EAC4b,3b cells were used as described with EAC1,4b cells (15). FJ also accelerates the decay of C3 convertase. When three concentrations (0.44, 1.1,and 2.2 pg/ml) of FJ were added, the

Inhibitory Effect of FJ on Alternative Complement Pathway

26020

A 30

- FJ ADDITION

-l--t HEPARIN

60

-

“c

CONTROL

50

--t

FJ (0.44 pghnl) FJ (1.1 pR/ml) FJ (2.2 pdml)

40

-x-

-

70

HEPARIN+FJ (4.4 pg/ml) HEPARIN+FJ (2.2 pghnl)

30

20 20

-

10 -

0

10

I

0

~

I

1

~

2

I

3

~

4

I

5

HEPARIN (pg/ml) FIG.4. Modification of the heparin inhibitory effecton A p by FJ. C3 convertase was generated in EAC4b,3b cells (107/tube) with B, D, and P in thepresence of increasing concentrations of heparin (0)or with the sameconcentrations of heparin and two doses of FJ (4.4 and 2.2 pg/ml (0and x, respectively)). The reaction was continued as described under “Materials and Methods.”

resulting curves demonstrated that theC 3 convertase without P was dose-dependently susceptible to decay dissociation (Fig. TIME (rnin) 3A). In addition, FJ accelerated the decay of C 3 convertase stabilized by P. H and FJ similarly acceleratedthe decay of both enzymes, and they didnot interfere with each other when added simultaneously (Fig. 3 B ) . Effect of FJ and Heparin on Alternative Pathway Convertase --t CONTROL Generation-The interaction of FJ with heparin was shown by -AH (0.022 pR/ml) the specific interaction of FJ with biotinylated heparin by dot -0- FJ (2.2 pdml) blot and by retention on AfE-Prep-heparin gel. As a consecapacity of heparin to quence, FJ was ableto interfere with the inhibit the generationof AP C3 convertase as shown in Fig. 4. In the presence of FJ alone at 4.4 pg/ml, there was 50% inhibition of convertase generation. Thus, in the presence of increasing amounts of heparin alone, there was a dose-related increase ininhibition of convertase generation, with 60%inhibition a t 4.4 pg/ml heparin. In contrast, when heparin was present with 4.4pg/ml FJ, the dose-response curve was modified significantly. At higher doses of heparin with FJ present, inhibition was still seen. However, at lower doses of heparin with FJ present, little or no suppression of convertase generation was observed. For example, 40% inhibition of convertase 30 generation was seen in tubes containing 2.2 pg/ml heparidtube in theabsence of FJ, andnone was seenwhen FJ was present. At very low heparin concentrations when FJ was present, inLJ hibition of convertase generation increased and approached the 0 10 20 50% seen when only FJ was present. Inhibition of More Stabilized Systems-When rabbit erythrocytes were used as an activator, FJ inhibited the generation TIME (rnin) of stabilized C 3 convertase in a dose-dependent manner as FIG.3. Decay of the C3 convertase in the presence of FJ. A, shown in Fig. 5A. When FJ was added to preformed C 3 converdecay of EAC3b,Bb convertase. C3 convertase was preformed by incu- tase with rabbit erythrocytes, we could not observe any inhibbation of EAC4b,3b (IO7 cellskube) with B (1pg) and D (15ng) for 2 min itory effect (data not shown). Similarly, inhibition of lysis was at 30 ‘C. Then, threeconcentrations of FJ were added (0.44,1.1,and 2.2 observed when C 3 convertase was stabilized by NEF serum.A pg/ml) in 225 pl of DGVB/Mg. The incubation was continued for different times, and the residual hemolytic sites were developed by adding dose-dependent inhibition was observed when FJ was present 300 pl of GPSEDTA and incubating for 60 min at 37 “C. Two ml of during or after C 3 convertase formation (Fig. 5 B ) . This effect saline was added to each tube to stop the reaction. The percentage of was much less evidentwhen FJ was added after theformation

B

-

I

lysis was calculated by measuring the absorbance of the supernatants at 414 nm and comparing with tubes that received water. The control curve corresponds t o the spontaneous decay of EAC3b,Bb convertase without FJ. B , decay of EAC3b,Bb,P convertase in the presence of F J andor H. F J (2.2 pg/ml), H (0.022 pg/ml), or a mixture of both inhibitors

(FJ, 2.2 pg/ml; and H, 0.022 pg/ml) was incubated with EAC3b,Bb,P cells (107/tube) in 200 111 of DGVBMg. The rest of the experimental procedure was similar t o that described for A.

~

Inhibitory Effect of FJ on Alternative Complement Pathway

1

1

0

2

4

3

Factor J (pdml)

?1, 4

4.8

2.

DURING

la AFTER

12

.6

0.3 0.15

Factor J (ps/ml)

0.075

OK37

26021

of the NEF-convertase complex. A dose-dependent inhibitory effect of FJ during generationof C3 convertase was also evident with guinea pig erythrocytes and CVF as activators as shown in Fig. 5C. In contrast, FJ did not inhibit when added to preformed CVF,Bb convertase. Furthermore, Fig. 6 shows a dosedependent inhibition induced by F J of the deposition of FITClabeled C3 and B. The results were obtained after analysis by flow cytometry of C3b and Bb deposition in rabbiterythrocytes in the presence or absence of FJ. Effect of FJ on the Generation of Convertase in Fluid-phase and Solid-phase Assays-Cleavage was specifically induced by B, as observed by the appearanceof the a' chain from C3b; the a' chain increased progressively with time (Fig. 7, lanes 36). In the presence of FJ, an important reduction in the appearance of a' was observed (lanes 7-9). Bands with higher molecular mass than the a chain could correspond to degraded or aggregated C3 forms, as have been previously described in C3 preparations (21). The band witha molecular mass lower than the p chain corresponds to hemopexin, a protein that copurifies with B. In another experiment, we analyzed the inhibition of C3 cleavage induced by B in the presence of different concentrations of FJ after 2 h of incubation. The percentage of C3 cleavage in each lane was defined as (a'/(a + a ' ) ) x 100. FJ concentrations up to33.3 pg/ml produced a maximal inhibition of 60% in a dose-dependent manner. Moreover, FJ reduced the cleavage in fluid phase of B by D in the presence of C3b (data not shown). In the solid-phase experiments, C3, C3b, and B interacted with coated F J on plates as shown by enzyme-linked immunosorbent assay. Dose-response curves were obtained and are evidenced when C3, plotted in Fig. 8. This same interaction was C3b, B, and Bb were immobilized on the plates and incubated with different amounts of FJ (data not shown). Modulation of the Inhibitory Activity by Surfaces Such as Hydroxylapatite and Silica Matrices-During the procedure of FJ purification, F J lost inhibitory activity in more stabilized systems(rabbit erythrocytes, CVF, or NEF)after passage through a n HPHT hydroxylapatite column (7.8 x 100 mm; BioRad). The same phenomenon was observed after loading onto and elution from a pBondapakm C,, column (3.9 x 300 mm; Millipore Corp., Waters Chromatography, Milford, MA). In contrast, FJ inhibition in CP and in the generationof EAC3b,Bb and EAC3b,Bb,P convertases was invariable after these interactions. In addition, FJ-mediatedinhibition in more stabilized systems wasrecovered when loaded onto and eluted from heparin-Sepharose or Sepharose columns. DISCUSSION

This study suggests thatfactor J may play a role in vivo in regulating complement activation by either theclassical and/or the alternativeamplification pathway. Experimental data support the view that FJ acts with the sameefficiency in C3 convertases of the classical and alternativecomplement pathways if FJ is preincubated with cellular intermediates. This fact may indicate an effect on the complement components bound to cells. However, if F J is added simultaneously with C3 convernormal human serum) and incubating the mixture for 10 min at 30 "C. The effect of FJ wasalsotested on preformedsheeperythrocytes/ C3b,Bb,NEF (AFTER). Sheeperythrocytes/C3b,Bb,NEF cells were Factor J (pg/ml> washed withGVBIEDTA, followed by the additionof FJ and incubation FIG.5. Inhibition induced by FJ in more stabilized systems.A, for 10 min more a t 30 "C. Then, rat serum diluted inEDTA was added inhibition of C3 convertase stabilized by rabbit erythrocytes. Rabbit to all the tubes, and the incubation was continued for a n additional 60 erythrocytes was incubated with 20 pl of normal human serum and withmin at 37 "C. C, FJ effect on C3 convertase stabilized by cobra venom different concentrationsof FJ for 30 minat 37 "C in GVBIEGTA. Then, factor. Guinea pig erythrocytes (107/tube) were incubated for 20 min a t the reaction was stopped by the addition of 2 ml of GVI3EDTA. B , 37 "C with guinea pig serum (20 pl), 900 ng ofCVF, and different inhibition of C3 convertase stabilizedby C3 nephritic factor. The effect concentrations of FJ in GVBEGTA. The reaction was stopped by the was tested by simultaneously including (DURING) FJ and the con- addition of 1 ml of cold VBS.In all cases, the percentageof inhibition vertase-forming reagents (sheep erythrocytes, NEF-positive serum, was and calculated as described in the legendof Fig. 1.

Inhibitory Effectof FJ on Alternative Complement Pathway

26022

FITC-C3 DEPOSITION

LOG FIAJORESCENCE ISTENSITY

FITC-I3 DEPOSITION POSITIVE CONTROL

NEGATIVE CONTROL

2.9 pdml

0.6 pdml

0.11 pdml

LOG FLUORESCENCE INTENSITY FIG.6. Deposition ofFITC-labeled C3 and B in rabbit erythrocytesin the presenceof FJ. FITC-labeled C3 was incubated with adsorbed C7-deficient serum and rabbit erythrocytes GVBmGTA in for 15 min a t 37 "C. Different concentrationsof FJ were simultaneously introduced with the reagents. After the incubation, cold GVBEDTA was added to each tube and washed. Each pellet was resuspended, and the tubes were analyzed byflow cytometry. FITC-labeled B deposition was analyzed in the same way. Negative fluorescence controls were included by incubation of FITC-labeled C3 or B in the absence of C7-deficient serum. Positivefluorescence controls correspondto tubes without FJ. The figure represents the obtained histograms with the acquisitionof 3000 individual cells.

F-

Oh

-FJ2h

lh

-k

200kDa

-i,

92.5 kDa

-

,

z

i 1 2

3

4

+FJ

5h

"-

Oh

lh

2h

1 1I

-

1

-a'

a

-B

"p

1

5

6

7

2.5

-

2.0

-

1.5

-

1.0

-

0.5

-

5h

E; c.(

a 7

8 9

FIG.7. C3 cleavage in fluid phase in the presence of FJ and time course ofC3 cleavage in the presence of FJ after SDSPAGE on 8% slab gel. C3b was generated by incubation of C3 (5 pg) with B(2.5 pg) inVBS containing 5 mM MgCI, a t 37 "C and subjected to SDS-PAGE under reducing conditions. The gel was finally stained with Coomassie BrilliantBlue R-250. Lane 1 containedmolecularmass markers (myosin, 200 kDa; and phosphorylase b, 92.5 kDa). Lanes 2-9 contained a C3 and B mixture with different incubation times: lanes 2 and 6,O h lanes 3 and 7 , l h; lane 4 and 8,2 h; lane 5 and 9 , 5 h. Lanes 6-9 contained 33.3 pg/ml FJ. FJ appeared as precipitated material in lanes 6-9.

C3IC3hlB (pmol) FIG.8. C3,C3b,B interaction with factor J. Wells of microtiter

were coated with purified FJ (0.15 pg/ml), and purified C3, C3b, tase components (C2 or B, D, and P), the amount of FJ neces- plates or B was added (10"-102 pmol). The reaction was developed with the sary to induce the same inhibition is -10-fold higher for AP appropriate antibodies (see "Materials andMethods"). than for CP. The lower efficiency for AP could be connected with possible competition of FJ for C3 (bound to thecells) and B (in interaction with C3b (37). Nevertheless, this property is not fluid phase). Thiscompetition is not evident in the classical C3 associated with all thecationic molecules, e.g. cytochrome c (PI 9.6; 5 1-18)does not inhibit complement activation (data not convertase due to thefact that FJ clearly disrupts C1 (15). The abilityof FJ to inhibitCP and A p could be related to the shown). Some similarities between H and FJ actions can be estabhigh PI of this protein. Other cationic proteins with regulatory effects in CP and AP have been described: eosinophil granule lished. For instance,FJ accelerates thedecay of C3 convertase major basic protein (14)and myelin basic protein (35, 36). Clq in a similar timecourse as H, and theeffects of both inhibitors other. Furthermore, FJ acts inUrnore (PI 9.3) has been related to the regulation of AP by direct do not interfere with each

Inhibitory Effect of FJ on Alternative Complement Pathway

26023

stabilized C3 convertases” stabilized by NEF, CVF, or rabbit C3b is protected from H, then preference is given to B, so that the convertase complex C3b,Bb is formed, and the particle beerythrocytes with effects analogous to those of H. In these systems, we have found some differences if FJ is added during comes an AP activator capable of producing more active C3b or after theformation of C3 convertase. Thus, FJ prevents the cleavage products. If C3b is accessible to H, then the complex C3b,H allows cleavage of C3b to iC3b by factor I, and the generation, but is not able to dissociate the C3 convertases stabilized by CVF or rabbit erythrocytes. Moreover, FJ hinders particle is inactivated(48). H binds less strongly to C3b immoC3 deposition in rabbit erythrocytes. B deposition is also af- bilized on particles that areAP activators (49).These observafected due to the absence of C3 deposition (flow cytometry ex- tions together indicate that a comparable mechanism for FJ periments). In addition, C3 convertase stabilized by CVF is inhibition in AP takes place. In conclusion, FJ is able to inhibit CP and AP. Regarding A P , completely resistant to H and I inactivation (38, 39). Rabbit erythrocytes havepreviously been shown to interfere with the FJ affects C3 convertase by interacting with C3 and B while it dissociation of this enzyme. effectiveness of the control by I and H, allowing unrestricted interferes with the generation and formation of C3 convertase (40). H is less efficient in NEF- This inhibitionis evident in thepresence of known activators of AP C3 convertase and can be modulated by some polyanions, stabilized convertase (41, 421, as is also true for FJ. hydroxylapatite and Enzyme-linked immunosorbentassayand fluid-phase ex- such as heparin, and surfaces, such as periments suggest that the observed FJ inhibition in hemolytic hydrophobic matrices. assays is not due to a nonspecific effect on the cellular interAcknowledgments-Factors B and D were a gift fromDr. Pilar mediates, but rather to an effect on the specifically bound Sbnchez-Corral(Department of Immunology, Fundacidn JimBnezDiaz, complement components. The possibility that FJ acts as an Madrid, Spain). We thank Dr. Michael K. Pangburn (University of inhibitor of D could not be ruled out with our assay because D Texas) for helpful discussions, Teresa Casado (Department of Immucleaves B only when B is ina Me-dependent association with nology,Fundaci6n Jimenez Diaz) for help in scanning the gels, and C3b (43). According to our data, FJ interacts with the compo- Angeles Saboya for expert technical assistance. We aregratefulto Drs. Jose Gonzalez-CastafioandJorgeMartin-PBrezforcritique nents of C3 convertase and prevents the generation of the of the manuscript. C3b,Bb complex. FJ is able to neutralize the inhibitory activity of heparin in REFERENCES AP. This effect was observed with other regulators such as 1 Liszewski, M. R,and Atkinson, J. P. (1993) in Fundamental Immunology eosinophil major basic protein, and charge neutralization was (Paul, W. E., ed) 3rd Ed., pp. 917-939, Raven Press, Ltd., NewYork 2 Pangburn, M. K., and Muller-Eberhard, H. J. (1984) Springer Semin. Zmmusuggested to explain this effect (14). Many charged molecules nopathol. 7, 163-192 are present in tissues, including heparin, protamine sulfate, 3 Fearon, D. T.(1978)Proc. Natl. Acad. Sci. U.S. A . 76, 1971-1975 4 Pangburn, M. K., and Muller-Eberhard. H. J. (1978) Proc. Natl. Acad. Sci. major basic protein,and thechondroitin sulfates. Thesehighly L? S. A. 76, 241G2420 charged molecules are found in areas where complement acti5 Joiner, K. A. (1988) Annu. Rev. Mzcrobiol. 42, 201-230 6 Kazatcbkine, M. D., Fearon, D. T., and Austen K. F. (1979) J. Zmmunol. 122, vation occurs. Therefore, complement activation may play an 75-81 important role in vivo in the immune and inflammatory re7 Kazatchkine, M. D., Fearon,D. T., Sibert, J. E., andAusten, K. F. (197915. Exp. sponse. The FJ concentrations used in most of these experiMed. 150, 1202-1215 ments are similar to those estimated in normal human serum8 Pangburn, M. K., Morrison, D. C., Schreiber, R. D., andMuller-Eberhard, H. J. (1980) J. Zmmunol. 124,977-982 (5.8 2 2.8 pg/ml) (171, suggesting thatFJ may playa role in the 9. Carrefio, M. P., Labarre, D., Maillet, F., Jozefowicz, M., andKazatchkine, M. D. (1989) Eur. J. Zmmunol. 19,2145-2150 regulation ofAPin vivo.We have observed that FJ is present in 10 Pangbum, M. K., Atkinson,M. A. L., and Men, S . (1991) J. Biol. Chem. 266, many cells (data notshown). FJ, heparin, and other regulators 16847-16853 probably act in conjunction. Furthermore, the interactions be- 11. Edens, R.E.,Linbardt, R. J., and Weiler, J. M. (1993) Complement Profiles 1, 96120 tween these charged molecules may modulate their mutual 12. Sahu, A,, and Pangburn M. K. (1993) Mol. Zmmunol. 30,679484 functional activities. The balance between activation and inhi- 13. Weiler, J. M. (1983) Zmmunopharmacology 6, 245-255 bition of complement could be finely controlled by the relative 14. Weiler, J. M., and Gleich, G. J. (1988) J. Zmmunol. 140, 1605-1610 15. Lbpez-Trascasa, M., Bing, D. H.,Rivard, M., and Nicholson-Welleq A. (1989)J. concentrations of the polycations and polyanions. Biol. Chem. 264,16214-16221 FJ loses its inhibitory activity in more stabilized systems 16. Nicholson-Weller, A., Rivard, M., Lbpez-Trascasa, M.,and Rynkiewicz, M. (1991) Complement 8, 198 after chromatography on hydroxylapatite and C,, matrices. 17. JimBnez-Clavero, M. A., Gonzalez-Rubio, C., Fontan, G., andLbpez-Trascasa, This fact could be connected to the interaction with certain M. (1994) Clin. Biochem. 27, 169-176 surfaces. Since the inhibitory activity reappears after interac- 18. JimBnez-Clavero, M. A., Gonzalez-Rubio, C., Fontan, G., andLbpez-Trascasa, M. (1993) Mol. Zmmunol30,21 tion with heparin or agarose, the effect is reversible. In con- 19. Sakai, K., and Stroud, R. M. (1973) J. Zmmunol. 110, 1010-1020 trast, we did not observe any changes in FJ inhibition in CP. 20. Gigli, I., and Austen, K. F. (1969) J. Exp. Med. 129, 679485 P., Antbn, L. C., Alcolea, J. M.,MarquBs, G., Sanchez, A,, and The mechanisms for modulation of FJ inhibitory activity by 21. Sbnchez-Corral, Vivanco, F. (1989) J. Zmmunol. Methods 122, 105-113 surface interactions are not clear. In thiscontext, the activation 22. Kerr, M. A. (1981) Methods Enzymol. 80, 102-112 of the alternative pathwayby an ordered surface configuration 23. Lesavre, P. H., Hugli, T.E., Esser,A. F., and Muller-Eberhard, H. J. (1979)J. Zmmunol. 123,529-534 of repeating polysaccharides, e.g. dextran or other polymeric 24. Ohi, H., Watanabe, S., Fujita, T., Seki, M., and Hatano,M.(1990)J. Zmmunol. Methods 131, 71-76 units, appears to be important. There is evidence to suggest 0.G., Arnaiz-Villena, A,, Iglesias-Casarrubios, P., Martinez-Laso, that hydroxylapatite crystals inmineralized bone will bind the 25. Segurado, J., Vicario, J. L., Fontan,G.,and Lbpez-Trascasa, M. (1992) Clin. Exp. third component of the complement system (C3) on exposure to Zmmunol. 87,410-414 plasma (44).Recently, it hasbeen suggested that C3 deposition 26. Hudson, L., andHay, F. C. (1989) Practical Immunology, pp. 49-50, Blackwell Scientific Publications Ltd., Oxford in mineralized bone surfaces mediatestherecruitment of 27. Lbpez-Trascasa, M., Marin, M. A., and Fontan,G. (1987) J . Zmmunol. Methods 98, 77-82 mononuclear osteoclasts to the site of deposition (45). C.-W., and Muller-Eberhard, H. J. (1984) J. Zmmunol. Methods 73, The consequences of the modulation of FJ inhibitory activity 28. Vogel, 203-220 by surface interactions could indicate that FJ might regulate 29. Vogel, C.-W., and Miiller-Eberhard, H. J. (1985) Deu. Comp. Immunol.9, 311325 C3 deposition under certain circumstances, e.g. the nature of 30. Hudson, L., andHay, F. C. (1989)Practical Immunology, pp. 34-35, Blackwell the surface binding of C3b is crucial as to whether or not the Scientific Publications Ltd., Oxford particle will be an AP activator. In thepresence of B and H (e.g. 31. Sanchez-Corral, P., Antbn, L. C., Alcolea, J. M., MarquBs, G., Sanchez,A,, and Vivanco, (1990) Mol. Zmmunol. 27, 891-900 in plasma), both factors compete for bound C3b (46). The out- 32. RQOS, M. H.,F.Mollenhauer, E., Demant, P., and Rittner, C. (1982)Nature 298, come of the competition is determinedby the surface (47).If the 854-856

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33. Lambris, J. D., and Muller-Eberhard, H. J. (1984)J . Biol. Chem. 259, 12685- 42. Weiler, J. M., Daha, M. R., Austen, K. F., and Fearon, D. T.(1976)Proc. Nutl. 12690 Acud. Sci. U. S. A. 73, 3268-3272 34. Laemmli, U.K. (1970)Nature 227,680-685 43. Lesavre, P. H., and Muller-Eberhard, H. J. (1978)J . Ezp. Med.148,1498-1509 44. Nilsson, U.R., and Muller-Eberhard, H. J. (1965)J. Exp. Med. 122, 277-298 35. Cyong, J.-C., Witkin, S. S., Reiger, B., Barbarese, E., Good, R. A,, and Day, N. K. (1982)J. Exp. Med. 155, 587-598 45. Mangham, D. C., Scoones, D. J.,and Drayson, M. T. (1993)J. Clin. Pathol. 46, 517-521 36. Koski C. L., Vanguri, P., and Shin, M. L. (1985)J. Zmmunol. 134, 1810-1814 37. Fishelson, Z., and Muller-Eberhard, H. J. (1987)Mol. Zmmunol. 24, 987-993 46.Kazatchkine, M. D., Feamn, D. T., and Austen, K. F. (1979)J. Zmmunol. 122, 75-81 Clin. Exp. Zmmunol. 21, 109-114 38. Lachmann, P. J., and Halbwachs, L. (1975) 47. Fearon, D. T., and Austen, K. F. (1977)Proc.Natl.Acud. Sci. U. S . A . 74, 39. Nagaki, K., Iida, K.,Okubo,M., and Inai, S. (1978)Znt. Arch.AllergyAppl. Zmmunol. 57,221-232 48. Pangburn, M. K. (1989)J. Zmmunol. 142,2759-2765 40. Pangburn, M. K., and Muller-Eberhard, H. J. (1978)Proc. Nutl. Acad. Sci. U. S. A . 75, 2416-2420 49. Horstmann, R. D., Pangburn, M. K., and Muller-Eberhard, H. J. (1985)J . Immunol. 134, 1101-1104 41. Daha, M. R., Fearon, D. T., and Austen, K. F. (1976)J. Zmmunol. 116, 1-7