CARL H. HAMMER, ANNE NICHOLSON*, AND MANFRED M. MAYER. Department of Microbiology, The Johns Hopkins University School of Medicine, ...
Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 5076-5080, December 1975
Immunology
On the mechanism of cytolysis by complement: Evidence on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte membranes (complement insertion into membrane bilayer/complement lesion in biomembrane/doughnut model of complement cytolysis)
CARL H. HAMMER, ANNE NICHOLSON*, AND MANFRED M. MAYER Department of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Communicated by Albert Lehninger, August 29,1975
ABSTRACT The doughnut hypothesis of cytolysis by complement [Mayer, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 2954-2958] describes an annular structure made up of C5b-9 (complement factors C5b, C6, C7, C8, and C9) which becomes inserted in the lipid bilayer of the cell membrane, thus creating a hole. We now present initial explorations of this hypothesis. EAC1-6 and EAC1-7 (sheep erythrocytes carrying rabbit antibody and complement factors Cl through C6 or Cl through C7, respectively), prepared with either 1251 C3 or 125I-C5 were incubated with trypsin and the release of bound 1251 was measured. In the case of 125I-C3, all of the radioactivity was released by trypsin from both intermediates. With 125I-C5, trypsin released all of the 125I from EAC1-6, but only 40-55% from EAC1-7. Possible reasons for resistance of the C5b subunit in EAC1-7 to tryptic digestion are discussed; in terms of the doughnut hypothesis it would be due to shielding by lipid molecules as a consequence of insertion into the lipid bilayer. In accord with this interpretation we have also found that C5b in EAC1-7, but not in EAC1-6, resists elution by 0.3 M NaCl. Similarly, we have found that 125I-C7 in EAC1-7 resists stripping by trypsin. Hence, we now propose the hypothesis that hydrophobic polypeptide chains from the C5b and the C7 subunits of C5b,6,7 complex become inserted in the phospholipid bilayer and that subsequent reactions with CS and C9 open a channel across the membrane.
the EAC1-7 stage. If so, '25I-C5b should be susceptible to stripping by trypsin or elution with 0.3 M NaCl at the EAC16, but not at the EAC1-7 stage. In addition to these experiments with 125I-C5b, we present studies of the effect of trypsinization on 125I-C7 in EAC1-7, and on 125I-C3 in EAC1-3, EACI-6, and EAC1-7. Preliminary reports have been presented (9, 10).
MATERIALS AND METHODS Solutions Buffer A++. A 5 X concentrated stock solution was prepared by dissolving 10.19 g of sodium veronal and 83.0 g of NaCl in 1500 ml of H20, adjusting to pH 7.3-7.4 with 1 N HC1, and making up to exactly 2 liters with H20. For use, 400 ml of the stock solution were diluted with H20 up to exactly 2 liters with incorporation of CaCl2 and MgCl2 to final concentrations of 0.15 mM and 1.0 mM, respectively, as well as 0.1% gelatin. Ionic strength = 0.147. Buffer B++. This was prepared by diluting 200 ml of the buffer A++ stock up to 2.0 liters with incorporation of glucose to a final concentration of 0.139 M, as well as CaCl2, MgCl2, and gelatin to the same concentrations as in buffer A++. Ionic strength = 0.074. Buffer C++. A 5 X concentrated stock solution was prepared by dissolving 10.19 g of sodium veronal and 24.57 g of NaCl in 1500 ml of H20, adjusting to pH 6.5 with 1 N HCl, and making up to exactly 2 liters with H20. For use, 400 ml of the stock solution were diluted with H20 up to exactly 2 liters with incorporation of glucose to a final concentration of 0.192 M, as well as CaCl2, MgCl2, and gelatin as in buffer A++. Ionic strength = 0.051. Buffer D++. Prepared like buffer C++, except that the pH was adjusted to 7.4. Guinea pig complement components C1, C2, and C9 were isolated as described in refs. 11, 12, and 13, respectively. For radioiodination, C3 and CS were prepared as described in refs. 14 and 15, respectively. For analytical purposes and for preparation of cell intermediates, CS was purchased from Cordis Corp., Miami, Fla. and CS was isolated by isoelectric precipitation, and chromatography on DEAE-cellulose DE-52 and CM-cellulose CM-52 (Reeve Angel and Co., Ltd., Clifton, N.J.). The product of this simpler procedure contained less than 4 units of C3, C6, C7, C8, or C9 per 10,000 units of C5, as judged from assays with at least 1000 units of C5. C6 and C7 were prepared by similar procedures. The products contained less than 1 unit of C3, C5, C8, or C9 in 50,000 units of C6 or C7. There was less than 1 unit of C7 in 10,000 units of C6, and less than 1 unit C6 in 10,000 units of C7. For radioiodination, C7 was
In a theoretical analysis of the mechanism of cytolysis by complement, one of us proposed the hypothesis that complement produces lesions in biomembranes by insertion of an annular structure composed of terminal complement components into the phospholipid bilayer (1). We now present radiochemical experiments in which the cell intermediates EAC1-6 and EAC1-7, carrying 125I-C5b, were treated with trypsin or 0.3 M NaCl and the extent of removal of 125I from the cells was measured. The choice of these cell intermediates was based on the fact that nascent CSb, 6, 7 complex (2-7), but not C5b, 6 (8), has the capacity to combine with plain erythrocytes (i.e., red cells not carrying antibody and complement components), and the resultant'hypothesis that insertion of the terminal complement components begins at Abbreviations: C, C1, C2, C3, etc. -C refers to complement, and the numbers indicate the components of the complement system. The letters "a" as in C2a, or "b" as in C4b, refer to fragments of the complement molecules. The bar in C1 or C4b,2a,3b indicates an enzymatically active component or complex. EAC1-3, EAC1-6, and EAC1-7 are shorthand designations for erythrocytes carrying antibody and components C1, C4, C2, and C3; C1, C4, C2, C3, C5, and C6; and C1, C4, C2, C3, C5, C6, and C7, respectively. SAC1-6 and SAC1-7 are shorthand designations for cell surface sites carrying antibody and the respective complement proteins; tma,c, the time at which SAC1,4b,2a per cell reach a maximum. Nomenclature of Bull. W.H.O. (1968) 39, 935-938. * Present address: Department of Medicine, Harvard Medical School, Robert B. Brigham and Beth Israel Hospitals, Boston, Mass. 02120.
5076
Immunology:
Hammer et al.
fractionated further on Sephadex G-200. For preparation of C8, the precipitate obtained at 2.0 M ammonium sulfate in the C9 isolation procedure was subjected to batch treatment on DE-52 and column chromatography on CM-52. The product had less than 1 unit of C.3 or C7 in 50,000 units of C8, and less than 1 unit of C5, C6, or C9 in 10,000 units of C8. lodination of C3, C5, and C7 with 125I The peroxidase (B grade, Calbiochem, San Diego, Calif.) procedure of Marchalonis was used (16). Na'25I (>350 mCi/ ml) was purchased from New England Nuclear, Boston, Mass. The iodinated complement components were isolated by chromatography on Bio-Gel P-60 and Sephadex G-200. These products contained between 0.06 and 1.1 atom 1251 per molecule of complement protein. Todination did not cause significant loss of activity. Cell intermediates General Procedures. EACi,4b, prepared according to ref. 17, were treated with the complement components, including the appropriate radioiodinated component, required for preparation of the desired intermediate, as described in detail in the following sections. After incubation, each of the preparations was cooled, a sample was removed for input count, and the cells in the sample were washed three times with ice-cold buffer and transferred to a clean tube for counting bound 125I. The preparation of each radioiodinated cell intermediate was accompanied by a control in which EAC1,4b of the same cell lot were treated with the radiolabeled complement component, but not the other components, under exactly the same experimental conditions as those used in the experiments proper. After measurement of the input count, the cells were washed three times, transferred to a clean test tube, and counted to measure nonspecifically bound 125I. Small variations in 1251 input of the controls, relative to the respective cell intermediates, were corrected proportionately. The nonspecifically bound 125I so obtained was subtracted from the bound iodine of each of the intermediates, yielding specifically bound iodine. EAC1,4b,2a,125I-3b. EAC1,4b (1.5 X 108/ml) were incubated for 1.5 times the tmax at 30' with an equal volume of buffer A++ containing 250 units/ml of C2 and 23 units/ml of '25I-C3 (3700 cpm/unit). Nonspecifically and specifically bound '25I-C3 were 1700 and 10,600 cpm/1.5 X 108 cells, respectively. EAC1,4b,2a,Z 5I-3b,5b,6 and EAC1,4b,2a,'2I-3b,5b,6/7. EAC1,4b (3.0 X 108/ml) were incubated for 13.5 min at 30° with an equal volume of buffer B++ containing 500 units/ ml of C2 and 180 units/ml of 125I-CS (35,000 cpm/unit). The mixture was cooled, diluted to 1.5 X 108 cells per ml, and a sample was taken for counting radioactivity input. The reaction mixture was centrifuged and the cells were washed once with ice-cold buffer C++. They were dispersed in buffer C++ and the suspension was divided into two equal portions each of which was then adjusted to 12 X 108 cells per ml. One of the portions was incubated for 22 min at 30° with an equal volume of buffer C++ containing 2000 units/ 'ml of C5 and 6000 units/ml of C6. The other portion was incubated with the same quantities of C5 and C6, plus 12,000 units/ml of C7. In the case of EAC1-6, nonspecifically and specifically bound IZI-C3 were 14,700 and 47,900 cpm/1.5 X 108 cells, respectively, and for EAC1-7, 14,700 and 47,200 cpm/1.5 X 108 cells, respectively.
Proc. Nat. Acad. Sci. USA 72 (1975)
5077
EACl,4b,2a.3b.125I-b,6 and EAC1,4b,2a,3b,25I-5b,6,7. Two lots of EACI,4b,2a,3b were made by incubating EACl,4b (3 or 6 X 108 cells per ml, lot I or II, respectively) for 13.5 or 11 min, respectively, at 30° with an equal volume of buffer B++ containing 300 units/ml each of C2 and C3 in the case of lot I, and 340 units/ml each of C2 and CS for lot II. The mixtures were cooled and the cells were washed once with ice-cold buffer B++ or C++ (lot I or II, respectively). After adjustment to 12 X 108 cells per ml, each of the two suspensions was divided into two equal portions. The two portions of lot I were converted to EAC1-6 and EAC1-7, respectively, by incubation for 22 min at 300 with an equal volume of buffer B++ containing 140 units/ml of 125I-C5 (4600 cpm/unit) and 2400 units/ml of C6, for the first portion, or with the same concentrations of these components, plus 4800 units/ml of C7, for the second portion. The two portions of lot II were incubated under the same conditions with an equal volume of buffer C++ containing 840 units/ml of 125I-C5 (2500 cpm/unit) and 8000 units/ml of C6, for the first portion, or the same concentrations of these components, plus 24,000 units/ml of C7, for the second portion. (C7 was used in excess to ensure conversion of all SAC1-6 to SAC1-7.) A third lot of each of these intermediates was made 1 month later in the same way as lot I; during this interval the radioactivity of 125I had decayed to 2900 cpm/unit. Nonspecifically and specifically bound '25I-C5 on EAC1-6 for lots I, II, and III were: 3900 and 9700; 600 and 8800; 600 and 3000 cpm/1.5 X 108 cells, respectively. For EAC1-7, the corresponding values were: 3900 and 11,600; 600 and 8200; 600 and 4400 cpm/1.5 X 108 cells, respectively. EAC1,4b,2a,3b,5b,6,'25I-7. EAC1,4b (6 X 108/ml) were incubated for 9 min at 300 with an equal volume of buffer D++ containing 400 units/ml of C2 and 632 units/ml of C3. The mixture was cooled, the cells were washed once with ice-cold buffer C++, and the sedimented cells were suspended smoothly to 6 X 108 cells/ml in buffer C++ containing 4000 units/ml of C5, 6000 units/ml of C6 and 1860 units/ml of 125I-C7 (9400 cpm/unit), followed by incubation for 35 min at 300. The cells were then converted to ghosts by suspension in distilled H20 to a concentration of 1.5 X 108/ml. In the washings required prior to determination of bound 125i, as well as in the subsequent experimental manipulations, the ghosts were centrifuged at 48,000 X g for 15 min to effect complete sedimentation. The EAC1,4b control used for determination of nonspecific binding was also converted to ghosts and treated in the same way. Nonspecifically and specifically bound 125I-C7 were 28,500 and 25,500 cpm/1.5 X 108 cell ghosts, respectively. Trypsinization. The various cell intermediates (4.5 X 107/ml) were treated with 0.1% trypsin (Calbiochem) at pH 7.4 and ionic strength 0.074. At the end of the digestion period, presence of trypsin was checked. During the trypsinization, samples were collected in ice-cold buffer containing a 3.5-fold excess of soybean trypsin inhibitor (Worthington). Cell-bound 1251 was measured after washing the cells three times. RESULTS Susceptibility of '251-C3b to Tryptic Removal from EAC1-3, EAC1-6, and EAC1-7. Fig. 1 shows that trypsin strips '25I-C3b almost completely from these cell intermediates. Susceptibility of 125I-C5b to Tryptic Removal from EAC1-6 and EAC1-7. Fig. 2 shows that trypsin strips 125I-
WO_~ EACI-7 ~ ~ ~ EACI-6
Proc. Nat. Acad. Sci. USA 72 (1975)
Immunology: Hammer et al.
5078
100
_
in
I
BUFFER, 27°c-
10,000[
en80 LEACI -7 in BUFFER, 27°C -
-J wJ
I
12,000
I RADIOLABEL on C7I
EACI-3 in BUFFER, 370C
0
z
60l
0
RADIOLABEL On
O0 \ F 20 z
20
"' 8,000 w
)i
0 z 0
C3|
,- 6,000 N
SPECIFIC
1251 on EACI-7 In TRYPSIN
mu 0
a0
4,000 I.
EACI-7 in TRYPSIN,270C EACI-6 in
EACI-3 in TRYPSIN, 37 0C
TRYPSIN,270C
80 40 60 MINUTES FIG. 1. Kinetics of tryptic removal of 1251 from EAC1-3 (-), EAC1-6 (0) and EAC1- 7 (-) carrying 125I-C3b. Buffer controls for each of these intermediates are shown by corresponding open symbols. The sudden initial release of 1251 in the controls was due to temperature and ionic strength shift. 20
C5b completely from EAC1-6, but not from EAC1-7. Susceptibility of 1251-C5b to Elution from EAC1-6 or EAC1-7 with 0.3 M NaCi. After washing twice in the cold, 1.5 X 108 cells of each type were suspended in 2.0 ml of buffer A++ (adjusted with NaCl to ionic strength 0.3) and incubated at 270 for 75 min. After centrifugal removal of cells and ghosts, the fluids from the EAC1-6 and EAC1-7 were found to contain, respectively, 61% and 14% of the 125I that was cell-bound prior to the elution. Elution with 0.15 M NaCl released 29% and 10% of the cell-bound '25I from EAC1-6 and EACL-7, respectively. EAC1-6 and EAC1-7
1I)
in BUFFER
-J~~~~~~~~~~~~~~~~~~~~~~.
t80
ACI 6iB;FER
0 ~~
In
~
~
~
~
~
0
LI-0
z
9~~~~~~.EC-
20
orEA17 - *
40
60
aryngl5ICb.BffrACI-6
in TRYSIN
8 in TRYPSIN
these intermediates are shown by the corresponding open symbols. Cell lots I are shown by hexagons and triangles; cell lots II by squares and circles.
2,000 NON-SPECIFIC 1251 on EACI-4 in TRYPSIN l
l
40 60 80 MINUTES FIG. 3. Kinetics of tryptic removal of 1251 from erythrocyte membranes in the state EAC1-7 carrying 125I-C7. This experiment was done with cell membranes (ghosts) prepared by water lysis, instead of intact erythrocytes. Since the extent of nonspecific uptake of '251-C7 on EAC1,4b was unusually high, these cells (in the form of cell membranes) were also subjected to trypsinization, as shown (3). The curve showing the stripping of 125I from radiolabeled EAC1-7 (0) represents data that have been corrected by subtracting the nonspecifically bound 125I from the total radioactivity on the cells. Accordingly, the curve showing stripping of EAC1-7 represents specific 1251 remaining on the cells. 20
that were treated with buffer of ionic strength 0.074 released 15% and 8% of the cell-bound 1251, respectively. Susceptibility of 1251-C7 to Tryptic Removal from EACI7. Fig. 3 shows that only part of the C7 was removed from the cell membranes by trypsin. Since the 1251-C7 showed unusually high nonspecific binding on EAC1,4b, these cells (in the form of membranes) were also subjected to trypsinization. As shown in Fig. 3, the nonspecifically bound 125I was removed rapidly and virtually completely. The same results were obtained in another experiment in which intact erythrocytes, rather than membranes, were used.
DISCUSSION The present experiments are based on the working hypothesis that insertion of the terminal complement components into the phospholipid bilayer of erythrocytes starts at the EAC1-7 stage. This concept was derived from the wellknown fact that nascent C5b,6,7 complex (2-7), but not C5b,6 (8), has the ability to combine with plain erythrocytes. The results shown in Fig. 2 are in accord with our working hypothesis, since trypsin removed all '25I-C5b from EAC1-6, but not from EAC1-7. Similarly, Fig. 3 shows that 1251-C7 in EAC1-7 was partially resistant to tryptic stripping. The salt elution experiment, in which 0.3 M NaCl solution released 61% of the cell-bound 125I from EACL-6 and only 14% from EAC1-7, is also in accord with the insertion hypothesis. Possible Mode of Insertion. Useful clues bearing on this question may be derived from studies of mellitin (18), a polypeptide in bee venom, or cytochrome b5 (19). Both of these substances have the capacity to become inserted in
Immunology:
Hammer et al. C5/67dC56789d
C56 -C56
c_ C567
C8 + C9
FIG. 4. Schematic diagram of the insertion of the C5b,6,7 complex into the cell membrane and reaction with C8 and C9 to form a transmembrane channel. As shown, the C5b,6 complex dissociates reversibly from the C4b,2a,3b enzyme on which it was generated. On reaction with C7, the C5b,6,7 complex is formed which has a half-life of less than 0.1 sec at 37°. If it does not become inserted in the phospholipid bilayer during its brief existence, it decays to an inactive form designated C5b,6,7d. The superscripts d indicate that the cytolytic reactivity has decayed (all letters designating fragments are omitted from this diagram, for simplicity), C8 and C9 may react either with the inserted C5b,6,7 to form a transmembrane channel or with C5b,6,7d to yield an inactive derivative of the cytolytic attack element.
phospholipid bilayers by virtue of the fact that their polypeptide chains contain domains of hydrophobic amino acids. On admixture of mellitin or cytochrome b5 with liposomes, their hydrophobic peptide chains enter into the interior hydrocarbon moiety of the phospholipid bilayer. By analogy, it appears possible that C5b and C7 have hydrophobic polypeptide chains which become exposed when C5b,6 reacts with C7 to form the C5b,6,7 complex. Such hydrophobic polypeptide chains would tend to associate with other hydrophobic molecules, rather than with water. Hence, they would have the capacity to enter the hydrocarbon environment of a phospholipid bilayer. In this context it should be recalled also that the activated state of nascent CSb,6,7 during which it is able to combine with plain erythrocytes has a half-life of less than 0.1 sec (4). It is not known how and why nascent C5b,6,7 loses its reactivity with erythrocytes so rapidly, but it appears possible that the decay may be due to a conformational change of the C5b,6,7 complex which buries the hypothetical hydrophobic peptide chains in its interior and thus removes them from the aqueous environment. Thus, we regard nascent C5b,6,7 as an unstable molecule which can attain a stable configuration either by insertion of hydrophobic chains into a phospholipid bilayer or by a conformational change which buries them in the interior of the complex (compare Fig. 4). Sequential Assembly of the Cytolytic Attack Element. The hypothesis that CSb, 6, 7 becomes inserted in the phospholipid moiety of biomembranes leads to a modification of current ideas on the assembly of the cytolytic attack element comprising C5,6,7,8,9 (20-22). As shown schematically in Fig. 4, the C5b,6 complex has the capacity to dissociate reversibly from the C4b,2a,3b enzyme on which it is generated. On reaction of C5b,6 with C7, the trimolecular complex C5b,6,7 is formed. Since the half-life of C5b,6,7 is less than 0.1 sec. it is expected that some of these complexes decay to the inactive form-C5b,6,7d before they get a chance at insertion. Presumably, the efficiency of insertion will depend on the availability of phospholipid bilayer close to the site of generation of CSb,6,7. The scheme in Fig. 4 also shows that C8 and C9 can react either with inserted C5b,6,7 or with the inactive derivative C5b,6,7d. The latter pathway yields the inactive cytolytic attack element C5b,6,7,8,9d which has been studied intensively by others (21, 22). While the present experiments are restricted to the question of partial insertion of C5b and C7 into biomembranes, it is interesting to speculate whether C8 and C9 also become inserted. A priori it seems unlikely that insertion of two
Proc. Nat. Acad. Sci. USA 72 (1975)
5079
polypeptide chains, one from C5b and another from C7, would suffice for formation of a channel across the phospholipid bilayer and, therefore, we believe it likely that CS and/ or C9 also become inserted. It is noteworthy that cells that have reacted up to and including C8 leak slowly, indicating the presence of a small hole and that reaction with C9 greatly increases the rate of leakage (23). Presumably, this means that C9 enlarges the hole, perhaps by becoming inserted in such a way that a wedge-like action creates an open channel across the cell membrane. Alternative Interpretations. Our interpretation of the present experiments is based on the concept that resistance to trypsin is due to shielding by membrane phospholipids. However, as an alternative interpretation, the possibility needs to be considered that C7 may shield CS, thus protecting it from tryptic attack. We have addressed this question in several different experiments. The first of these is the experiment in Fig. 3, which shows that about one-half of the 125I-C7 was stripped from EAC1-7 by trypsin, indicating that C7 is at least partly susceptible to proteolysis by this enzyme. The second argument against shielding by C7 derives from an experiment which showed that the hemolytic activity of C7 is destroyed by trypsin. The third argument is based on an experiment in which tryptic digestion of soluble C5b,6,7 complex containing 125I-C5b produced breakdown of the C5b subunit to the extent that most of the radioactivity was associated with material that sedimented more slowly than C5b on ultracentrifugation in a sucrose density gradient. Conversely, this observation could also serve as an argument against the possibility that the C5b subunit in C5b,6,7 shields the C7 subunit against tryptic attack. Another alternative interpretation can be derived from the recent work of Nilsson who showed that human C5 comprises two chains, a and ,B, 140,000 and 80,000 daltons, respectively, which are linked by one or more disulfide bonds (24). On tryptic digestion of native C5, the a chain was degraded to several small fragments, but the 13 chain remained intact, as judged by electrophoresis of the reduced and alkylated material in the presence of sodium dodecyl sulfate. Assuming that guinea pig C5 has properties that are similar to those of human C5, the hypothesis might be constructed from these observations that the C5b subunit in EAC1-6 is bound via the a chain, whereas in EAC1-7 the C5b subunit is bound via bonds involving the a and ,B chain. If so, it would be expected that C5b would be partially resistant to tryptic stripping in EAC1-7, but not in EAC1-6. It is evident from these alternative interpretations that the present observations, though compatible with the insertion hypothesis, do not represent decisive support. We regard the insertion concept as a working hypothesis which needs to be explored by a variety of experimental approaches. The present elution and stripping experiments are our first attempt in this direction. We thank Ms. Yvonne I. Fisher for her excellent technical assistance. This work was supported in part by grants from the National Science Foundation no. GB38628 and U.S. Public Health Service no. 5R01 AI-02566-16. C.H.H. was supported by a Postdoctoral Fellowship from the Andrew W. Mellon Foundation. A.N. was supported by the Stetler Research Fund for Women Physicians.
1. Mayer, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 29542958. 2. Thompson, R. A. & Lachmann, P. J. 629-641.
(1970) J. Exp. Med. 131,
5080
Immunology: Hammer et al.
3. Lachmann, P. J. & Thompson, R. A. (1970) J. Exp. Med. 131, 643-657. 4. Gotze, 0. & Muller-Eberhard, H. J. (1970) J. Exp. Med. 132, 898-915. 5. Goldman, J. N., Ruddy, S. & Austen, K. F. (1972) J. Immunol.
109,353-359. 6. Goldman, J. N. (1974) Transplant. Proc. 6,21-25. 7. McLeod, B., Baker, P. & Gewurz, H. (1974) Immunology 26, 1145-1157. 8. Goldlust, M. B., Shin, H. S., Hammer, C. H. & Mayer, M. M. (1974) J. Immunol. 113,998-1007. 9. Mayer, M. M. (1974) in Progress in Immunology II, eds. Brent, L. & Holborow, J. (North-Holland Publishing Co., Amsterdam), Vol. I, p. 301. (Proceedings of the Second Interna-
tional Congress of Immunology, Brighton, U.K.) 10. Hammer, C. H., Nicholson, A. & Mayer, M. M. (1975) Fed. Proc. 34, 965. 11. Nelson, R. A., Jr., Jensen, J., Gigli, I. & Tamura, N. (1966) Immunochemistry 3,111-135. 12. Mayer, M. M., Miller, J. A. & Shin, H. S. (1970) J. Immunol. 105,327-346.
Proc. Nat. Acad. Sci. USA 72 (1975) 13. Tamura, N. & Shimada, A. (1971) Immunology 20,415-425. 14. Shin, H. S. & Mayer, M. M. (1968) Biochemistry 7, 29912995. 15. Cook, C. T., Shin, H. S., Mayer, M. M. & Laudenslayer, K. A. (1971) J. Immunol. 106,467-472. 16. Marchalonis, J. J. (1969) Biochem. J. 113,299-305. 17. Borsos, T. & Rapp, H. J. (1967) J. Immunol. 99,263-268. 18. Williams, L. C. & Bell, R. M. (1972) Biochim. Biophys. Acta 288,255-262. 19. Robinson, N. C. & Tanford, C. (1975) Biochemistry 14, 369378. 20. Kolb, W. P., Haxby, J. A., Arroyave, C. M. & Mfiller-Eberhard, H. J. (1972) J. Exp. Med. 135,549-566. 21. Kolb, W. P. & Muller-Eberhard, H. J. (1973) J. Exp. Med. 138,438-451. 22. Kolb, W. P. & Mfiller-Eberhard, H. J. (1975) J. Exp. Med. 141,724-735. 23. Stolfi, R. L. (1968) J. Immunol. 100, 46-54. 24. Nilsson, U. R., Mandle, R. J., Jr. & McConnell-Mapes, J. A. (1975) J. Immunol. 114,815-822.
