Proc. NatL Acad. Sci. USAVol. 79, pp. 3290-3294, May 1982Immunology

Complement activation by isolated myelin: Activation ofthe classical pathway in the absence of myelin-specificantibodies

(Cl activation)

PADMAVATHY VANGURI*, CAROL L. KOSKIt, BARRY SILVERMAN*, AND MOON L. SHIN**Departments of *Pathology and tNeurology, University of Maryland School-of Medicine, Baltimore, Maryland 21201

Communicated by Manfred M. Mayer, February 19, 1982

ABSTRACT Many pathological conditions of the central ner-vous system involve damage to and removal of myelin membrane.Very little is known about initiation of this membrane damage andthe mechanisms of disposal of the damaged tissue. We are inter-ested in the interaction between complement (the components ofcomplement are designated C1, C2, C3, etc.) and myelin mem-branes and the possible role of complement in amplifying myelindamage and in the disposal of damaged myelin in vivo, becauseactivation of complement generates both membrane-attack com-plexes and opsonin(s). In this study, we found that isolated rat orhuman myelin consumes complement in the absence of specificantibodies. Activation ofcomplement was demonstrated by show-ing C3 cleavage in fresh serum incubated with myelin. Incubationof central nervous system myelin with C2-deficient serum pro-duced no C5 consumption and only minor factor B conversion, thusexcluding the alternative pathway of activation. Involvement ofthe classical pathway was shown directly by the Cl fixation andtransfer assay. Myelin incubated with C2-deficient serum or withpurified CI and then washed contained Cl activity that could lysesheep erythrocytes sensitized with anti-Forssman IgM antibodyand carrying C4, together with C2 and C3-C9. Membranes inbrain tissues other than myelin (heavy membrane fraction ob-tained on sucrose density gradient centrifugation) were unable toactivate Cl.

Myelin membrane damage has been observed in various path-ologic conditions of the central nervous system. This damageoccurs in primary demyelinating diseases, such as multiple scle-rosis, and as a consequence of extramyelin tissue damage inconditions such as infarct, trauma, and infection (1-4). Thereis much speculation on how myelin breakdown is initiated.Furthermore, it is not understood how the damaged myelinmembranes and the myelin-associated proteins are disposed of.Breakdown products of myelin basic protein and its -fragmentshave been found in the cerebrospinal fluid (5-7) and in the sys-temic circulation (8-10).

In this context, it is of interest to investigate the role ofcom-plement (the components of complement are designated C1,C2, C3, etc.) since its activation generates a membrane attackcomplex that could attack myelin. Membrane attack by com-plement is initiated when C5 is cleaved into C5a and C5b byC5 convertases generated by either classical or alternative path-way activation (11, 12). When C5b binds to C6, a stable complexis formed that is capable ofcausing membrane damage togetherwith C7, C8, and C9 (13). Also, activation ofcomplement gen-erates nonspecific opsonins. 'Thus, in complement-mediated.phagocytosis, covalently bound C3b, a cleavage product of C3,

on the membrane surface promotes efficient phagocytosis viaC3b receptors on the phagocytic cell membrane (14, 15).

Since the blood-brain barrier is -broken in most pathologicconditions in which extensive myelin loss occurs, central ner-vous system tissue is exposed to an abundance of complement.This raises the possibility of its activation either with or withoutspecific antibodies to brain tissue components. If so, comple-ment might amplify the tissue damage. Also, complement couldparticipate in the subsequent healing process. Accordingly, wehave studied whether and how myelin itself could activate thecomplement system in the absence of myelin-specific antibodies.

MATERIALS AND METHODSBuffers. Barbital-buffered saline (pH 7.4; g, 0.15) (Barb/

NaCl) was prepared by diluting a stock solution (16) 1:5 withwater. G/Barb/NaCl was Barb/NaCl/0.1% gelatin/0.15 mMCaCl2/1.0 mM MgCl2. D/G/Barb/NaCl was barbital-bufferedsaline (pH 7.4; IL, 0.075)/25% dextrose/0.1% gelatin/0. 15 mMCaCl2/1.0 mM MgCl2. EDTA/Barb/NaCl was.prepared bymixing 9 vol of Barb/NaCl with 1 vol of 0.1 M EDTA.Complement Components. Guinea pig C2 and C5-C9 and

human'Cl were purchased from Cordis Laboratories (Miami,FL).

Antibodies. Rabbit anti-human 'C3c (IgG) was purchasedfrom Dako Chemicals (Hicksville, NY). Rabbit anti-human fac-tor B was obtained from Behring Werke (Marburg, FederalRepublic of Germany).

Preparation of Intermediate Complement Complexes onErythrocytes. Sheep erythrocytes sensitized with anti-Forss-man IgM (EA) and carrying C1, C4b, C2a, and C3b(EAC1,4b,2a,3b) were made with fresh serum treated with K-76 monocarboxylic acid as described in ref. '17. K-76 monocar-boxylic acid abolishes the functions of C5 and C3b inactivator(18, 19). Sheep EAC4b,3b were prepared by incubatingEAC1,4b,2a,3b at 37°C for 2 hr in EDTA/Barb/NaCl.

Isolation of Myelin and Heavy Membrane Fraction. Myelinwas prepared from human spinal cord and from rat brain asdescribed (20). In brief, the tissue was homogenized and thehomogenate was fractionated on a discontinuous sucrose densitygradient. Myelin was collected at the interface of 0.32 M and0.85 M sucrose, washed to remove sucrose, and subjected to

Abbreviations: C1, C2, C3, etc., components of complement; C2-Dserum, human serum congenitally lacking 'C2; EAC1,4b,2a,3b andEAC4b,3b, sheep erythrocytes sensitized with anti-Forssman IgM an-tibody carrying C1, C4b, C2a, and C3b, and C4b and C3b, respectively;Barb/NaCl, barbital-buffered saline; G/Barb/NaCl, Barb/NaCl con-taining gelatin; D/G/Barb/NaCl, Barb/NaCl containing dextrose andgelatin; EA, erythrocytes sensitized with antibody.t To whom reprint requests should be addressed.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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osmotic shock twice. A second density gradient centrifugationproduced purified myelin. Its characteristic protein pattern byNaDodSO4/polyacrylamide gel electrophoresis was the sameas that in ref. 20. The pellet containing the heavy membranefraction was treated in the same way. This separation of myelinmembranes from the heavy membranes is based on the differ-ence in their protein content (20). The myelin and heavy mem-brane fractions were washed and stored in Barb/NaCl/2 mMphenylmethylsulfonyl fluoride/0.02% sodium azide at 40C untiluse. The membrane suspensions were standardized in terms ofwet weight of membrane sediment obtained in a BeckmanMicrofuge, model B. At the time of experiment, requiredamounts of membrane were washed in G/Barb/NaCl by cen-trifugation in a Beckman Microfuge, model B. The dry weightsofa myelin membrane and a heavy membrane preparation were35 and 18 pug/mg of wet weight, respectively, after removal ofsalt by dialysis and drying to constant weight at 800C.Cl Fixation and Transfer. The C1 transfer assay was per-

formed according to Rapp and Borsos (21) with minor modifi-cation. Myelin was incubated with various amounts of humanserum congenitally lacking C2 (C2-D) for 45 min at 370C andwashed with G/Barb/NaCl. An equal volume ofEAC4b,3b (1.0X 108/ml) was added to this myelin suspension and the mixturewas incubated at 30TC for 15 min. The suspension was then in-cubated with excess guinea pig C2 for 10 min at 300C. This wasfollowed by incubation with a 1:40 dilution of guinea pig serumin EDTA/Barb/NaCl for 60 min at 370C. A similar experimentwas performed with isolated human C1. In this case, the myelinsuspension was incubated with human C1 instead of C2-Dserum.

Immunoelectrophoresis. Crossed immunoelectrophoresiswas carried out with anti-C3 in the second dimension accordingto Laurell (22).

RESULTSInactivation of Complement by Myelin. Various amounts of

rat central nervous system myelin were suspended in 0.2 ml ofBarb/NaCl and incubated with 0.2 ml of a 1:50 dilution offreshnormal human serum, as a source of complement, for 45 minat 370C in 1.5-ml Microfuge tubes to facilitate rapid pelletingof the myelin at the end of incubation. Portions (0.2 ml) of thesupernates were assayed for residual complement by hemolytictests with 0. 1 ml of EA (1.5 X 108/ml). As shown in Fig. 1, re-sidual complement activity diminished progressively as theamount of myelin increased. Similar results were obtained withhuman myelin.

Effect of Removing Possible Traces of Antimyelin Antibodyfrom Normal Human Serum. For this purpose, undiluted nor-mal human serum was treated with an equal volume of packedrat myelin for 15 min strictly at 0°C (to avoid complement ac-tivation) and then centrifuged to remove the myelin; this ab-sorption treatment was repeated twice. The absorbed normalhuman serum and untreated normal human serum (control)were diluted 1:30 with G/Barb/NaCl and 0.2-ml portions ofeach were incubated for 45 min at 37°C with 0.2 ml of G/Barb/NaCl containing 0.85 mg (wet weight) of rat central nervoussystem myelin. Then, the myelin was removed by centrifuga-tion, and 0.2-ml portions of the supernates were assayed forresidual C5 activity with EAC1,4b,2a,3b as described (23). Theloss of C5 was 42% in the absorbed normal human serum and41% in the control. This shows that antimyelin antibody is notrequired for consumption of C5 by myelin. Furthermore, thisexperiment indicates that myelin activates the classical or al-ternative pathway enzymes of the complement system thatcleave the C5 molecule. Since this cleavage activates membraneattack by the terminal complement proteins, myelin mem-

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1 2 3 4 5Myelin (wet weight), mg

FIG. 1. Consumption of total hemolytic activity of complement byrat myelin. Residual hemolytic complement activity is shown in termsof the Z value, which represents the average number of lytic channelsper cell [Z = ln(l - y), where y = fraction of cells lysed]. Quantitiesof myelin are shown as wet weight. Dry weight -3.5% of wet weight.

branes may have the capacity to initiate complement-mediateddamage to themselves. (With respect to experimental design,this experiment shows that the centrifugal removal of myelinis complete; otherwise, the supernatant serum wouldhave con-tained.membrane material capable of interfering with the lysisof EACl,4b,2a,3b,5b by C6-C9.)

Identification of the Activation Pathway. To determinewhether classical or alternative pathway activation was in-volved, the following experiments were carried out. Rat myelinwas incubated with various amounts of C2-D serum for 45 minat 370C. The supernates were then tested for CS consumptionon EAC1,4b,2a,3b. As shown in Fig. 2, no C5 consumption bymyelin was observed. Thus, activation ofcomplement by mye-lin requires C2, a component essential for the classical pathway.

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0.25 0.5 1.0C2-D serum, relative concentration

FIG. 2. C5 titration of C2-D serum after incubation with myelin.Portions (0.25 ml) of a suspension containing 0.43 mg (wet weight) ofrat central nervous system myelin were incubated with various con-centrations of C2-D serum in a final volume of 0.4 ml, and 0.2-ml por-tions of the supernates were assayed for residual C5 activity on EAC1,4b, 2a, 3b. o, Incubation of C2-D serum with myelin; o, incubation ofC2-D serum without myelin. Relative concentration of C2-D serum:1.0 = 1:188 dilution.

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FIG. 3. Immunoelectrophoresis for factor B conversion. A 1:2 di-lution of anti-human factor B was applied to the horizontal slits andthe supernates (0.02 ml) from the incubation mixtures were placed inthe round wells. (A) Patterns: a, C2-D serum/myelin; b, C2-D serum/zymosan; c, normal human serum/zymosan; d, C2-D serum. (B) Pat-terns: a, normal human serum/zymosan; b, normal human serum/myelin; c, normal human serum; d, normal human serum/zymosan.

To evaluate alternative pathway activation, 0.04-ml aliquotsof normal human serum. or C2-D serum were incubated for 45min at 370C with 3.4 mg (wet weight) ofrat central nervous sys-tem myelin. and analyzed by immunoelectrophoresis with a 1:2dilution of anti-human factor B. Cleavage of factor B was de-tected by observing the conversion from a to y mobility. Aspositive controls, 0.04-ml portions of normal human and C2-Dsera were incubated for 45 min at 370C with 40 j.g of zymosanwashed with Barb/NaCl. As seen in Fig. 3, complete conver-sion was produced by treatment of normal human serum withzymosan (pattern c in group A and patterns a and d in group B).In zymosan-treated C2-D serum, conversion was partial, pre-sumably because only the alternative pathway was operative(pattern b in group A). Very little factor B conversion occurredwhen C2-D serum was incubated with myelin (pattern a ingroup A). It follows from these results that myelin activated theclassical pathway.

*Demonstration ofC3 Cleavage by Crossed Immunoelectro-phoresis with Anti-Human C3. This experiment was carried outto show that classical pathway activation leads to generation ofthe C3 convertase C4b,2a and consequent C3 cleavage. Myelinwas incubated with normal human serum for 45 min at 370C andthe serum was analyzed by crossed immunoelectrophoresis. Asshown in Fig. 4A, C3 conversion to C3b occurred when normalhuman serum was incubated with myelin. whereas incubationof normal human serum in the absence of myelin caused onlyslight conversion (Fig. 4B).

Activation of C1 by Myelin. Activation of complement viathe classical pathway presumably involves binding and activa-tion ofC1 by myelin. We.have tested this interpretation by the

A B

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FIG. 4. Cleavage of C3 in myelin-treated normal human serum.(A-) Normal human serum (0.04 ml) was incubated at 370C for 45 minwith 3.4 mg (wet weight) of rat central nervous system myelin. Con-version of C3 to C3b was shown by crossed immunoelectrophoresis.One percent rabbit anti-C3 IgG was used in the second dimension. (B)Control normal human serum incubated with buffer for 45 min at 370Cshowed slight spontaneous conversion.

0.25 0.5 1.0C2-D serum, relative concentration

FIG. 5. C1 activation by myelin. Human central nervous systemmyelin [1.675 mg (wet weight)] in 0.25 ml was incubated with 0.15 mlof various dilutions of C2-D serum for 45 min at 37TC and washed.Myelin was resuspended to 0.25 ml in G/Barb/NaCl and incubatedwith 0.25 ml of EAC4b,3b (1.0 x 108/m}) for 15 min at 30TC. Excessguinea pig C2 was added and the mixture was incubated for 10 min at30TC. The cells were lysed with a 1:40 dilution of guinea pig serum inEDTA/Barb/NaCl as a source of C-C9. Relative concentration ofC2-D serum: 1.0 = 1:250 dilution.

C1 transfer assay. Ifactivated C1 is present on myelin previouslyincubated with C2-D serum, it can lyse EAC4b cells togetherwith C2 and C3-C9 (21). Fig. 5 shows that human myelin canactivate C1 in C2-D serum. As would be expected, no C1 ac-tivity was observed on myelin that had been incubated withC2-D serum and then treated with EDTA/Barb/NaCl (resultsnot shown). Direct C1 binding was also demonstrated by usingpurified C1. In this experiment, rat myelin suspensions wereincubated with various amounts ofpurified human C1, washed,and then tested by the C1 transfer assay. As shown in Fig. 6,the amount of hemolytically active C1 bound to myelin was lin-early related.to the input ofpurified C1. Experiments with hu-man central nervous system myelin gave identical results.

Comparison of Myelin Membrane and Heavy MembraneFractions of Rat Brain for Ability to Activate Complement. Toexamine whether other membranes in the central nervous sys-tem can activate complement, the myelin and heavy membranefractions obtained by sucrose gradient centrifugation ofrat brainwere examined for capacity to activate complement by mea-suring C1 activation. We incubated various amounts of myelinor heavy membranes with different concentrations of C2-D

1.25 2.5 5.0 10.0C1, units

FIG. 6. C1 binding by myelin. This experiment was like those inFig. 5 except that 0.852 mg (wet weight) of rat myelin was incubatedwith human C1 instead of C2-D serum.

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serum. Membranes were washed and C1 activities on mem-branes were measured by C1 transfer assay as described above.Fig. 7 shows that membranes from the heavy membrane frac-tion did not carry significant C1 activity whereas myelin boundand activated C1 to an extent that was dependent on the amountof myelin and the input of C2-D serum as a source of C1.

DISCUSSIONOur results indicate that isolated human or rat central nervoussystem myelin activates the complement system. This was dem-onstrated indirectly in terms of decreased total hemolytic ac-tivity and loss of C5 in fresh normal serum. In the latter ex-periment, adsorption of normal human serum with rat myelindid not affect C5 consumption, which indicates that antimyelinantibody is not required. That loss of complement activity re-flects complement activation was shown by detection ofC3 con-version to C3b during incubation of serum with myelin (Fig.4). Both rat and human central nervous system myelin activatedguinea pig complement with an efficiency similar to that of hu-man complement activation (results not shown). Involvementof the classical pathway is evident from the finding that com-plement consumption and significant factor B conversion didnot occur when myelin was incubated with C2-D serum (Figs.2 and 3).

Diverse substances other than antigen-antibody complexes,such as the lipid A component of lipopolysaccharide (24), C-re-active protein complexes (25, 26), and RNA viruses (27), alsoactivate complement by binding C1 through its Clq subunit.The binding of C1 triggers a series of intramolecular changesof subunits Cir and Cls, resulting in enzymatically active Clswhose substrates are C4 and C2 (28-30).

In our experiments, involvement of C1 was demonstrateddirectly. Thus, C1 in C2-D serum was bound to myelin (Fig.5). Identical results were obtained when purified C1 devoid ofantibodies was used instead ofC2-D serum (Fig. 6). In this case,the C1 molecules bound to myelin may include previously ac-tivated C1, since spontaneous activation ofC1 can occur duringpurification (31).

Previous studies with liposomes have shown that comple-ment activation is not a general property of bilayer membranes(32, 33). This was also observed in the present work. Thus, non-myelin membranes of brain obtained in the heavy membranepellet from high-speed centrifugation of brain homogenate

A B1.5 -

1.0

co 0.5-

2 4 2 4Membranes (wet weight), mg

FIG. 7. Measurement of C1 activity on rat brain membranes. Mye-lin membranes (Left) and heavy membranes (Right) were tested for C1activation as described in Fig. 5. While myelin binds and activates C1,no detectable C1 activity was observed with the nonmyelin mem-branes. C2-D serum dilutions used: *1:1,000; *, 1:3,000; A, 1:9,000.

were ineffective in activating C1 (Fig. 7) or C5 (results notshown). Also, preliminary experiments with myelin from hu-man peripheral nerve did not indicate binding and activationofC1 (not shown). Furthermore, living central nervous systemexplant cultures do not spontaneously activate complement(34). At present, the factor(s) in the myelin membrane that ac-tivates C1 is unknown.

Myelin is a plentiful substance, comprising 25% of the dryweight of whole brain in adult rat (20). Since the blood-brainbarrier is impaired in many pathologic lesions in which myelinmembrane damage is observed, our demonstration of comple-ment activation by myelin raises the possibility that tissue dam-age may be produced via membrane attack by C5b-9. Thus, itis conceivable that complement can amplify myelin damageafter an initiating event. In turn, myelin membrane damage bycomplement may facilitate subsequent injury by other factors,such as proteolytic enzymes. In the absence ofdirect evidence,these concepts are speculative.

In addition, it is possible that complement may play a rolein the healing process. Thus, the efficient disposal of centralnervous system myelin followed by healing with gliosis has beenobserved in diseases that affect primarily myelin and those thataffect brain tissue indiscriminately, such as infarct, trauma, andinfection (1, 2). Such brain damage is associated with extensivephagocytosis of myelin by astrocytes and macrophages and re-lease of their breakdown products into the cerebrospinal fluidand systemic circulation (3-8). Effective phagocytosis can bemediated by the complement-derived opsonin system, thusenhancing proteolytic digestion ofmyelin by phagocytes. A sim-ilar role of complement involving C1 activation by C-reactiveprotein in the absence of antibody has also been suggested forremoval of damaged tissue (25).

We thank Dr. J. Winkelstein for his generous gift ofC2-D serum andDr. M. Mayer for his critical evaluation of this study. This work wassupported by U.S. Public Health Service Grant 1 RONS 15662 andby Multiple Sclerosis Society Grant RG1374-A-1.

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