VB, veronal-buffered saline, ph 7.2; VB2+, VB containing mm CaCl2 and 0.5 mm MgCl2; GVB2+, VB2+ containing 0.1%

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 75, No. 5, pp , May 1978 Immunology Complement C3 convertase: Cell surface restriction of fi1h control and generation of restriction on neuraminidase-treated cells (alternative pathway/properdin/cell membranes/protein-protein interactions/c3b inactivator) MICHAEL K. PANGBURN AND HANS J. MOLLER-EBERHARD Department of Molecular Immunology, Research Institute of Scripps Clinic, La Jolla, California Contributed by Hans J. Muiller-Eberhard, February 27,1978 ABSTRACT The alternative or properdin pathway of complement is primarily controlled by the endopeptidase C3b inactivator (C3bINA) and the nonproteolytic glycoprotein j1h. The molecular mechanisms of control were investigated by performing binding studies of radiolabeled complement proteins to C3b bearing sheep erythrocytes (EsC~b) C3b was found to have distinct binding sites for P1H, C3bINA, Factor B, and pro erdin. jb1h binding increased C3bINA binding 30-fold, while Factor B binding prevented C3bINA action on C3b and was competitive with P1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the #1H and C3bINA sites. Activators such as rabbit erythrocytes (ER) have previously been shown to interfere with the effectiveness of the control by C3bINA and P1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of control does not occur on the surface of Es, a nonactivator of the alternative pathway. On the basis of comparative binding studies, restriction of control is explained entirely by reduced binding of P1H to ERC3b relative to EsC3b. Access of properdin, Factor B, C3bINA, and the Fab fragment of anti-c3 to the two cell types was unrestricted. Restriction of j1h control could be generated on the surface of Es by removal of cell-surface sialic acid with neuraminidase (acylneuraminyl hydrolase; EC 3±2.1.18) This enzymatic treatment converted Es from a nonactivator to an activator of the alternative pathway. The alternative pathway C3 convertase (C3bb) is formed by the proteolytic action of Factor D on the bimolecular complex consisting of C3b and Factor B (CMb,B). Upon cleavage of Factor B, the Ba fragment is dissociated, while the active-sitebearing Bb fragment is retained in the complex. CS convertase cleaves its substrate, native CS, into the fragments CMa and C&b. A C3b molecule thus produced can combine with a molecule of Factor B and, with the aid of Factor D, form a second molecule of CS convertase (1). As this process of positive feedback continues, the originally small signal provided by the first C3b molecule is greatly amplified. Since C3b can anchor itself firmly on the surface of particles, including cells, the entire process of amplification can take place on surfaces. As the multiplicity of surface-bound CMb molecules increases, immune adherence reactivity and C5 convertase activity are generated. Through the action of C5 convertase on C5, complement-dependent cytolysis is initiated. Regulation of the amplification phase of the alternative pathway is exerted by multiple mechanisms: (i) intrinsic decay of CS convertase, (Hi) stabilization of this enzyme by properdin, (iii) disassembly of this enzyme by the serum glycoprotein,b1h (2, 3), (iv) inactivation of C3b by the CMb inactivator (CMbINA) and,b1h (4), and (v) protection of C3 convertase from the action of these control proteins afforded by the surface properties of certain cells and other activators of the alternative pathway (5, 6). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact The purpose of this communication is to show that restricted control of CS convertase is entirely due to reduced accessibility of,b1h to surface-bound C3b, and to report an example of generation of restriction by enzymatic modification of the outer membrane of intact cells with neuramninidase (acylneuraminyl hydrolase; EC ). The neuraminidase-treated cells also acquired the ability to activate the alternative pathway. These studies were presented in part at the Seventh International Complement Workshop (1977), St. Petersburg Beach, FL (7). MATERIALS AND METHODS Preparation of Alternative Pathway Components. Highly purified human CS (8), Factor B (9), Factor D (10), activated properdin (P) (10), C3bINA, and j1h (4) were isolated as described. (31H, C3bINA, Factor B, P, and anti-c3 Fab were radiolabeled by using insolubilized lactoperoxidase at 00 (11) or by the Bolton-Hunter method (New England Nuclear, Gardena, CA) (12). These proteins were labeled to specific activities ranging between 0.4 and 1.5 Ci/g. The IgG fraction of a monospecific rabbit anti-cs was used to prepare anti-cs Fab (13). Buffers and Reagents. The following buffers were used (14): VB, veronal-buffered saline, ph 7.2; VB2+, VB containing 0.15 mm CaCl2 and 0.5 mm MgCl2; GVB2+, VB2+ containing 0.1% gelatin; Mg-GVB, VB containing 0.5 mm MgCl2 and 0.1% gelatin; GVBE, GVB2+ containing 10 mm EDTA; DGVB2+, one-half diluted VB2+ containing 0.1% gelatin, 2.5% (wt/vol) dextrose, and 0.01% sodium azide. C4-depleted normal human serum was prepared by passing serum containing 20 mm EDTA over an immune adsorbent column prepared by coupling the IgG fraction of a monospecific goat antiserum to human C4 to CNBr-activated Sepharose 4B according to the manufacturers directions (Pharmacia Fine Chemicals, Inc., Piscataway, NJ). The eluting protein was pooled, concentrated to its original volume, and dialyzed exhaustively against VB. This C4-depleted serum contained no detectable C4 and did not lyse hemolysin-sensitized sheep erythrocytes (Es), indicating an inoperative classical pathway. It contained normal levels of CS, Factor B, P, (31H, and C3bINA, as assessed by radial immunodiffusion. Preparation of EC3b and Neuraminidase-Treated EC3b. EC3b cells were prepared by incubating 1010 erythrocytes with 5 mg of purified CS and 50,ug of trypsin in 1 ml of GVB2+ for 3 min at 37. The resulting C3b-bearing cells were washed thoroughly in cold Mg-GVB and stored at 00 in DGVB2+. EC3b cells possessing varying amounts of C3b per cell were prepared by incubating 1010 EC3b cells prepared with trypsin with 100,ug of Factor B, 2 Mg of Factor D, and excess partially purified Abbreviations: CMbINA, C3b inactivator; VB, veronal-buffered saline; P. properdin; P. activated properdin; ER, rabbit erythrocytes; ES, sheep erythrocytes.

2 60 Immunology: Pangburn and Mfiller-Eberhard 50', I 30 C0?. UP0 V 'D 0.0 I 44 Proc. Natl. Acad. Sci. USA 75 (1978) )00 50,000 75,000 Number of C3b per cell 100,000 FIG. 1. Dependence of jt1h binding on the number of C3b molecules per cell. Bolton-Hunter labeled 1256-#lH (0.4 Ci/g) was incubated at 0.8 Mg/ml with 5 X 107 Es bearing various numbers of C3b on their surface in a final volume of 100 Al of DGVB2+. The cells plus bound #1H were then centrifuged through 20% sucrose. The amount of C3b per cell was determined by the amount of purified, soluble C3b required to inhibit by 50% the binding of 125I-anti-C3 Fab fragment to the EC3b cells. This number was consistently higher than the estimate of C3b deposited calculated from 125I-C3 uptake. nephritic factor (15) in 1 ml of Mg-GVB for 10 min at 37'. The washed cells possessing the nephritic-factor-stabilized C3 convertase on their surface were incubated with varying amounts of purified C3 in 1 ml of Mg-GVB for 2 hr at 37. After overnight decay of the nephritic-factor-stabilized enzyme at 00 in GVBE, the cells were washed and stored in DGVB2+. In some experiments a small amount of 125I-labeled C3 was added to unlabeled C3 in order to compare the amount of C3b uptake by sheep and rabbit erythrocytes. Neuraminidase (Behring Diagnostics, Somerville, NJ) treatment was accomplished by suspending cells in 50 mm sodium acetate/0.9% NaCl/0. 1% CaC12, ph 5.5, containing 10 units of neuraminidase per ml. The reaction was complete after 60 min at 37. The treated cells were washed and stored in DGVB2+. Sialic acid determinations on supernatants and cells were performed by the method of Warren (16). Binding Assays. All of the binding studies were performed at room temperature, except C3bINA binding, which was examined at 00. The preincubated (5 min) mixture of cells and radiolabeled protein in 100,ul of half-isotonic buffer (DGVB2+) was layered on 300 ul of 20% sucrose in DGVB2+ in a microfuge tube. The cells were pelleted rapidly, in approximately 15 sec, the bottom of the tube was cut off, and radioactivity was determined in cell pellets and supernatants. In '25I-labeled f1h (125I-fl1H) binding assays, the functional activity of radiolabeled #I1H was determined by competition with unlabeled #1H. /#1H labeled by either the lactoperoxidase (11) or the Bolton-Hunter method (12) retained 80% of its activity in the binding assay, while 125I-(#1H bound to and subsequently eluted from ESC3b was' 100% active. RESULTS Interaction of 61H with cell-bound C3b Competition between OIH and Factor B. Fig. 1 shows the proportionality between 125I-(B1H binding to EC3b and the number of C3b molecules bound per cell. The relationship is linear until the amount of bound (1H significantly reduced the,31h concentration in the fluid phase. Binding of fl1h was competitively inhibited by fluid phase C3b. It was abrogated by prior treatment of EC3b with C3bINA and #I1H under 1i2 4 1gg P or B added FIG. 2. Competition between P, Factor B, and #1H for binding to cell-bound C3b. EsC3b cells (5 X 107) were added to a solution (DGVB2+) containing the indicated amounts ofp or Factor B and 0.08,ug of 125I-ft1H in a final volume of 100 Ml. The reaction mixtures were allowed to reach equilibrium at room temperature and the cells were centrifuged through 20% sucrose. The percent bound was calculated by taking as 100% the proportion of #1H bound in the absence of P or Factor B (54% of the total 125I-01H added). A, EC3b + P; 0, EC3b + Factor B; 0, EC3b P + Factor B. conditions that cleave and inactivate C3b, indicating that #I1H cannot bind to inactivated C3b (CMbj). Prior treatment of EC~b with #l1h alone had no effect on subsequent 125I-fl1H binding. Similar results of,b1h binding studies were obtained independently by Ruddy et al. (17). #1H accelerates the rate of dissociation of Bb from EC3b,Bb as well as from the properdin-stabilized CS convertase EC3b,P,Bb (2, 3). Since Bb cannot rebind once dissociated, competition studies could not be performed with the active enzyme. Therefore, the nonactivated complex was used, which is formed by the interaction of EC3b and native Factor B in the absence of Factor D (18). Fig. 2 shows that native Factor B competes with 01H for binding to EC3b. A 70-fold molar excess of Factor B was required over (B1H in order to reduce /31H binding by 50%. This competition proved to be independent of the presence of activated properdin (P). The small increase 25I-C3bINA to EC3b in the presence in,1h uptake by EC3b in the presence of P (Fig. 2) was probably due to aggregation of EC3b by P and trapping offlh. There does not appear to be any competition between the P and B1H binding sites on C3b. Dependence of C3bINA Binding on O1H. At 00 the proteolytic action of C(bINA is slowed sufficiently to allow demonstration of binding of of #1H. C3bINA binding was proportional to the amount of fb1h bound and it approached equimolar amounts at saturation of the C3bINA binding sites. C3bINA binding to EC3b was up to 30-fold enhanced in the presence of 01H; however, there was a small but definite amount of binding of C3bINA to EC3b in the absence of the cofactor. Availability of C3b on Es and ER for binding of complement proteins A study comparing C3b molecules on sheep (Es) and rabbit (ER) erythrocytes was undertaken by the methods described above for measuring (31H and C3bINA binding. 01H, C3bINA, Factor B, P, and the Fab fragment of anti-cs were radiolabeled and their interactions with cell-bound C3b were compared (Table 1). Both ERC3b and EsC3b were prepared so that they 8

3 2418 Immunology: Pangburn and Muller-Eberhard Table 1. Relative binding of complement proteins to C3b on ES and ER Relative binding Protein offered Binding site on E on ER*,B1H C3b 0.1 C3bINA C3b, 01H 0.8 P C3b 1.2 Factor B C3b, P 1.2 Anti-C3 Fab C3b 0.8 * Relative to binding on Es. contained approximately 100,000 C3b molecules per cell. Binding of Fab anti-c3 was examined as a measure of the general availability or exposure of C3b on the surface of the two cell types. From low concentrations of Fab anti-c3 to concentrations approaching saturation, binding of the antibody fragment to ERC3b and ESC3b was similar. Properdin binding was slightly higher on ERC3b than ESC3b, and binding of Factor B to preformed C3b,P sites was also slightly higher on ER than on Es. (31H, however, bound only 10% as effectively to C3b sites on ER relative to ES. 131H binding was compared over a wide range of concentrations ( ,ug of 125I-#lH per ml). C3bINA binding, previously shown to be dependent on the amount of fl1h bound to ESC3b, was also found to be proportional to the amount of,b1h bound to ERC3b. Based on the number of,b1h molecules bound per cell, C3bINA binding was similar for the two cell types. However, since (i1h binding was lower on ERC3b than on ESC3B, the actual C3bINA binding was proportionately decreased. Neuraminidase treatment of Es Restriction of P1H Control and Generation of Ability to Activate the Alternative Pathway. Normal Es do not belong to the group of cells and substances that are activators of the alternative pathway. Restriction of,b1h control associated with ER could be generated on the surface of Es by treating ESC3b with neuraminidase (see Materials and Methods). These cells, from which 70% of membrane-bound sialic acid was removed, exhibited a 60-80% reduction in,b1h binding compared to untreated ESC3b (Table 1). Furthermore, Es treated with neuraminidase prior to C3b deposition restricted,b1h binding to subsequently deposited C3b to the same extent. Neuraminidase-treated Es also acquired the ability to activate the alternative pathway. Fig. 3 shows that in normal human serum immunochemically depleted of C4 to block the classical pathway, neuraminidase-treated Es were lysed, while normal Es were unaffected. Treatment of ER with neuraminidase did not alter the behavior of these cells. Es treated with 10 mg of trypsin or chymotrypsin per ml for 60 min at 370 showed no evidence of susceptibility to alternative pathway lysis, but could still be converted to an activator by neuraminidase. The lysis of neuraminidase-treated ES shown in Fig. 3 required higher concentrations of serum than did rabbit cell lysis. This effect may be related to the difference in the ability of ER and neuraminidase-treated Es to restrict f31h control. DISCUSSION The ability of the alternative pathway to proceed on the surface of activating particles depends entirely on the initial deposition of C3b (19, 20). The mechanism by which this initial C3b is deposited is uncertain. The following hypotheses have been advanced regarding the early molecular events of the pathway that precede C3b deposition. Properdin has been implicated _ 2' C4-depleted serum,,ll 100 FIG. 3. Activation of the alternative pathway by neuraminidase-treated Es. Erythrocytes (5 X 107) were incubated with the indicated amounts of C4-depleted normal human serum for 10 min at 37 in a total volume of 170 Ml of Mg-GVB. The reaction was stopped with 0.83 ml of cold GVBE and the released hemoglobin was determined after centrifugation. 0, ER; 0, neuraminidase-treated Es; *, untreated Es. in initiation (21, 22) but has since been shown not to be involved in this reaction (20). Because of antibody-like specificity (23, 24) observed by some investigators to be associated with the pathway, immunoglobulin has been considered part of initiation. That human IgA (25), certain IgG type antibodies (26), and guinea pig gamma-l-globulin (27) may trigger the pathway has been demonstrated. A functional analogue of Clq was postulated in this laboratory to fulfill the function of recognizing activators and of assembling the initial C3 convertase (20). Others envisaged spontaneous CS turnover with random attachment of C3b to nearby particles as the initial events (28). 1+) Synergistic 1-1 Antagonistic Proc. Nati. Acad. Sci. USA 75 (1978) C3b CHbINA FIG. 4. Schematic representation of interactions between binding sites on the C3b molecule. As described in the text, P binding enhances Factor B binding and fl1h has a similar effect on C3bINA binding. Factor B and C3bINA binding are strongly antagonistic if not mutually exclusive, while j#1h binding and Factor B binding are competitive. Bb

4 Unrestricted NIH Control Restricted,81H Control Immunology: Pangburn and Miiller-Eberhard AM AC3J0- +C3klNA +cs Cl- Lysis FIG. 5. Schematic representation of differential control by,31h of cell-bound C3b: dependence on cell-surface properties. Cell surfaces (Upper) such as Es allow unrestricted j#1h control of bound C3 convertase. ER and neuraminidase-treated ES (Lower) restrict,b1h binding and allow amplification of the alternative pathway. This model lacks a focussing entity that directs the spontaneously forming C3b toward an activator rather than a bystander on nonactivator surface. If, however, the surface of an activator is capable of restricting the control by the regulatory proteins of the pathway, then focussing of C3b deposition becomes possible by default. Fearon and Austen demonstrated deregulation of alternative pathway control by activators like rabbit erythrocytes, Escherichia coil, and yeast cell walls (5, 6, 29). Thus, much of the available data suggest that initiation may occur by default of control of the pathway imposed by the surface characteristics of activators. The present report probes the molecular interactions of cell-bound C3b with the control proteins as well as with the other alternative pathway proteins. The observations indicate that activating surfaces possess structures that interfere specifically with the fulh-c3b interaction while permitting uninhibited access of other proteins to bound CAb. Potentially similar structures exist on a nonactivating substance, but their expression is concealed by neuraminic acid residues. Alternative pathway control mechanisms Radiolabeled,B1H was used as a macromolecular probe to characterize the interactions of complement proteins with cell-bound C&b. The binding of fu1h to C3b (EsC3b) led to the generation of an efficient binding site for C3bINA. A stable complex, EsC(b,fulH,C3bINA, was demonstrated at 00. A weak interaction between ESC3b and C3bINA was also detected in the absence of fu1h, suggesting separate but cooperative binding sites for,b1h and C3bINA on C3b. The binding of properdin did not significantly affect fuih binding (Fig. 2), and, as shown by Medicus et al. (19), occupation of the properdin binding site on C3b did not inhibit C3bINA cleavage of C3b, suggesting that this site is distinct from and independent of the,r1h and C3bINA sites. Properdin binding appears to influence only the Bb site on C&b. The binding sites for Factor B and fu1h appear to be physically distinct but competitive. The evidence for this conclusion is the following: (i),81h increased the decay rate of preformed C3 convertase by causing dissociation and thus inactivation of Bb, and (ii) both Bb and fu1h bind directly to C3b.,B1H was found to enhance binding of C3bINA 30-fold. contrast, Bb was shown to protect C3b from cleavage by the inactivator (19). The latter observation suggests the existence of interacting sites for C3bINA and Bb. These observations allow a more detailed description of,#1h L In Proc. Natl. Acad. Sci. USA 75 (1978) 2419 and C3bINA control of the alternative pathway CS convertase (Fig. 4). Factor B and fl1h compete for binding to C3b. The binding of Factor B followed by Factor D cleavage produces the CS convertase EC3bBS. Although this complex is protected from direct C3bINA action, it has a short intrinsic decay rate. Formation of the hypothetical, transient trimolecular complex EC3b,Bb,#lH probably lowers the binding constant between C3b and Bb leading to more rapid decay. The product remaining after Bb decay (ECt3bjH) can subsequently be inactivated by C3bINA, which abolishes further participation in the complement sequence. Alternatively, #1H may dissociate, leaving the C3b unaltered and capable of reforming a C3 convertase site. The presence of properdin on the cell-bound C3 convertase renders it considerably more stable, but it does not alter the sequence of possible events described above. Restricted control by ft1h of C3 convertase bound to particulate activators Any event that generates C3b, even a nonspecific, proteolytic event, may initiate alternative pathway amplification. The existence of such amplification necessitates stringent control except where it is biologically advantageous for the host. Fearon and Austen have proposed (5, 6) that certain activators of the alternative pathway possess the ability to protect the C3 convertase from regulation, thus allowing localized amplification. For such a mechanism to function, the activator must allow access of CS, Factor B, Factor D, and of the terminal complement proteins while circumventing regulation. The comparison of an activating surface (ER) with a nonactivating one (ES) in terms of the freedom of interaction of cell-bound C3b with various complement proteins has demonstrated (Table 1) that only the effectiveness of the regulatory proteins was restricted on ER relative to ES. In fact, the rabbit erythrocyte surface selectively restricts the binding of #I1H to C3b. Only secondarily is C3bINA function decreased due to its strong #l1h dependence. It has been reported (6) that preformed CS convertases on ER and ES have an identical intrinsic decay rate. That leaves restriction of,b1h control as the only difference thus far detected which accounts for stabilization of ER bound C3 convertase (Fig. 5). Restriction of #1H control and generation of ability to activate the alternative pathway by neuraminidase treatment of a nonactivator Treatment of ES with neuraminidase removed a large proportion of membrane-bound sialic acid. By implication, chemical groups were revealed that are normally concealed on the cell surface. Galactose residues may belong to these structures because sialic acid is usually linked to this carbohydrate. The possibility exists that certain protein or lipid constituents also were revealed by removal of sialic acid. It may be assumed that specific surface structures are responsible for the restriction of #1H accessibility to the surface-bound C3b. These structures, which may tentatively be referred to as,b1h antagonists, may function by direct interaction with bound C3b, reducing its affinity for j#1h, or by blocking of the /31H binding site of C3b. Alternatively, they may act by excluding,b1h selectively from the microenvironment of the cell surface. Whether the cell surface j#1h antagonist is also responsible for alternative pathway activation is not known and is presently under investigation. Findings related to these results were made by Koethe et al. (30), who demonstrated activation of the alternative pathway by neuraminidase-treated erythrocytes from patients with paroxysmal nocturnal hemoglobinuria and by normal human

5 2420 Immunology: Pangburn and Mfiller-Eberhard erythrocytes after treatment with both neuraminidase and glutathione. Since certain bacteria and viruses produce neuraminidase, host cell-surface alteration may occur during infections with resultant alternative pathway activation and complement-dependent tissue injury. This is publication no from the Research Institute of Scripps Clinic. This investigation was supported by U.S. Public Health Service Grants Al and HL M.K.P. is supported by Fellowship AI from the National Institute of Allergy and Infectious Diseases. H.J.M.-E. is the Cecil H. and Ida M. Green Investigator in Medical Research of the Research Institute of Scripps Clinic. 1. Mfiller-Eberhard, H. J. & Gotze, 0. (1972), J. Exp. Med. 135, Weiler, J. M., Daha, M. R., Austen, K. F. & Fearon, D. T. (1976) Proc. Nati. Acad. Scd. USA 73, Whaley, K. & Ruddy, S. (1976) J. Exp. Med. 144, Pangburn, M. K., Schreiber, R. D. & Muller-Eberhard, H. J. (1977) J. Exp. Med. 146, Fearon, D. T. & Austen, K. F. (1977) Proc. Nati. Acad. Sci. USA 74, Fearon, D. T. & Austen, K. F. (1977) J. Exp. Med. 146, Pangburn, M. K., Schreiber, R. D. & Muiller-Eberhard, H. J. (1978) J. Immunol. 120, 1791 (abstr.). 8. Tack, B. F. & Prahl, J. W. (1976) Biochemistry 15, Gotze, 0. & Muller-Eberhard, H. J. (1971) J. Exp. Med. 134, 90s-108s. 10. Gotze, 0. & Muller-Eberhard, H. J. (1974) J. Exp. Med. 139, David, G. S. & Reisfeld, R. A. (1974) Biochemistry 13, Bolton, A. E. & Hunter, W. M. (1973) Biochem. J. 133, Proc. Natl. Acad. Sci. USA 75 (1978) 13. Nisonoff, A., Wissler, F. C., Lipman, L. N. & Woernley, D. L. (1960) Arch. Biochem. Biophys. 89, Mayer, M. M. (1961) in Experimental Immunochemistry, eds. Kabat, E. A. & Mayer, M. M. (Charles C Thomas, Springfield, IL), pp Schreiber, R. D., Medicus, R. G., Gotze, 0. & Muiller-Eberhard, H. J. (1975) J. Exp. Med. 142, Warren, L. (1959) J. Biol. Chem. 234, Ruddy, S., Carlo, J. & Conrad, D. (1978) J. Immunol. 120,1794 (abstr.). 18. Medicus, R. G., G6tze, 0. & Mfiller-Eberhard, H. J. (1976) J. Exp. Med. 144, Medicus, R. G., Schreiber, R. D., Gotze, 0. & Muller-Eberhard, H. J. (1976) Proc. Natl. Acad. Sct. USA 73, Schreiber, R. D., Gotze, 0. & Mfiller-Eberhard, H. J. (1976) J. Exp. Med. 144, Fearon, D. T. & Austen, K. F. (1975) Proc. Nati. Acad. Sci. USA 72, Pillemer, L., Blum, L., Lepow, I. H., Ross, 0. A., Todd, E. W. & Wardlaw, A. C. (1954) Science 120, Wedgwood, R. J. (1960) Fed. Proc. Fed. Am. Soc. Exp. Biol. 19, 79 (abstr.). 24. Phillips, J. K., Snyderman, R. & Mergenhagen, S. E. (1972) J. Immunol. 109, Spiegelberg, H. L. & Gotze, 0. (1972) Fed. Proc. Fed. Am. Soc. Exp. Biol. 31, 655 (abstr.). 26. Joseph, B. S., Cooper, N. R. & Oldstone, M.B.A. (1975) J. Exp. Med. 141, Sandberg, A. L., Oliveira, B. & Osler, A. G. (1971) J. Immunol. 106, Lachmann, P. J. & Halbwachs, L. (1975) Clin. Exp. Immunol. 21, Fearon, D. T. (1978) J. Immunol. 120, 1772 (abstr.). 30. Koethe, S., Hause, L. & Rothwell, D. (1978) J. Immunol. 120, (abstr.).