Proc. Natl. Acad. Sci. USAVol. 75, No. 12, pp. 6078-6082, December 1978Cell Biology

Autosomal locus regulates inverse relationship between sialic acidcontent and capacity of mouse erythrocytes to activate humanalternative complement pathway

(01H regulatory protein/amplification C3 convertase)

URS E. NYDEGGER, DOUGLAS T. FEARON, AND K. FRANK AUSTEN*Departments of Medicine, Harvard Medical School, and the Robert B. Brigham Division of the Affiliated Hospitals Center, Inc., Boston, Massachusetts 02120

Contributed by K. Frank Austen, September 18, 1978

ABSTRACT The observation that mouse erythrocytes (Em)from 21 inbred strains had variable capabilities to activate thehuman alternative complement pathway permitted the dem-onstration that membrane sialic acid content was inversely re-lated to activating capacity and was regulated by codominantalleles of a single autosomal locus. Linear regression analysisalso demonstrated a significant inverse correlation between thesialic acid content of Em from four inbred strains and the con-centration of #1H required for decay-dissociation of the pro-perdin-stabilized amplification convertase on the Em. Em fromF1 hybrids derived from strains with high and low alternativeathway activating capacities and from their backcrosses ex-

hibited the alternative pathway activating capacities expectedif the activity were regulated by alleles of a single autosomallocus. That this same locus predominantly regulated the sialicacid content of Em was established by the significant inversecorrelation between the sialic acid content and the alternativepathway activating capacity.of Em from mice of the F1 andbackcross generations. Although the fluid phase interaction ofC3, B, andTD continuously generates C3b in a reaction aug-mented by properdin, it is the covalent attachment of C3b tobystander surfaces deficient in sialic acid that activates thealternative complement pathway at that site because of im-paired binding of ,B1H to C3b on such surfaces. Thus, discrim-ination between activating and nonactivating surfaces occursafter C3b deposition, and sialic acid deficiency represents themolecular basis for our earlier finding that activating particlescircumvent the regulatory actions of the control proteins of thealternative pathway.

The alternative pathway of human complement serves as aprimary recognition mechanism in plasma of the nonimmunehost for various microbial polysaccharides such as zymosan fromyeast (1), lipopolysaccharides from Gram-negative bacteria (2,3), and teichoic acid from pneumococci (4). It also responds tocertain mammalian cells such as erythrocytes and lymphocytesfrom rabbits (5) and mice (6, 7) and some cultured humanlymphoblastoid cell lines (8). The interaction of native C3, B,and D (9) continuously generates C3b in a reaction that isaugmented by properdin (10); the C3b may covalently (11)attach to bystander surfaces and, depending on the characterof the surface, may or may not lead to amplified C3 cleavageat that site (12, 13). C3b (14) complexes with B, and cleavageby D generates C3b,Bb (15), the amplification C3 convertase.Transition to the amplified C3 cleavage is achieved because thesurface characteristics of an activating particle protect C3bfrom inactivation by the C3b inactivator (C3bINA) in thepresence of (1H and retard decay-dissociation, by 01H, of theunstabilized convertase and the properdin-stabilized amplifi-cation convertase (P,C3b,Bb) (12, 13).

Natural activating surfaces such as zymosan (16) and rabbit

erythrocytes (Er) (17) have diminished or no sialic acid residuescompared to nonactivating surfaces such as sheep erythrocytes(Es). However, when the sialic acid residues on Es are removedenzymatically with sialidase (18, 19) or are converted to theirheptulosonic acid derivative by limited oxidation and reduction(18), these cells become activators of the human alternativepathway. The molecular basis for activation by the altered cellsis a decreased association constant of 031H for C3b on the desi-alated membrane without any change in the binding constantof B for C3b, a circumstance that facilitates formation of theamplification convertase and diminishes decay-dissociation ofthe convertase by f1H (18, 20).The observation that mouse erythrocytes (Em) are lysed in

human serum because they activate the alternative complementpathway (6) provided an opportunity to establish the inverserelationship between membrane sialic acid content and acti-vating capacity of Em from different strains. The inverse rela-tionship was due to impaired l01H function when membraneswere relatively deficient in sialic acid. The capacity of Em toactivate the alternative complement pathway was controlledby a single non-H-2 linked autosomal locus that regulated themembrane content of sialic acid.

MATERIALS AND METHODSMice. Inbred strains of mice were purchased from the

Jackson Laboratory. The congenic mice were a gift from M.Dorf and H. Cantor (Harvard Medical School, Boston, MA).Female mice, ages 6-10 weeks, were used throughout the studyexcept that for the genetic studies male offspring were alsoincluded. The animals were anesthetized with ether, and Emwere collected by orbital sinus puncture into Veronal-bufferedsaline containing 0.1% gelatin (GVB) and 0.04 M EDTA(GVB-EDTA). After an initial centrifugation for 10 min at 1300X g at 250C, the buffy coat was removed and the Em werewashed four times in GVB and stored in this buffer at 40C forno longer than 24 hr. Before being used, the Em were washedonce in GVB containing 2 mM Mg2+ and 8 mM ethylene glycolbis(f-aminoethyl ether)-N,N,N',N'-tetraacetic acid (GVB-Mg-EGTA) and adjusted to a concentration of 1 X 108 cells perml with a model 2F Coulter Counter.

Abbreviations: C3b,Bb, amplification C3 convertase formed from themajor cleavage fragments of C3 and B; P,C3b,Bb, properdin-stabilizedamplification C3 convertase; Em, mouse erythrocytes; Er, rabbiterythrocytes; Es, sheep erythrocytes; EC3b, erythrocytes bearing C3b;EC3b,Bb, erythrocytes bearing the amplification convertase, C3b,Bb;EP,C3b,Bb, erythrocytes bearing the properdin-stabilized amplifi-cation convertase; GVB, Veronal-buffered saline containing 0.1%gelatin; DGVB2+, half-isotonic GVB containing 2.5% dextrose, 0.5 mMMg2+ and 0.15 mM Ca2+; GVB-EDTA, GVB containing 0.04 MEDTA; GVB-Mg-EGTA, GVB containing 2 mM Mg2+ and 8 mMethylene glycol bis(f-aminoethyl ether)-N,N,N',N'-tetraacetic acid.*To whom reprint requests should be addressed.

6078

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

Proc. Natl. Acad. Sci. USA 75 (1978) 6079

Activation of Human Alternative Complement Pathwayby Em. Activation of the human alternative pathway -wasmeasured by direct lysis of Em in dilutions of normal humanserum from a single donor. The serum was diluted -withGVB-Mg-EGTA to prevent any contribution by the classicalcomplement pathway (21-23). Em (1 X 107 in 0.1 ml ofGVB-Mg-EGTA) were added to 0.25 ml of GVB-Mg-EGTAcontaining decreasing amounts of serum. The reaction mixtureswere incubated for 45 min at 37'C with agitation, 1.25 ml of0.15 M NaCl was added to each sample, and the percentage lysisof the Em was determined. The reagent blank showed no lysis;100% lysis was obtained by addition of distilled H20 to 1 X 107Em. The amount of serum required for lysis of 50% of the cellswas estimated with a Hewlett-Packard 9805A calculator bylinear regression analysis of the plot of percentage lysis versus-In (Al serum). To allow comparison of results to a standardactivating principle, Er obtained from a single New Zealandalbino rabbit were assayed in each experiment for the amountof human serum required for 50% lysis in the same manner asEm. The amount of human serum required for 50% lysis of Erdivided by the amount of serum required for 50% lysis of eachstrain of Em gave a ratio of activating capacity for Em relativeto the standard Er. Activation of the alternative pathway by Erwas arbitrarily assigned an activity of 100 units, and activatingactivity of each of the various strains of Em was expressed rel-ative to that standard. In those experiments in which alternativepathway activating capacity was to be correlated with jBlH-mediated decay-dissociation and to sialic acid content of Em,incubation of both Em and Er in the human serum dilutions wasextended to 60 min.

Decay-Dissociation of EC3bBb by ,#1H. Em were obtainedfrom three female mice of each of the following strains:BUB/BnJ, C57BL/6J, A/WySn, and DBA/2J. The Em of eachstrain were pooled and washed three times in half-isotonic GVBcontaining 2.5% dextrose, 0.5 mM Mg2+, and 0.15 mM Ca2+(DGVB2+). Then, 1 X 1010 Em of each strain as well as 1 X 1010Es were converted to their respective EC3b intermediates bya sequential two-step procedure for C3b deposition by a fluidphase C3 convertase and by a cell-bound convertase. C3 (24),B (25), properdin (12), D (26), and fl1H (27) were prepared freeof detectable contaminating proteins and quantitated as de-scribed. Initial C3b deposition was accomplished by incubating1 X 1010 Es and Em in 2 ml of DGVB2+ with 6.6mg of C3, 1 mgof B, and 5 itg of D5 for 30 min at 300C. The C3b-bearing cellswere washed twice in GVB containing 0.5 mM Mg2+ and 0.15mM Ca2+ and once in DGVB2+ and resuspended with 100,ugof B and 5 ,ug of D in 1.5 ml of DGVB2+ containing additionalMg2+ (10 mM). After incubation for 30 min at 30°C, the in-termediates bearing C3b,Bb were washed in ice-cold DGVB2+and resuspended with 130 Ag of C3 in 0.4 ml of DGVB2+. Afterincubation at 30°C for 60 min, the cells were washed threetimes in DGVB2+, twice in 0.04 M GVB-EDTA, and twiceagain in DGVB2+ to yield EC3b. In order to determine theamount of B required to form two hemolytically effectiveP,C3b,Bb sites per erythrocyte bearing C3b (EC3b), 1 X 108EC3b of each cell type were incubated with 2,ug of properdin,10 ng of D, and doubling amounts of B (1-32 ng) in 0.2 ml ofDGVB2+ for 30 min at 300C. After addition of rat serum di-luted 1:30 in GVB-EDTA and further incubation of the cellsfor 60 min at 37°C, the percentage hemolysis was determinedand the average number of hemolytic sites per cell was calcu-lated. The amounts of B required for formation of approxi-mately two hemolytic sites per cell were: 70 ng of B for EsC3b,21 ng for EmC3b of BUB/BnJ, 42 ng for EmC3b of C57BL/6J,66 ng for EmC3b of A/WySn, and 10 ng for EmC3b of DBA/2J.The capacity of 013H to decay-dissociate the membrane-

bound P,C3b,Bb was assessed with intermediates prepared withquantities of B that yielded approximately two effectiveP,C3b,Bb sites per cell. One-tenth milliliter of DGVB2+ con-taining 1 X 107 erythrocytes bearing P,C3b,Bb was directlyadded to 0.1 ml of ice-cold GVB-EDTA alone or containingdoubling amounts of ,31H ranging from 0.8 to 64 ng. The re-action mixtures were incubated for 30 min at 30'C and theresidual convertase sites were then developed by addition of0.3 ml of rat serum diluted 1:30 in GVB-EDTA and incubationfor 60 min at 370C. The concentration of fBlH required todecay-dissociate 50% of the P,C3b,Bb sites on Es and Em fromthe four mouse strains was calculated.

Determination of Sialic Acid Content in Em Membranes.Erythrocyte stroma were prepared from strains BUB/BnJ,ST/bJ, C57BL/6J, DBA/2J, and B6D2 and backcrosses of theF1 hybrid to the parental strains. Em (3 X 109) were hemolyzedwith 1 mM Na acetate at pH 5.4, and the stroma were washedeight times by repeated sedimentation at 8000 X g for 20 min(SS-34 rotor, Sorvall RCB2) and resuspension with 1 mM Naacetate at pH 5.4. The pelleted, washed stroma were resus-pended and hydrolyzed in 1 ml of 0.1 M HCI for 60 min at80°C. The residual insoluble debris was sedimented for 10 minat 8000 X g, and the supernatant was removed and assayed forsialic acid by the thiobarbituric acid method (28). Sialic acidwas calculated by comparison to a standard reference curveestablished with synthetic N-acetylneuraminic acid (Calbio-chem) and expressed as nmol/109 Em.

RESULTSActivation of Human Alternative Complement Pathway

by Em from Various Mouse Strains. Em from 21 differentmouse strains were examined for their capacity to activate thehuman alternative complement pathway as assessed by directlysis of the erythrocytes. The mean values of five individualfemale mice of each strain are represented in Table 1 in rankorder from a high of 85 units to a low of 26 units of activatingcapacity. Lysis by activation of the human alternative com-plement pathway was dose-related for Em of all strains. This

Table 1. Activation of human alternative complement pathwayby erythrocytes from various inbred strains of mice

ActivatingH-2 capacity,

Strain haplotype units*

BUB/BnJ q 85 ± 1C57BL/KsJ d 75 8C58/J k 70 5C57BL/6J b 69 ± 6C57BL/1OJ b 66 ± 7B1O.A a 63+5C57BL/6By b 62 + 3SWR/J q 54±5CBA/HN k 53 ± 3BALB/cJ d 49 ± 2A/WySn a 42 ± 3A/J a 42±5129/J bc 39 ± 2DBA/1J q 33 ± 2DBA/2J d 33 ± 2RF/J k 33 2AKR.B6 b 31 2AKR/J k 28 + 3RIII/2J r 28 ± 6P/J p 27±2STlbJ k 26 4

* Data shown as mean ± SD.

Cell Biology: Nydegger et al.

6080 Cell Biology: Nydegger et al.

relationship is illustrated in Fig. 1 for four strains that exhibitedhigh (BUB/BnJ and C57BL/6J), intermediate (A/WySn), andlow (ST/bJ) activating activity. Em from strains with a partic-ular H-2 haplotype were randomly distributed throughout therange of activating capacities. In addition, the activating ca-pacity of Em from B10.A mice resembled that of theC57BL/10J background strain rather than that of the A/J straindonating the H-2 haplotype. Similarly, Em from AKR.B6 micehad the same activating capacity as Em from AKR/J, indicatingthe predominant effect of genetic loci not linked to the H-2complex (Table 1).

Relationships among Alternative Complement PathwayActivating Capacity, Susceptibility of Cell-Bound Convertaseto Decay-Dissociation by P1H, and Membrane Sialic AcidContent of Em. Em from four strains of mice (BUB/BnJ,C57BL/6J, A/WySn, and DBA/2J) differing in their capacityto activate the human alternative complement pathway wereconverted to their cellular intermediates bearing P,C3bBb andassessed for the capacity of j31H to decay-dissociate the con-vertase. #lH-mediated decay-dissociation of 50% of theerythrocyte-bound P,C3b,Bb required (per ml) 178 ng of #1Hfor DBA/2J, 240 ng for A/WySn, 324 ng for C57BL/6J, and395 ng for BUB/BnJ. The alternative pathway activating ca-pacities of Em from the same bleedings are compared to thesusceptibility of EmP,C3b,Bb to decay-dissociation by ,B1H inFig. 2. The correlation between the alternative pathway acti-vating capacity and the concentration of ,B1H required todecay-dissociate 50% of the convertase sites on Em from eachstrain is significant (P < 0.001; r = 0.99). Only 60 ng of j31H wasrequired to decay-dissociate 50% of the convertase sites on Es,and these cells do not have the capacity to activate the humanalternative complement pathway.

Stroma prepared from ET from four inbred strains of micevarying in their capacity to activate the human alternativepathway were analyzed for their sialic acid content. Theanalysis was carried out on Em from four individual mice ofstrains BUB/BnJ, C57BL/6J, and DBA/2J and from eight in-dividual mice of strain ST/bJ. Linear regression analysis re-vealed a significant inverse correlation (r = -0.95; P < 0.001)between the sialic acid content of Em from 20 individual miceof the four strains and their respective capacities to activate thealternative complement pathway (Fig. 3). There was also aninverse relationship between the mean content (per 109 Em) ofsialic acid (Fig. 3) of Em from BUB/BnJ (10.2 nmol), C57BL/6J(13.2 nmol), and DBA/2J (25.4 nmol) and the amounts of #1H

Serum input, gi

FIG. 1. Lysis of Er (A) and Em from mouse strains BUB/BnJ (0),C57BL/6J (0), A/WySn (A), and ST/bJ (o) by incremental amountsof human serum diluted in GVB-Mg-EGTA. For Em, each pointrepresents the mean ± SD of lysis from five individual female mice;for Er, the points represent the mean + SD for five replicate deter-minations.

a0> 0B-= 80

s.5 0o ._606

o05 A

I-0r,+ 20-

150 200 300 400P11 H, ng/ml

600

FIG. 2. Correlation between the alternative pathway activatingcapacity ofEm from strains BUB/BnJ (-), C57BL/6J (0), A/WySn(A), and DBA/2J (O) and the concentration of j31H required todecay-dissociate 50% of the P,C3b,Bb sites on Em from each strain.Each point represents results obtained with a single pool ofEm fromthree mice of each strain.

required for 50% decay-dissociation of P,C3b,Bb bound to Emof these strains (Fig. 2).

Segregation Analysis of Relationship of Sialic Acid Con-tent and Capacity of Em to Activate Alternative ComplementPathway. A strain whose Em had a high activating capacity andlow sialic acid content (C57BL/6J) was crossed with a strainwhose Em had a low activating capacity and a high sialic acidcontent (DBA/2J). The F1 generation was backcrossed to theparental strains and intercrossed to yield the F2 generation. Thedata for alternative pathway activating capacity of the parentalstrains, the F1 and F2 generations, and the backcrosses arepresented in Fig. 4. Em from 12 F1 hybrids exhibited alternativepathway activating capacities that were intermediate to anddid not overlap with those of Em from mice of either parentalstrain. The alternative pathway activating capacity of Em fromthe parental DBA/2J (range, 25-34 units) was defined as low,that of Em from the parental C57BL/6J strain (range, 55-70units) was designated high, and that of Em from the F1 hybrids(range, 36-54) was termed intermediate. Analysis of Em from78 individual mice of the F2 generation revealed 25 with low,37 with intermediate, and 16 with high capacities to activatethe human alternative pathway. The x2 value calculated withregard to expectations from a one-gene, two-codominant-allele

.= 80

.*- 70Q

CL

cm 60r._4)

._

u 50

U

'300)40

M 20

CLCD

40.

;;~20

10 15 20 25Sialic acid, nmol/109 Em

30

FIG. 3. Correlation between the alternative pathway activatingcapacity and membrane sialic acid content ofEm from individual miceof strains BUB/BnJ (A), C57BL/6J (0), DBA/2J (v), and ST/bJ(-).

A

0

* O

O

* v* v

Proc. Nati. Acad. Sci. USA 75 (1978)

Proc. Natl. Acad. Sci. USA 75 (1978) 6081

DBA/2J F C57B L/6J

4F 0 An~~~~~~~~~~~~.......ni l l. ..4H~~~~~~~~........7....

6c

._

42H

Sr4,-

H" ...." ...'.' .'.......-"' ..... ''''...',,'.'

F X C57BL/6J

8-~~~~~-...

4r--...... -..

40 20 30 40 50 60 70Alternative pathway

activating capacity, units

FIG. 4. Distribution of alternative pathway activating capacityof Em from individual mice of strains DBA/2J and C57BL/6J, theB6D2 F1 generation, the F2 generation, and the backcrosses of the F1generation to DBA/2J and C57BL/6J.

hypothesis is 2.28 (0.3 < P < 0.4). The -individual analyses of49 F1 backcrosses to the parental DBA/2J demonstrated 21mice to have Em with low capacity and 28 to have Em with in-termediate capacity to activate the human alternative pathway(x2 = 1.0; 0.3 < P < 0.4). The individual analyses of 28 F1backcrosses to the parental C57BL/6J revealed 13 mice to haveEm with high, 14 mice to have Em with intermediate, and 1

mouse to have Em with low capacity to activate the human al-ternative pathway (X2 = 0.14; P t 0.7). The mode of inheri-tance in the F2 and backcross generations of the capacities ofEm to activate the human alternative pathway is indicative ofregulation by a single autosomal locus with codominant al-leles.To confirm that membrane sialic acid content determined

the capacity of Em to activate the alternative pathway, thesetwo characteristics were assessed for Em from four B6D2F1hybrids and nine B6D2F1 backcrosses to each of the parentalstrains. Linear regression analysis revealed a highly significantinverse correlation (r = -0.91; P < 0.001) between the sialicacid content of Em from these hybrid mice and their capacityto activate the alternative pathway (Fig. 5).

DISCUSSIONModulation by surface sialic acid of the capacity of a particleto activate the human alternative pathway was recognized instudies in which nonactivating Es were converted into activatorsby enzymatic deletion or chemical modification of this carbo-hydrate moiety (18, 19). The interaction of native CS, B, andD in the fluid phase continuously generatesC3b (9) in a reactionthat is augmented by properdin (10). Covalent attachment (11)of C3b to bystander surfaces deficient in sialic acid leads to

15 20 25Sialic acid, nmol/109 Em

FIG. 5. Correlation between alternative pathway activating ca-pacity and membrane sialic acid content of erythrocytes from indi-vidual mice of B6D2 F1 generation (A), the backcross to C57BL/6J(3), and the backcross to DBA/2J (c).

amplified CS cleavage at that site because of impaired bindingof 31H to C3b on such surfaces (19, 20). This circumstance fa-vors binding of B to C3b to form the bimolecular complex,C3b,B, which D converts to C3b,Bb, and retards decay-disso-ciation of C3b,Bb by fl1H (18). Thus, discrimination betweenactivating and nonactivating surfaces occurs after C3b depo-sition (12, 13), and a relative sialic acid deficiency representsthe molecular basis for the earlier finding that activating par-ticles circumvent the regulatory actions of the control proteinsof the alternative pathway (12, 13, 18). This view that the al-ternative pathway of complement involves only six plasmaproteins-namely, C3, B, ID, and properdin to form the stabi-lized amplification convertase and C3b inactivator and #1H(29) to direct the reaction to an activating surface (12, 13)-hasrecently been confirmed in studies in which the previouslystated requirement for an additional principle termed "ini-tiating factor" (30-32) was withdrawn (33).The observation that Em activates the human alternative

pathway afforded an opportunity to examine whether natural,possibly strain-related, variations in the sialic acid content wouldconfirm the conclusions regarding mechanisms of activationthat had'been based on deletion of cell surface sialic acid byenzymatic and chemical approaches (18). There was a 3-foldrange in the activating capacity of Em from 21 different inbredstrains as assessed by their direct lysis in whole serum dilutedin GVB-MgEGTA (Table 1). The capacity of Em from fourstrains to activate directly the human alternative pathway inwhole serum increased in proportion to the concentration offl1H required to decay-dissociate 50% of the P,C3b,Bb formedon these Em (Fig. 2). Linear regression analysis revealed a sig-nificant inverse correlation between the respective capacitiesof Em from 20 individual mice of four strains to activate thehuman alternative complement pathway and the sialic acidcontent of these Em (Fig. 3). Thus, there was an inverse rela-tionship between the mean value for membrane sialic acid andthe capacity of fl1H to decay-dissociate cell-bound amplifica-tion convertase, a finding completely in accord with that ob-tained by experimental deletion of sialic acid from a nonacti-vating particle (18).The evidence for an inverse relationship between membrane

sialic acid and the capacity to activate the alternative pathwaycould be rigorously tested by determining the segregation ofthese two traits in inbred strains. Em from F1 hybrids derivedfrom a strain with high activating capacity (C57BL/6J) and a

Cell Biology: Nydegger et al.

6082 Cell Biology; Nydegger et al.

strain with low activating capacity (DBA/2J) exhibited an in-termediate activating capacity that did not overlap with thatof Em from the parental strains (Fig. 4), Analysis of Em from78 individual mice of the F2 generation revealed a distributionamong low, intermediate, and high activating capacities thatis consistent with a one-gene, two-codominant allele geneticmode of inheritance. Individual analysis of 49 mice of the F1backcrosses to the parental strain with low activating capacity(DBA/2J) demonstrated Em with either low or intermediateactivating capacity, whereas mice of the F1 backcross toC57BL/6J, the parental strain with high activating capacity,had Em with either high or intermediate activating capacity(Fig. 4). The alternative pathway activating capacity of the Emfrom the backcross to each parental strain was as expected ifthe activity observed were regulated by alleles of a single au-tosomal locus. That this locus also predominantly regulated thesialic acid content of Em was established by the highly signifi-cant inverse correlation between the sialic acid content and thealternative pathway activating capacity of Em from 22 indi-vidual mice of the F1 and backcross generations (Fig. 5).

It has been observed that the relative electrophoretic mo-bilities of Em, a characteristic that in other cell types reflectstheir content of sialic acid (34), show strain-specific variationsthat are regulated by alleles of a single autosomal locus (35). Emwith high electrophoretic mobility (DBA/2J, AKR/J, andRIII/J) were those with a low capacity to activate the humanalternative complement pathway and Em with a low electro-phoretic mobility (C57BL/6J, C57BL/10, C57BL/KsJ, C58/J,and B1Q.A) were, with the exception of ST/bJ and RF/J, those,having high alternative pathway activating capacity (Table 1).Susceptibility to Friend leukemia virus is controlled by severalgenetic loci, one of which, Fv-2, is a non-H-2-linked diallelicautosomal locus that determines absolute resistance or suscep-tibility to development of leukemia (36, 37). It is of interest thatfive of five resistant strains had Em that were good activatorsof the human alternative pathway, implying a low sialic acidcontent, and six of six susceptible strains had Em with low ac-tivating capacities, implying high sialic acid content.

This work was supported by National Institutes of Health GrantsAI-07722, AI-10356, AM-05577, and RR-05669 and by a grant fromthe New England Peabody Home for Crippled Children. U.E.N. wasa Fellow of the Swis National Research Foundation. D.T.F. is a Re-search Career Development Awardee (KO4 AI-00245-02) of the Na-tional Institutes of Health.

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