Immunologically mediated membrane damage: The mechanism of complement action and the similarity of lymphocyte-mediated cytotoxicity

IMMUNOLOGICALLY MEDIATED MEMBRANE DAMAGE: THE MECHANISM OF COMPLEMENT ACTION AND THE SIMILARITY OF LYMPHOCYTE-MEDIATED CYTOTOXICITY MANFRED

M. MAYER,

CARL H. HAMMER, MOON L. SHIN

DAVID

Department of Microbiology. Johns Hopkins Baltimore.

Unlcersity. MD 21205. U.S..i\.

The destruction of cells by immune mechanisms proceeds via humoral routes, i.e. by soluble factors, as well as by cellular pathways. Among the former, the most prominent and best known is the complement system. As a consequence of investigations spanning about 100 years. it has been learned that the complement system is made up of 14 proteins, not counting inhibitors and regulatory enzymes. The system can be divided into two pathways of activation and a common cytolytic pathway, as shown in Fig. 1. Five of the proteins (Clq, Clr. Cls. C4* and C2) belong to the classical pathway of complement activation which is usually initiated specifically by antigen-antibody complexes. Three of the complement protelna (B. D and P) belong to the alternative pathway which may be activated specifically or non-specifically. Another complement protein, C3, functions in the classical and the alternative pathway. The other five proteins (CS, C6, C7, C8 and C9) belong to the final common pathway which mediates the cytolytic function of complement by damaging the membranes of targets such as bacteria, protozoa, fungi and cells of higher organisms. In addition to the cytotoxic action, activation of the complement system, either via the classical or the alternative pathway, generates fragments of C3 and C5, by enzymatic cleavage, which mediate a variety of biological activities, notably opsonization (C3b), chemotaxis (C5a), release of

C7 or C8-deficient human scum: E. erythrocytes: A. antierythrocyte antIbody; EACH-6 or EACI-7. erythrocytes carrying antibody and C proteins Cl through C6 or C7. respectively: LACI-6. liposomes carrying antibody and C proteins Cl through C6: MCSt+X and MC5b-9. membranes carrying C5k4 or CSb-9. respectively: BLM. black lipld membranes: CVF. cobra venom factor. an analog of C3. which activates C via the alternative pathuay: PC. phosphatidyl choline: GMO. glycerol monooleate: SDS. sodium dodecyl sulfate; ADCC. antibody-dependent. cellmediated cytotoxicity

School

and

of Mcdiclne.

allergic effecters. such as histamine (C3a and CSa) and activation of B lymphocytes and macrophages (C3b). For details of the activation pathways, the reader is referred to reviews by Ruddy et al. ( 1972), Vogt ( 1974). Miller-Eberhard (1975) and Mayer (1978). The first part of this article describes recent studies of the mechanism of attack by C5b-9 on the lipid bilayer of cell membranes. This attack produces two distinct effects; one of these is the formation of transmembrane channels by insertion of complement peptides. The other involves the removal of phospholipid from the bilayer which is interpreted to be due to abortive insertion of hydrophobic peptides from the terminal complement proteins. Through biochemical and physico-chemical studies of these effects, the way has now been opened to a better understanding of the mechanism of the cytolytic action of complement. Recently, some progress has also been made in elucidating the mechanism of cytotoxic reactions mediated by lymphocytes. Comparison of complement-mediated and lymphocyte-mediated cytolytic processes has revealed several characteristic features in which they resemble one another (Mayer, 1977). Consequently. efforts are now under way to determine whether their mechanisms are indeed similar. These initial explorations will be described briefly in the second part of this article. PART THE

*Abbreviations used: C. complement: GPS. guinea pig berum: C4D-GPS. C4-deficient GPS: C7D-HS or CXD-HS.

W. MICHAELS

CI’TOTOXIC

A<‘TION

I OF COI\lPI.EVENT

As outlined schematically in Fig. I. the final common pathway. which mediates the membrane attack, is initiated when complement protein C5 is cleaved into C5a (ca. 11.000 daltons) and C5b (cu. 200,000 daltons). either by the classical pathway enzyme C4b.2a.3b or the alternative pathway enzyme P.C3b,.Bb. The C5b fragment has thecapacity to bind C6, C7 and C8 successively, thus forming the C5b-8 complex. In turn, the C8 sub-unit of this complex binds C9, resulting in formation of C5b-9 (Kolb et al., 1973). It is important to note that these binding studies of the terminal reaction sequence were performed in

IMembrane

tvVXib-9~tvlC5b-8-C8MC5b,6,7

an aqueous tTledlLlm and. tkrckm. the molrcLllar arrangement thus formed is probably not the same as that which is assembled in the hydrophobic milieu ofa lipid bilayer. The concept that the lipid bilayer of cell membranes is the target of complement attack has its origins in studies by Kinsky and associates (Haxb) 01 t/l.. 1968: Kinsky. 1972). These workers demonstrated that phospholipld hposomes release trapped water-soluble markers when trcatcd with complement. On the basis of their experiments and in light ol‘thc one-hit theory of’ cytolysis by complement (Mayer. I961 ). the hypothesis wah proposed that the terminal complement proteins become inserted and a\semblcd in the lipid bllaycr so as to form an annular structure. like a doughnut. which penctratcs through the bilayer and thus provides u stable tram-mcmbranc channel (Mayer. 1973). Such a channel may well resemble those f’ormed by antibiotic peptides like gramicidln (Hladky & Haydon, 1972) or alamethicln (Gordon & Haydon, 1972). Cell membranes damaged by complement display behavior that indicates the presence of channels whvzh are large enough to permit exchange of \mull molecules and ions between the cytoplasm of the cell and the extracellular milieu, but too small to permit release of macromolecular cytoplasmic constituents like hemoglobin. Due to the Donnan effect. water enters through such channels causing the cell to swell and burst (Green et u/.. 1959, 1959~). Thus. lysis bq complement is thought to proceed in two stages. In the first stage. trans.membrane channels are fbrmed. and in the second stage, the cells swell and burst. The present discussion of’ cytolysis by complement is concerned only with the first stage. i.e. formation 01‘ from the late-acting tram-membrane channels complement proteins C5b. C‘6, C7. C‘X and c‘9.

Bilayer

t

With the exception of the studies of the ‘reactive Iysis system’ (Thompson & Lachmann. 1970; Lachmann & Thompson, 1970) to be described shortly, information on the terminal reaction sequence is derived from experiments in which the late complement proteins were allowed to react with one another in an aqueous milieu. On the other hand, the trans-membrane channel hypothesis describes a process in which the terminal complement proteins react with one another in close association with ;I bilayer membrane. Part of’ the process ,is believed to take place within the hydrocarbon interior 01‘ the mcmbranc. In such a membrane-azsociated process it is likely that the inserted complement peptides interact with one another during channel formation in \uch :I way that their hydrophobic domains tace out toward the hydrophobic parts 01‘the mcmbranc Ilpids. while their hydrophilic regions fhrm a central core on the internal face ofthccomplex. Thisconfiguration would satisf’y the thermodynamic requirement of minimizing the area of hydrophobic and hydrophilic contact. It would also be in concordance with a relevant precedent. the known structure of the channel Ihrmer gramicidin (Urry. I97 1 ). On the other hand. when the terminal complcmcnt proteins interact with one another in the aqueous phase it is likely that the hydrophobic domains on different peptides will face toward one another. an arrangement which would produce a collapsed structure rather than B channel. Indeed. the cytolytic effect ofCSl+) is expressed only when this complex is Formed in close association with a membrane: C5t~9 formed in the aqueous phase lacks cytolytic activit). As pointed out 111the section on electron microscop). this functional difference ia rellected in the morphology of c‘5&9. Only when the complex IS I’OI-mcd in aszociatlon v.ith ;I membrane. docx it ha\e

Immunologically

Mediated Membrane

the appearance of a channel (cf. Figs. 14-16). There is no direct structural information on hydrophobic domains of the late complement proteins since sequence data are not available at present. Meaningful progress in elucidating the reactions of the terminal complement proteins with biological and artificial membranes was initiated by the discovery that erythrocytes can bind CSb.6.7 complex that has been freshly generated in thrir presenw on incubation of C5b.6 with C7. The resulting cell intermediate EC5b,6.7 can be lysed by C8 and CY (Thompson & Lachmann. 1970; Lachmann & Thompson, 1970). Parallel observations have also been made with bacteria (Goldman & Austen. 1974) and with liposomes (Lachmann PI ul.. 1970). Thus. sequential application ofC5b,6 + C7 + C8 + C9. the so-called reactive lysis system, will damage membranes nonspecifically. In several ofour studies, to be described in later sections. the reactive lysis system was used. Since it does not require antibody and the early complement proteins. this system greatly facilitates the execution and interpretation of experiments on the mechanism of membrane damage. Before proceeding. it is necessary to explain why the C5b,6 complex is used for initiation of the reactive lysis system. Specitically. it is necessary to consider the role of the C6 sub-unit of this complex. Since the C5b sub-unit carries the receptors which bind C7 and C8. C6 does not appear to be involved directly in the aggregation process. Nonetheless, C6 serves an essential capacity. The nature of its function emerged from studies by H. Shin ct (11.(1971) showing that C5b decays to a cytolytically unreactive form immediately after its release from the generating enzyme (C4b,Za.3b) into the fluid phase. The mechanism of this decay is unknown. However. if C5b combines with C6 while it is still associated with the generating capacity becomes stabilized enzyme. its cytolytic (Goldlust C’Iul.. lY74). As a consequence, when CSb.6 complex dissociates from the generating enzyme and diffuses into the fluid phase. its C5b sub-unit remains In the ‘activated’ state, i.e. capable of initiating the cytolytic process. Hence. C5b.6 may be regarded as a stable form of ‘activated’ C5b. A convenient method for preparation of CSb.6 involves removal of C7 from human serum by chromatographic fractionation and subsequent incubation of the C7-dcticient serum with zymosan particles that have been previously subjected to brief incubation with whole human serum_____~ so as to generate the alternative pathway enzyme P.C3b,Bb on their surface. The resulting CSb.6 can be purified by salt precipitation and chromatography (Yamamoto & Gewurz. 1978). The product can be stored for a long time without loss of activity.

Our studies on the assembly of complement complexes on and within lipid bilayers and the resultant membrane damage will be presented in five sections. The first section deals with the insertion of peptide chains from the terminal complement proteins into the bilayer as studied by enzymatic stripping and clution experiments (Hammer c’f ul.. 1975, 1977). The second section concerns ot the question

Xl5

Damage

hydrophobicity of these peptides. which is a prerequisite for their insertion into the hydrocarbon interior of a bilayer. This issue has been investigated by studying the transfer of phospholipid from bilayer membranes to the fluid phase during interaction with the complement proteins (M. Shin car (I/.. 1977). The third section deals with the problem of assembly of the complement peptides in the bilaycr and the properties of the resultant channels, as revealed by electrical conductance measurements across planar lipid bilayers during treatment with the terminal complement proteins (Michaels 1’1(11.. 1976; Mayer, 1978). The fourth section addresses the relation between the structure of membrane lipid!, and the susceptibility to damage by complement (M. Shin rt (11.. 1978). In the final section we will present some recent electron micrographs prepared in other laboratories which show the channel structure.

If complement components were bound on the surface ofthe erythrocyte membrane without insertion into the phospholipid bilayer, appropriate proteolytic treatment would be likely to remove them. On the other hand. if hydrophobic polypeptide chains from the components were to become inserted in the phospholipid bilayer. it would not be possible to strip off all of the complement protein by proteolytic digestion since the inserted polypeptide chains would be shielded by lipid. This general approach is based on studies of integral and peripheral membrane proteins (Juliano, 1973). It should be noted that failure to strip does not necessarily indicate insertion; conversely. successful stripping definitely rules out insertion, provided the lipid bilayer is not destroyed by the enzymatic treatment. The following sections describe

EACI-7

mm

8” BUFFER,

4 EACI-3

in BUFFER.

37’C

: g Z

60.

& 5 40-

RADIOLABEL on C3

27°C

Xl6

MANFRED

M

VIAYER

(‘I t/l

a series of enzymatic stripping experiments with compared with respect to the susceptibility of the celldifferent radioiodinated complement proteins and bound C5b to trypsin. The results, shown in the upper erythrocyte intermediates. left panel of Fig. 3. indicate that practically all of the Enzymatic stripping experiments ti,ifh radioiodinated radioiodine associated with C5 was removed from c’3. Since C3 is not a part of the cytolytic attack subEACI-6. but only about one-half could be stripped system it was reasoned that this complement from EACI-7. This result is in accord with our component does not become inserted in the working hypothesis since it indicates that part of the C5 molecule became resistant to enzymatic digestion. hydrocarbon moiety of the phospholipid bilayer. If so, it should be susceptible to enzymatic attack. In order presumably by insertion. when the intermediate EACI-7 was formed (Hammer rt ul.. 1975). to examine this question. the intermediates EACI-3. As shown in the other three panels of Fig. 3. tryptic EACI-6 and EACI-7 were prepared with radiostrippingexperimcntswithintermediatescarryingradioiodinated C3. (The choice of these intermediates will Iabelled C7 or C‘X or C9 also indicated substantial be discussed in the next paragraph,) In each case, as resistance to enzymatic attack (Hammer (‘t ul.. 1975. shown in Fig. 2, it was found that treatment with 1977). In the case of radiolabelled Cc). after treatment trypsin removed practically all the radioactivity from with protease, the EACI-9 membranes were treated the cells. indicating that the iodinated part of C3 was with sodium dodecyl sulfate and the solubilized not inserted in the lipid moiety (Hammer r’l al.. 1975). C9 Enzymufic stripping r.\-/~l~rir,lrrlr.,with rrr~lioro~lirlutc~~~material was found to contain a radioiodinated fragment of 18,000 daltons. which corresponds C5. C7. C8 or C9. The experiment with radioiodinated approximately to one-quarter of the C9 molecule C5 was designed on the basis of the assumption that (78.000 daltons). Essentially identical results were insertion of peptide chains from terminal complement obtained with trypsin and chymotrypsin. proteins starts when C7 reacts with EACI-6. (For a C’ommrnt. The main limitation of the stripping recent modification of this assumption refer to a later experiments arises from the possibility that a cellSection on studies of ion tlow across planar lipid bound complement protein might be resistant to bilayers.) This concept was derived from the proteolysis due to shielding by another complement demonstration that nascent C5b,6,7 combines with component or by lipid that was removed from the erythrocytes (Thompson & Lachmann, 1970; bilayer on activation of C5b-9, as described below. In Lachmann & Thompson, 1970). In light of the parallel this context, it should be noted that the 18,000 dalton experiments with liposomes (vidc supru Lachmann PI peptide that resisted proteolysis greatly exceeds the al.. 1970) it was assumed that the binding of C5b.6,7 length required to span a bilayer: actually. a peptide of by erythrocytes involves the lipid bilayer of the cell about 2000 daltons would suffice. In view of this, there membrane. While it seemed possible that C5b,6.7 is the possibility that only part of the 18,000 dalton combines only with the head groups of the peptide was inserted in the bilayer and that the phospholipids, we preferred the idea that hydrophobic remainder may have resisted proteolysis due to lack of polypeptide chains from nascent C5b,6.7 penetrate susceptible bonds or because of shielding or due to into the hydrocarbon interior of the phospholipid penetration across the membrane into the cytoplasmic bilayer because the resultant increase in bond strength space (Rauterberg, 1978). would account for the fact that the intermediate EAC l-7. unlike its precursors EAC l-5 and EAC l-6. Elution e.xperirnent.s. It is known that the C‘Sb.6 complex can be eluted readily from EACI-6 by is stable (Inoue & Nelson, 1965. 1966). the intermediates EACI-6 and elevation of temperature or ionic strength (Goldlust (J/ Accordingly. ~1.. 1974). Accordingly. experiments were performed EACI-7. prepared with radiolabelled C5, were

EACI-7

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5 loo*,

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RADIOLABEL

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20

-.

40

60

80

20

40

60

80

MINUTES (0) or EACI-7 (0) carrying ““I-C‘5b. Fig. 3. Kmetics of tryptic removal of lzsl from EACI-6 (A). or from EACI-Y carrying EACI-7 carrying ““I-C7 (Cl). or from EACl-X carrymp “‘I-C8 (v). (Adapted from Hammer 6’1 rd.. 1975. 1977.)

or from “‘I-C9

Immunologically rahlc

I. Elutlon of [‘?‘I] C’Sb from EAC‘I-6

[“ill c‘5 Input. total count\ “;I in IlLlId phaw. “,, ‘.‘
l’rom Hammer

Mediated

or EACI-7

EAC l-6

EAC l-7

3327 61 37

4100 I4 85

ml,,

<‘I
to compare the elution of radioiodinated C5b from EACI-6 and EAC‘I-7. It was reasoned that elution of C5b from EACl-7 should be impeded if a hydrophobic polypeptide chain from this sub-unit of the C5b.6,7 complex becomes inserted in the hydrocarbon moiety of the phospholipid bilayer (Hammer c’r cl/.. 1975). The results of this experiment. shown in Table 1. were in accord with our expectation since 0.3 M NaCl solution readill eluted radioiodinated C5b from

of [‘L’l] CY from EACI-Y

Sol\ent

0.02 91 EDTA-I I’,,, srx

(Adapted

I WI Y7Y 107x I222

‘$1 NaCl

from Hammer

or SDS CY eluted (counts. min)

101x 40

_ 0 Y?Y

1055 x3

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(‘I u/.. lY77.)

0

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CY on cells After elution Before elution (count5 min) tcountsmin)

.O .2{ NaCl

0.0’ \I FD7A-I.0 I” II L SDS

x17

Damage

EACI-6, but not from EACI-7. While there are possible alternative interpretations. we believe that this experiment lends support to the insertion hypothesis because the dramatic difference between EACI-6 and EACI-7 with respect to elution of C5b parallels the striking difference between these intermediates with respect to tryptic digestion (Fig. 3). Similar experiments with erythrocyte membrances treated with the whole complement system. published by Bhakdi er ul. (1975), also support the insertion hypothesis. Presumably. these membranes carried all of the complement proteins. Their results indicate that bound C3 can be eluted from such membranes, whereas bound C5 and C9 are resistant to elution. We have also performed elution experiments with EACl-9 carrying radioiodinated C9. The results, shown in Table 2. indicate that treatment of EAC l-9 did not release any radioiodinated C9 from the cells. On the other hand. essentially all of the C9 could be with SDS (sodium dodecylsulfate) recovered

by

)

Table 7. Elutlon

Membrane

______

- __-__-----

Release

Release

of “Rb+ ------

of

“C

(Ab + C) ___-_-----

_--_

(Ab+C)

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25

of “Rb+(C solo) --------------_ _ _ - - - - - _ _ --

1_-_-_-_-_-_-_-_-_ (C4D? 100

0

Ab) or (Ab solo)or(Heated

GPS)

- - - _ ,” GPS) zoc

s.rum tuo t-lg 4. Release of h”Rba and lJC-PC from llposomes as a function of the quantity of guinea pig serum. Llposornes were made from Cl43 lecithin, cholesterol and dicetyl phosphate (molar ratios 2:1.5:0.2). Forssman hapten and ‘*C-phosphatidyl choline (PC) were incorporated in the lipid bilayer. and ““Rb’ was trapped in the aqueous compartment. Liposomes were treated with rabbit anti-Forssman IgM at 27 C for 30 min. folloucd by incubation with GPS at 37 C for 30 min. The released markers were separated from the lipowmes on a 50-m column of agarose. The cross-hatched area represents lJC-PC release attributable to complement activation. (0). GPS (guinea pig serum) solo: (0). antibody plus GPS: ( n ). heated GPS: (0). antibody plus heated GPS (30 min. 56 C); (A), C4D GPS, C4-deficient gumea pug serum: (A). antibody plus C4D GPS. (From Shin <“Icrl.. 1977).

XIX

MANFRED

M

(Hammer (‘1ul., 1977). These results are in agreement with those of Bhakdi et ul. (1975) and thus support the insertion hypothesis.

The insertion hypothesis involves the assumption that interactions among the late-acting complement proteins lead to exposure of hydrophobic polypeptide domains. It should be possible to substantiate this assumption by hydrophobic binding experiments. Through this reasoning our attention was drawn to several publications in which it was reported that complement releases phospholipid from the membranes of bacteria, liposomes and erythrocytes (Wilson & Spitznagel, 1968. 1971: Inoue et al., 1977: Kinoshita ef ui.. 1977: Giavedoni 81 Dalmasso. 1976). In light of the insertion hypothesis, it seemed to us that this phenomenon might be due to phospholipid binding to hydrophobic sites that are exposed on complement proteins during the terminal reactions. Accordingly. we have made a quantitative study ofthe complement-mediated phospholipid release from liposomes, including an investigations of the role of individual complement proteins (M. Shin et cd., 1977). Our experiments were performed with liposomes made from dimyristoyl lecithin. cholesterol and dicetylphosphate (molar ratios 2: I .5:0.2). Forssman hapten and “C-phosphatidyl choline ( IqC-PC) from algae, as marker. were incorporated in the lipid bilayer. “ORbC‘I was used as marker in the aqueous compartments. After incubation with rabbit antiForssman IgM and guinea pig serum as a source 01 complement, the liposomes were separated from the aqucou\ pha~ on ;I 50 m column ot‘aparose. and the released ‘“C-phosphoiipid and H”Rb + wcrc measured. ~uu?7tit~lfi~~, .srr(uf~~ of’ thr rritwsi~ CI/ “Y-PC anti KbRb’ hr ~~~ltib~~~~l, wd ~~3~~~~~~3~~~. This series ot experiments is shown in Fig. 4 in uncorrected form, i.e. without subtraction of the blanks. With respect to X6Rb ‘. the background release was 4.9 1 0.7”,,, regardless of whether the liposomes were incubated with buffer, with antibody alone, or with C4D-GPS (Crl-deficient guinea pig serum), either alone or with antibody. Slightly higher release of N6Rb+, 7.7 2 0.7”,,. was observed with normal GPS aione. presumably due to slight complement activation. On incubation with antibody and GPS, 66.0 f 2.2”,, of H6Rb’ was released from the liposomes on the plateau of the dose-response curve. After subtraction of 4.9”,, background release. this leaves 61. lo,, specific release of “6Rb* in the plateau region. In the case of- 14C-PC, non-specific release on the plateau was substantial, 25.7 & 1.9”,, in the controls run with GPS alone. or with C4D-GPS. either alone or with antibody. The plateau release of ‘“C-PC mediated by antibody and normal GPS was 44.3 + 1.7”,,. After subtraction of non-specific release of 25.?“,, this leaves a specific release value of k&6”,, in the plateau region. The large release of phospholipid on incubation of liposomes in aqueous media containing substantial amounts of protein is believed to be due to binding by hydrophobic sites on protem molecules (Tanford. 1973). In theabsence of protein. only 0.7 to 3”,, of the

radioactivity has been found in the aqueous phase; part of this may represent impurity. It is of considerable interest to compare the nonspecific release to the specific release with respect to the ratio of released phospholipid to protein. While the actual quantity of specifically released radiolabeiled phospholipid IS ahout the same as that which IS removed non-specifically. the respective ratios of bound phospholipid to protem differ widely because the terminal complement protems represent only a small part of the total serum proteins. While precise data on serum cor~centration of C5&9 are not available. we estimate that C5b-9 binds and removes about IOOtimesas much phospholipid per unit protein as the serum proteins at large. This suggests that the mode of binding by the exposed hydrophobic complement peptides may be one ofcomicellization, a structure in which hydrophobic peptides become entirely surrounded by a contiguous array of phospholipid molecules that are oriented with their acyl chains toward the peptide and the head groups to the aqueous phase. Such structures have been considered for the interaction of the hydrophobic segment of the membrane protein cytochrome b, with phosphatidyi choline liposomes (Robinson & Tanford, 1975). 7%~ c:flkt c!/ itluc‘tiwtioif r!/. compicwrnt hj. cohrir wtwm fwior on remowl c!f pho.sphol@itl ,fkm lipc~.wme.s. This experiment was done in order to learn

whether the phospholipid-releasing capacity OI activated complement is short-lived like the ability of activated late-acting compIement components to become inserted in erythrocyte membranes (cf. Giitze ;G Miiller-Eberhard, 1970; Goldman Et ul.. 1972). GPS was treated with the cobra venom factor (CVF) which activates the alternative pathway and thus inactivates complement components. As a consequence of the CVF treatment, the hemolytic activity of C3 was reduced to 8”,, of that in a control sample of GPS

Immunologically

M~d~atcd

incubated with buffer. The results of a series ot experiments, shown in Fig. 5, indicate that inactivation of GPS with CVF reduced release of ‘Y-PC from 33.X”. to 16.6”,, i.e. to the non-specific release level. as observed with GPS alone (1X.2”,,). It is evident from these data that treatment of GPS with CVF eliminates the hydrophobic sites that are involved in specific release of ‘“C-PC by activated complement. just as it abolishes the cytolytic activity. Presumably, this loss is due to exposure of hydrophobic complement peptides on activation with CVF. followed by their removal from the aqueous phase by refolding. aggregation or binding of serum protein, such as the S-protein (Podack PI (II.. 1977). The fact that the phoypholipid binding \itcs are eliminated by treatment with CVF establishes an important parallel with the behavior of the terminal components in hemolytic experiments (Ghtze & Miiller-Eberhard, 1970: Goldman r’t al.. 1972; Kolb bz Miller-Eberhard, 1973) and with their action on planar lipid bilayers (Michaels LJIal., 1976).

Pho.y~holipid releasr uttrihutahle C8 und,or C-9. These experiments

to C5h,6,7

und to

were performed in order to determine whether the phospholipid removal from liposomes is attributable exclusively to the terminal complement proteins and to what extent the C5b.6,7 complex and the other terminal components are involved in this phenomenon. The first of these experiments (Fig. 6) was performed with C7-deficient human serum (C7D serum) which permits formation of the liposome intermediate LACI-6, but does not allow assembly of the C5b,6,7 complex. It is evident from the results that C7D serum did not mediate removal of phospholipid above the non-specific level. On the other hand, when purified guinea pig c‘7 was added to C7D serum, the capacity to remove phospholipid from liposomes was restored. In order to determine to what extent the

Membrane

Damage

x I ‘)

phospholipid release is attributable to C5b,6,7 and to C8 and, or C9. an experiment was performed with CXdeficient human serum (CXD serum) As shown in Fig. 6. treatment of liposomes with antibody and CXD serum caused release of a moderate amount of phospholipid above the non-specific level. bui much less than that produced by normal human serum. Addition of purified guinea pig CX to the CXD strum restored the phospholipid release to its full extent. From these data. out of l6”,, overall specific removal. to C5b.6.7 while the 6°C) was found attributable remainder was due to CX and;or C9. In all of these experiments ““Rb’ was used a5 a second marker. No release of ““Rb’ was observed with C7D serum and antibody, but reconstitution with purified guinea pig C7 fully restored release of ‘“Rb *. A very small amount of X”Rb’ was released by CXD serum and antibody. Addition of purified guinea pig CX restored release to its full extent. Comnzrr~r. The demonstration of phospholipid removal from liposomes by activated complement lends support to the trans-membrane channel hypothesis since it indicates that hydrophobic peptidcs are exposed on interaction of the terminal complement proteins. This concept is also supported by the demonstration that the terminal complexe\ bind deoxycholate (Podack (‘I u/.. 197X). We beliae that some of the hydrophobic peptides become inserted 111 the membrane bilayer and form channels. Others interact with the membrane. but do not nmaln inserted. It is these that are thought to mediate the phospholipid removal from liposomcs. Apart from their relevance to the trans.membrane channel these experiments habe drawn our hypothesis, attention to a new biological activity of activated to insertion ol complement. Thus. in addition hydrophobic peptides into membranes and resulting channel formation. complement may cause changes 111

x20

MANFRED ---r

--

7

M

-I

r--f i

10

20

30

40

50

60

70

80

I

90

MINUTES

Fig. 7. Sequential action ofcomplement protelnb on lecithin BLM. The aqueous phase contamcd 150 m.44 KCI and 5 mM histidine buffered at pH 7.0. The BLM was formed from egg lecithin In n-decane (membrane thickness = 6.0 nm). The vertxal arrows mark the time of each protein addition. The total units of activity added in each instance are given III parentheses. The abscissa is elapsed time in minutes: the ordinate is the specific membrane conductance. (From

Michael\ (‘t ul.. 1976.) the lipid moiety of membranes. This new biological function of complement is, perhaps, significant in inflammatory pathological processes involving modification of lipid and lipoprotein distribution. 3. Assembly andproperties of’the complement channels as studied by measuring ion flow acro.ys planar hilayer membranes Planar

lipid

bilayers,

or

er ul

(BLM), represent an attractive model system because the morphology of BLM is known to be biomolecular and because changes in membrane permeability to ions can be measured electrically. Several investigators have reported changes in ion flow across BLM after treatment with antigens, antibodies and fresh serum as a source of complement (Barfort et al.. 1968; Mueller & Rudin, 1970; Wobschall & McKeon. 1975). As described below, our studies were performed with the reactive lysis system (C5b,6 + C7 + CX + CO). Experiments with phosphatidyl choline (egg lecithin) BLM: (6 nm thickness,. As shown in Fig. 7, sequential treatment of BLM wjith the terminal complement components produced characteristic increments of ion flux as measured electrically. After treatment with C5b,6 + C7 + C8 a modest increase in conductance was observed. Subsequent addition of C9 greatly amplified this change. As shown in Table 3, no permeability changes occurred when components were added individually to the membrane, or when used in paired combinations. Also. no permeability change was observed when C5b,6 complex, C7 C8 and C9 were admixed prior to addition to the BLM; under these conditions the terminal attack sequence decays before its reaction with the membrane. These observations establish a significant parallel between the ion flux changes in the model membrane and damage produced in biological membranes by the CSb-9 attack sequence (Michaels et N/.. 1976). Of considerable importance is the observation that only bilayer membranes were susceptible to the terminal complement components: thick lipid films membranes

BROKE

0

MAYER

so-called

black

lipid

Table 3. Sybtcmatlc anuiy\is of the xtion of the terminal complement components on 6 nm leclthin B1.M conductance First addition

(‘9

None C‘5b.6 (‘7 C‘X C‘Sb.6 + Cl C‘Sb.6 + C‘X c‘7 + c‘x C5b.6 + c’7 + C‘X C5b.6.7d c‘5b-Xd The table summarizes the effect on membrane conductance 01 individual complement proteins and different combinations of components. For each set of experimental conditions iit lcubt three experiments were completed. the loNest and highest readings are shown. ‘N.D.’ means not done. The open spaces reprewlt redundant and nonsense combinations. The data arc giLen ;ib relative conductance increments [(G G,,)- I]. where Go is the BLM conductance prior to an) addition and ti 1s the tinal membrane conductance The experiments were performed by adding wquentially, with minimal intervals of 3 min for equilibration. the components listed in the left hand column. pausmg IS min for BLM conductance to stabilize and tinally adding the component lIsted across the top of the other columns. A wide range of component quantities w:(s used in these studies: C‘5b.6 (It&IOU unit\). (‘7 (lOt~2500 units). CX ( IO@3000 units). CY (0.3-60 units). In the case ofC5b.h.7dand C5tG3d. the components were admixed ma test tube and incubated at 37 c‘ for 30 min before addition to the membrane.

The superscript ‘d’ indicates decay. (Adapted from Mlchaels (‘I rri.. 1976.)

Immunologically

Mediated

Membrane

Damage

E 302 : 2 z 20E X “0 _

IO -

1 T coo,-

-5

0

7 “r” ~T??!PE__L1

20

f=

40 MINUTES

60

80

100

Fig. 8. The el’rcct of appliedvoltage and polarltj on complement dependent conductance ol’leclthin BLM. The experimental conditions here the same as those in Fig. 7. In the tirst half of the experiment (0) the mcmbranc uas held at 0 mV except for a brief IO mV pulse at I min Inter\& to measure conductance. The second hall’ofthe experiment (01 was performed Hith the membrane potential held constant at IO mV. The abscissa is time 111min. the ordlnste ih the \pecttic membrane conductance. (From Michaels c’, r/l.. 1976 ) were unaffected. This argues against a diffuse disordering of membrane lipid. e.g. by detergent action, and is in favor of the trans-membrane channel concept. We have also observed that the conductance increments were always accompanied by increased current noise and discrete current steps were often discerned at low protcin.lrvels. All of these features have been reported in BLM studies of putative channel farmers (Haydon & Hladky. 1972). The ejfeec,t qf upplicd wltup~ and polarity on ~~omplemc~nt-depend~,r,t~,o~I(JI~(.IoI?(.(‘, of lecithin BLM. It is of interest that the conductance of lecithin membranes treated with CSb,6 + C7 + C8 to form the membrane intermediate MCSb-F-X was affected markedly by the polarity of an applied electric field. Application of a constant positive voltage ( + V, Fig. 8) was found to cause a substantial increase in conductance. Reversing the polarity of this applied potential (-V) decreased the conductance to an intermediate level. When a positive potential was again applied (+ V) the conductance increased to an even higher level. At this point of the experiment, C9 was added and the conductance increased rapidly to a level 50 times above the base line. After the conductance had stabilized. the polarity of the applied potential was switched, but the conductance did not change. Thus, the ion flux of MC’5b--8 is sensitive to electric polarity. while that of MC5b--9 is not. The sensitivity of MC5b8 ion flux to electric polarity indicates that the number of conducting channels, or the ion permeability of individual channels is influenced by the applied electric field. Such a process can, conceivably, take place in two ways: one originating from the aqueous phase and the second occurring predominantly in the membrane phase. The first mechanism would entail formation of additional channels due to electrophoresis of protein toward BLM, followed by insertion. when the membrane potential is positive on the complement

side. In terms of this interpretation, when the direction of the electric field is reversed. some of the protein wsould be ejected from the membrane. thus decreasing the number of channels. The antibiotic monazomycin. another putative channel former in BLM, appears to display such an electrophoretic mechanism (Muller & Finkelstein, 1972). There is insufficient information as to the physico-chemical properties ofC5b,6 C7 and C8 to evaluate this possibility. However, ejection of C5b,6.7 or C5b-8 complexes from the membrane by electrophoresis is unlikely. If these complexes were shuttling between the membrane and the aqueous phase, the conductance of MC5b-8 should have decreased continuously under the influence of a negative potential due to decay of the complexes to an inactive form in the aqueous phase. This effect was not observed; instead, switching the voltage polarity in repeated cycles increased BLM conductance. We prefer the second type of mechanism, which involves the concept that the electric field can cause conformation changes in the proteins associated with the membrane, or can affect an electrostatic association between two or more components within the membrane. In light of the respective increase and decrease of conductance following application of a positive and negative potential to BLM. such changes must be regarded as reversible. Conceivably. electrically mediated changes of this kind could be so extreme as to close down existing channels or to open up new channels depending on the direction of the electric field. Precedent for this type of mechanism is provided by the action of alamethicin on BLM (Gordon & Haydon, 1972; Eisenberg et al., 1973). The fact that the MCSb9 channel is not dependent on polarity presumably means that its structural stability is much greater than that of the MC5b8 channel. E.vprir?wrt.s wit/l O.Yrdiccd c~holc~strrol (4 nm) or ultra-thin kcirliirr t~wt~lhrunes (.Michac4c et al., 1978. 1978a). These experiments were designed to

MANFRED Tahlc 4. Systcmattc complement protetns

M MAYER

analysts of the action of the terminal on the conductance of ‘ko nm oxiditcd cholesterol BLM Second

Ftrst addttion None Cl C5h.h + C‘7 C’5b.6 + C7 + C’S C‘.Sb.O,7d C‘5b-Xd

6’1II/

C 5h.h

(‘7

1-100 %I X0

(W. I

addition (‘S

(&(I 0-o. I 75-500 o-0

The e.xnrrnnents were nrrlhrmed presentation of the data I\ the same. quantities u\ed rn these studtes \\a\ (1~1000 untts). C‘X(I(&1000 units) and from Michacls VI LI/.. 197X.1

assess both the requirement for phospholiptd in the complement attack sequence and the potential importance of membrane thickness. In addition, the oxidized cholesterol and lecithin systems represent extreme and opposite examples of membrane fluidity since lecithin BLM are much more fluid than BLM made from oxidized cholesterol. Unlike the results with ‘normal’ lecithin (6 nm) BLM shown in Fig. 7, sequential treatment ofoxidized cholesterol BLM with C5b,6 + C7 + C8 + CY produced an increment of conductance at each step. A representative experiment is shown in Fig. 9. The results of a series of tests. with Individual reagents and with various combinations, are summarized in Table 4. In contrast to the tests on 6 nm lecithin BLM shown in Table 3. the present series with oxidized cholesterol revealed that C5b,6 as well as Co increase membrane conductance. indicating that these proteins can interact with membrane lipid. Equally important, Table 4 shows that the BLM conductance after the addition of all four terminal componcnt5

as In Table

3. and the The range of component C‘5b.6 (0.2-20 untts). (‘7 CO (5-50 untts). (.\Japtcd

(C5b.6 + c‘7 + CX + Cc)) is j-10 times higher than the simple additive effect expected for individual C5b-F-X and C9 conductance states. Thus. the cooperative interaction between components to produce a functional C5b-9 state previously shown with ‘normal’ 6 nm lecithin BLM is also observed with the 4 nm thick oxidized cholesterol BLM. As shown also in Table 4. when C5b.6 and C7 were mixed /W/&K addition to the membrane. no change in conductance was observed. Similarly, there was no conductance increment when C5b.6, C7 and CX were mixed with one another before addition to the membrane. Experiments of this type show that interaction of the terminal components in the fluid phase leads to the formation of complexes that are inactive with respect to BLM. As in the studies with 6 nm lecithin BLM that were described in the previous section, these result? establish an important parallel between the HLM

L_-

--A

I

5 MOLE

0

20

40

60

80

100

120

MINUTES

Fig. Y. Sequential action of terminal components on conductance of oxidized cholesterol BLM. Experimental conditions were described tn Fig. 7. The BLM was formed from oxidized cholesterol in n-decane + n-hexadecane (3:I ): the membrane thickness was 4.0 nm. (Stmtlar results. not shown here. were obtained with GM0 BLM.) (From Michaels er ol.. 1978.)

I-l 30 N D. N D. ?Oo-1000 N.D. (LO.3

10

PERCENT

L

I5

:o

HEXADECANE

Fig. IO. Effect of membrane thtckness on the reactton 01 C5b.6 and C5b.6.7 with lecithin bilayers. The aqueous phase contained IO0 mM KCI + 5 m.M histidine buffered at pH 7.0. The BLM were prepared from dioleyl lecithtn dissolved in a mixture of decane + hexadecane of varying molar ratio. The abscissa shoNs the mole per cent of hexadecane in the membrane-forming solution. The left hand ordinate shows the relattve BLM conductance increment (G G,,) obtained tn response to either CSb.6 complex (I 8 units ml) or to CSb,6.7 (C5b.618 units;ml: C7. 25 unitsml). The right hand ordinate shows the bilayer thickness (dashed line through the triangles) calculated from the specific electrical capacttancc of the membrane. (Each experimental point represents the average of three to five measurements. The total range is shown by a vertical bar through each pomt.) (Michael5 & Mayer. lY7G.)

Immunologically

Mediated

experiments and those with biomembranes. In addition, the data show that phospholipid is not required for the action of C5b-9; instead, the only is for a hydrophobic milieu of requirement bimolecular dimension. One argument against the above conclusions and biomembranes is that oxidized analogies to cholesterol is ‘non-physiological’ and complement somehow display anomalous behavior may characteristics. However. ultra-thin BLM prepared from either lecithin (vi& in/w) or glycerol monoolein solutions displayed the same response behaviour to the C5b-9 sequence as that shown in Fig. 9 and Table 4. The problem of membrane thickness has been studied in greater depth with a serves ofdioleyl lecithin BLM that were made from solutions of this phospholipid in n-decane,n-hexadecane mixtures. As shown in Fig. IO. membrane thickness can be changed continuously by varying the ratio of decane to hexadecane in the carrier solvent (Fettiplace et al., 1971). It is evident from the results in Fig. 10 that the response of the BLM to C5b.6 and C5b.6.7 was affected markedly by membrane thickness. Thus. C5b,6,7 began to elicit a conductance response when the membrane thickness fell below 4.4 nm (width of the hydrocarbon region), while C5b.6 began to give a signal when the membrane decreased below 4.0 nm. These results support the view that peptides from C‘5b.6.7 become inserted more deeply in the membrane than those from C.Sb.6. In conjunction with the data in Figs. 7 and 9. these observattons show that membrane thickness is a critical factor in the formation of trans-membrane channels by the C5b-9 reaction sequence. Furthermore, the experiments in Figs. 9 and IO, as well as those in Table 4, extend the enzymatic stripping and salt elution experiments of Hammer c’t ul. (1975) by showing that the process of insertion begins at the C5b.6 stage. Commc~t. Since the results of the BLM experiments are closely similar to comparable experiments with cells, the relevance of the BLM model isclear. Like the liposome experiments of Kinsky (1972). the BLM experiments indicate that the lipid moiety ofcells is the target of attack by complement. Furthermore, since membranes of oxidized cholesterol are susceptible to complement attack. phospholipase action can be ruled out, thus confirming the observation of Kinsky et ul. (1971). For certain types of experimentation, the planar lipid bilayers offer distinct advantages over liposomes. For example. the sequential interactions of the terminal complement proteins can be followed kinetically which facilitates studies of the mode of channel assembly. An item of new information derived from BLM experiments is the demonstration that the process of membrane interaction starts at the C5b,6 stage, not when C5b.6.7 is formed, as had been thought in the initial stages of our work on the basis of enzymatic stripping and salt elution experiments. However, stable insertion in cell membranes begins at the C7 stage. 4. Eftrct n~n&runc

o/

rncmhranc lipid structure on trunsforrnution undphospholipid removal

c~l7unnc~l

AS shown in Sections terminal proteins on

I and 2, activation of the biological or artificial

Membrane

823

Damage

membranes produces two effects. namely. channel formation and phospholipid removal. Since both of these phenomena involve lipid-protein interactions, the chemical properties of the lipid bilayer would be expected to influence them. Accordingly, it was decided to evaluate the effect of length and unsaturation of the acyl chains of synthetic lecithins and the effect of cholesterol concentration on channel formation and phospholipid removal (Shin et ul.. 1978). lZffi,ct of uc,~,l chuin length .on relrasr 01’H6Rb + and ’ “C-PC /k/n7 Iiposonws. Figure I I shows the effect of lecithin acyl chain length on release of HhRb’ and from liposomes containing removal of ‘Y-PC Forssman hapten in their bilayer by anti-Forssman antibody and whole guinea pig serum as a source of complement. All of the liposomes contained 50”/,, molar cholesterol in order to minimize the fluidity differences in bilayers of lecithins of differing acyl chain length. Two experiments are shown, one with 2 PI of guinea pig serum, the other with 30 nl ofserum. In each of these experiments Cl4:0, Cl6:O and Cl8:O lecithins were compared. It is evident that XhRb’ release decreased with increasing acyl chain length. Also, less “T-PC was removed from Cl 8:O than from Cl4:O lecithin. Since this might be attributable to an effect on antigen presentation on the liposomes experiment was (Alving et ul.. 1974). another performed in which the effect of acyl chain length on R6Rb+ release from liposomes was examined by use of the reactive lysis system. which excludes any possible effects on the antigen-antibody reaction or the early complement sequence. As shown in Fig. 12. the release of H6Rb+ by C5b-9 decreased progressively with increasing acyl chain length. Similarly, M6Rbf release by C5b-8 also diminished. It should be noted that the difference between release by C5b-8 and C5b-9 is attributable almost entirely to a kinetic effect because

70

%b* 0 ‘%PC

.

60 t

Fig. Il. Effect of leclthm acyl chain length on release ol ““Rh’ or ‘Y-PC‘by Ah + GPS. The release ofn”Rb’ (open bars) and ‘lc‘-PC (closed bars) is shown as specific relcasc which was calculated by subtracting the non-specific release ofmarkers by GPS from the total released by Ab + GPS. The llposomes were made from Cl4:O. or Cl6:O or Cl83 lecithins, dicetyl phosphate and Forssman hapten; 50 mole “,, cholesterol was Incorporated in order to diminish the differences of membrane lluidity due to variation in acyl was not measured in the chain length. (Release of “C-PC experiment with the Cl6:O liposomes.) (From Shin <‘I rrl.. 197X.)

824

MANFRED

M. MAYER

vt ul

Release of eeRb* from Liposomes Effect

of Acyl Chain Length

IO

Phospholipid Acyl Chain Length

Fig. 12. Effect of leathin acyl chain on release of “*Rb+ h) C5b-7. C5b-F-8 or C5t+9. Liposomes made from Cl4.0. Cl 5:0. C I6:O or C I8:O lecithins. 50 mole ‘I(, cholesterol. and dicetyl phosphate were treated at 0 C successlvcly uith CSb.6 for IO min and C7 for 5 min. C8 and C9 were added at 0 C. the mixtures were then incubated at 37 C for 30 min. ““Rb ’ release by buffer (m). by decayed complement complexes (admixture of components and incubation at 37 C for 30 min prior to Incubation with liposomes) (B), by sequential addition of C5b.6 + C7. CSb.6 + C7 + CX, or C5b.6 + C7 + CR + C9 tB%Zi) (From Shin (‘I cl/.. 1978.)

the rate of ion flow through CSb-8 channels is very much slower than that through C%-9 channels, due to the smaller size and instability of the former channels. It is evident from the results in Figs. 11 and 12 that variation of acyl chain length exerts a direct effect on the process of trans-membrane channel formation. Presumably, this is attributable to the changes in membrane thickness that are associated with variation in acyl chain length (M. Shin et al., 1978). Effect of cholesterol concentration. On the basis of experiments with liposomes which showed that increase of cholesterol concentration in the membrane diminishes trans-membrane channel formation (M. Shin et al., 1978), we have examined the effect of cholesterol incorporation into erythrocyte membranes on hemolysis by the reactive lysis system. The results of this experiment, shown in Fig. 13, indicate that incorporation of cholesterol interferes with transmembranechannel formation, in accord with the prior experiments with liposomes. Comment. While the effect of acyl chain length on the efficiency of channel formation is readily explicable in terms of membrane thickness, in line with corresponding BLM experiments, the effect on removal of 14C-PC from liposomes is not so easily explained. Presumably, we are dealing with a diminution in the extent of abortive insertion when the acyl chains are longer, but it is not clear why this should be the case. Since cholesterol decreases membrane fluidity it has been thought that its effect on channel formation may be attributable to the change of this parameter. However, we are not so sure that the depression of 86Rb+ release by cholesterol can be attributed entirely to a change in fluidity because it is also known that the introduction of unsaturation of acyl chains produces the same effect on complement-mediated *“Rb+ and “‘C-PC release from liposomes as the addition of

Cell

Population

Fig. 13. tffect of cholesterol mcorporatton lnlo sheep erythrocytes on hemolysis by c‘5b0. Sheep erythrocytes were incubated uith various dilution\ ofcholeaterol-enI-ichcd medium containing trace amounts of “‘C-cholesterol. The cells uere then tested for their su\ceptlbilir to Iysis by c‘5t+9. The per cent hcmolysis shown by cros\-hatched bar\ refer\ to erythrocytes that wcreenrlched with cholesterol. The percent hemolysis shoun by open bar\ rcfcr\ to control erkthrocyter that were incubated ulth the modification medium ~lthoul cholesterol. The uptake ofchole\terol IS 5hoa.n bq solld bars. (According to G. J. N&on. Bloc /~/HI h/~q~/~.~~r .Ic,irr 144, 22 I Cl9671. thecholesterolconcentratlon ol’thc\hceperqthrocytc membrane is about 6.23 /rg IO’ cells.) I.rom Shin VI rri.. 197X

cholesterol. Yet it is well known that the effect of unsaturation on fluidity is opposite to that of cholesterol. Therefore, we believe that it is necessary to take into consideration the role of lipid-protein and protein-protein interactions, in addition to membrane fluidity, in attempting to explain the effect of the physical and chemical characteristics of lipid bilayers. Probably, it will be necessary to study the interactions of individual lipids with the terminal complement proteins in order to elucidate the factors that influence the insertion of complement peptides into membrane bilayers and their subsequent assembly into channels, as well as the transfer of phospholid from membranes to the fluid phase. 5. Electron microscopy

of c~omplement chunnc~lt

It has been known for about ten years that the reaction of complement with membranes produces characteristic lesions visible by electron microscopy (Humphrey & Dourmashkin, 1969). When these are examined on membranes lying flat on the grid, uniform rings of approx 10 nm internal dia are observed. It is now believed that these rings are top views of the annular C5b-9 structure that forms the trans-membrane channel. Recently. profile views of this structure have also become available. One of these (Fig. 14) which we obtained through the courtesy of Dr. Robert R. Dourmashkin (1978). shows the C5b-9 channel on the rolled edge of an erythrocyte membrane that had been treated with the reactive lysis system. Another picture (Fig. 15). which we obtained through the kindness of Drs. Tranum-Jensen and Sucharit Bhakdi, shows a closely similar structure on the edge of erythrocytes that had been treated with antibody and whole serum as a source of complement. In this case, the erythrocyte membranes carrying

immunologically

Mediated

Membrane

Damage

825

Fig. 14. Top and profile view5 of complement channels on erythrocyte ghosts treated with C5b-9. Formalinlzed human erythrocyte ghosts treated with AET (aminoethylisothiouronium HBr) were used. Many typical complement lesions arc seen on the negatively stained surface. Cylindrical structures appear along the rolled edge of the membrane which is visible as an electron-transparent border. Thecircular lesions on the surface are top views and the cylindrical structures on the edge are profile views of the C5b-9 channel. .4t the base of these cylindrical structures the folded edge appears to be less dense than elsewhere on the border of the cell. Magnification x 420,000. (Kindly supplied by Dr. R. R. Dourmashkin.) (Dourmashkin, 1978.) C5b-9

were subjected

to tryptic

treatment

in order

to

remove the ‘fuzz’ on the edge of the erythrocytes. (This fuzz is seen on erythrocytes following treatment with antibody and whole serum. but not after treatment with the reactive lysis system.) It has been shown by Tranum-Jensen and Bhakdi that the tryptic treatment cleaves off a fragment from C5b-9, but this does not noticeably affect its channel-like appearance by electron microscopy. (It is not known at present whether these observations are in conflict with the enzymatic stripping experiments by Hammer et al., 1975, 1977.) Figure 16, also by Tranum-Jensen and Bhakdi, shows an electron micrograph of trypsin-treated CSb-9 channel structures that were eluted from erythrocyte membranes with and Triton deoxycholate. By immunoelectrophoretic analysis it was shown that these structures contain C5 and C9 (Tranum-Jensen et al., 1978).

Complement

attacks

lipid

bilayer

membranes

in

two ways. The first of these involves formation of trans-membrane channels by insertion of the lateacting complement proteins C5&9. The process of interaction begins at the C5b.6 stage and becomes very

much firmer at the C5b,6,7 stage. Penetration through membranes of about 6 nm thickness becomes evident after the C8 reaction and a stable trans-membrane channel is formed at the C5b-9 stage. For thermodynamic reasons, the C5b-9 channels must be a structure in which the hydrophobic peptide domains of the terminal complement proteins face outward towards the lipids of the bilayer and a hydrophilic region is oriented toward the central core. The process of progressively deeper insertion and channel formation is outlined schematically in Fig. 17. Electron micrographs of the C5b-9 channel are shown in Figs. 14, 15 and 16. The second element of membrane attack by complement involves the removal of phospholipid from the membrane on activation of the terminal attack sequence. This is believed due to abortive insertion, i.e. insertion followed by withdrawal from the membrane of terminal complement complexes together with phospholipid, probably in the form of co-micelles. The removal of phospholipid, as well as the process of channel formation, is influenced by the structure of the membrane lipids. Initial studies have shown that the membrane concentration of cholesterol, the length of the acyl chains and the presence of unsaturation in influence both elements of the phospholipids

MANFRED

826

M. MAYER

ct trl

Fig. 15. Top and profile views of complement channels on erythrocytes treated with antibody and complement. In order to remove the obscuring ‘fuzz’ on the rolled edge ofthe erythrocyte. the prcpara Cons were treated ulth tqpsln. Magnification x 203,000. (Kindly supplied by Dr. S. Bhakdi.) (Tranum-Jent ;cn (‘I al.. 197X.)

Fig. 16. Electron and deoxycholntc.

mwograph of complement lesiona extracted Magnltication x 130.000. (Kindly supplied 197X.)

from crqthrocq te mcmbrsnca with 7 ‘rlton bq Dr. S. Bhakdl.) (Tranum-Jensen c‘I C/l..

Fluid C5b

i6

,,-

Phase

observed

CSb.6.7~-C5b~8~-_C5b-9d I

/'

I' ,

1'

: C5b.6.7

incrcmcnts

to

hypothetical

interpretations.

01‘

c9

CB

conductance

attributed

ADCC

trans-membrane

hypothesis article

was also

describing

that

both

osmotic lipid

certain

similar

membrane

On this

and

possibly

complement of hydrophobic

attack

lipid-protein mechanism

In the lipid

trans-membrane process.

emphasize

These the

and protein-protein of complement l’4HI

action

reaction

importance ot interactions in the on lipid

bilayers.

2

The from

postulated

betueen

suggested

interactlons

cell

may

technique

and Blumenthal for

lymphocyte-mediated

( 1975)

studying

have

used the BLM

antibody-dependent

cytotoxicit!

(ADC‘C‘).

,o~5rc--

-----

; ,------Ant!-Tnp lo*:

i

1OlC

27lc

The!

insertlon

Icadlnp

tarset

to

cell and form

01‘ hydrophobic

membrane require

the plaxrn;i

toward

insertion

concept came from contact.

as

into

very

membranes

stimulus

mcdiatcd

cytotoxicity

suggested

a

Iqmphotouin

putative

which then become

ol‘thc

a maior

requlrcment

cell membranes.

that

lymphocytes.

components,

the

Gatclq inhlbitcd

peptides

the target

close

ol‘these

studlcs

cell

apposition cells.

development

Indeed. of

by Martz

the

( 1977)

well 3s our own investigation

on the el’fcct 01‘ anti-lqmphotoxin Henkart

u

between

activate of

pcptidcs

bllayer

hould

on ccll-ccll

may involve

channel\.

the lymphocyte

membrane

the concept wax

was

protein\

exposure

the

01‘ complement-medinted

It

target

colloidcases

has been implicated

basis.

specific

membrane-associ~tted

cyfolysis

and

in both

b> lymphocytes

damage.

lymphocyte

membrane

one-hit

Moreover.

to that

immunologically

characteristics

betueen

It was noted in thih article

exhibit

that cytolysis

This

theoretical

lymphocyte-mediated

of the cell membrane

mechanism

inserted

similarities

1977).

the target ol‘attack.

proposed

be

several

formation.

in a recent

and

characteristics,

could

among them the concept

analyzd

processes

bilayer

that suggested

channel

complement bl cytotoxicity (Mayer.

as

and

(Gately

antibody C/ (I/..

for close apposition c>tL/I. (1976) the

cytolyjis

on cell-

1076)

found

which of both

that anti-

01‘ ovalbumin-

coupled

target

spleen

cells.

m their

ccllh

by

but did

native

allogeneic

form

of

there

are

thcsc

immune

at

t\\\o

involves

secretIon

which

probabl\,

rcquira

mcmhranc:,

The

present hill

(Henkart from

lipsomes

and

(3) cytotoxicity

path\hay\

by

target

v+hich

cells. and

between

contact

target

and

01‘ the 01

on channel 1975).

(‘I oi.. 1977. 0 I’

inhlhition by phc)\pholiplcl

killer

one

another the

cells.

tran+mcmbrane

I)mphocb

to three

the that

01‘ I> mphotoxin

hc Iimitcd

(Juy

On

postulated

dcstro>

diwu\Gon

Kc Hlumcntbal.

to either

Intimate

01‘ the

( I ) expcrlments

namely.

WI\

distinct

hypothcsi\

cytotoxicity

nnmunc

ccl1 antigens. it

can

which

channel

cells

target

ohscr-\atton\. least

of target cells

the lqaia

by spleen

IymphocyW

plasma

inhibit

or seno~cneic

basis

guinea pig

OL;I~~~II~~~~~-II~~II~III~C

not

tc-mediated

relevant

elements,

I’ormation (2) marker

M. Shin

in BLM rcleasc

VI r/i., 197Xtr)

I\mphoc\,te-medi~ited (Free R F;iou. 1975).

I. E:‘lPc~f~/cYr/ 111(‘~1,\111’(‘/)1(‘Hi.\ o/ ,011//OH i,~‘l’O.S.\ HL.21 cllrl?lr,~c1rrtrd h_l,otltillorll~ ~/ml 11w,/‘/1(wl-/~~\

A

serlcs

01‘ e~perlmcnt~ a5 cl‘l’cc~~~rs v.;i\ Blumenthal ( 1975) with

01’

lymphocytes and

cholesterol’

nicnibranc3

\\ I11c‘h

in

this

pcrl~ormcci

t\pc h\

with

Hrnkart

of ‘oidizcd

I>?rl’-pho~phatid4I

hilaqer was hapten: subsequently. normal Iymphoid wlls from human peripheral blood v+erc added and allo~cd to settle on the membrane. Ah shown 111 the middle pan4 of Fig. 18. electrical conductance incrca\ed manq hundredfold after treatment of the mcmbranc MIX antibody and lymphoid cells. This el’l’cct MU\ obscried only when the membranes acre trcatcd xitll antibody and lymphoid cells. As shnmn In the bottom panel. no substantial Increahc ol‘conductance M;I\ produced b\ lymphoid cells alone. Nor did treatment of the membranes with antibody alonc‘changc t hclr electrical conductance (not show,n-in I-+.. IS). Another control involbcd the UK 01‘ b(ab). Instead ot‘~n~act antlbod~. Treatment of membran& uith this t’ragment pluh lymphocytes did not incrc‘lsc the ti.an~-membrane conductance. This Indlcatch that the l‘c \cgmcnt ofthe antibody i\ necessary. prcsumabl> for activation ol lymphocytes by binding to the Fc receptor. It is also ofintcrcst that the change ol‘conductancc produced by antibody and lymphocyte< itas found to be dependent on electrical polarity. a hwn in the top panel of Fig. IX. The satnc phenomenon \\:i\ observed In our studies of the effect of complement on the electrical conductance ol‘ planar leclthln bllayers (Fig. X). This probably indicate\ that the electric tieid can in the protclnz cause conformational changes associated with the membrane. or ;ln clcctrostatlc interaction between two or more components uithin the membrane. Conceivably. clcctricallq mediated changes of this kind could bc w cxtrcmc as to close down existing channels or to open LIP IKW channels. depending on the direction 01‘ the clcctrlc ticld. In light of our studies on the I’orrnatlon of transmembrane channels b\ In\crtlon 01‘ complement peptides. a mechanism i-or the Hcnkart-Blumenthal experiments was suggested (Mayer. 1977) which postulates channel formation by cooperative action 01 several molecules that bccomc Inserted in the target cell membrane. It ih thought that these molecules ma! ethanolamine

was

trcatcd

antibody

wth

incorporated. afalnst

The

the

TNP

be hydrophobic peptides that become extruded from the lymphocyte membrane by an activation mechanism.

-. -I .WrrI.l\eV,_&rr.Wjiom I;p”.w??r.F Lymphocyte-mediated marker release from sphingomyelin or lecithin liposomes has been investigated in order to approach more closely the behavior of natural cell membranes than the BLM cxperimcnts of Hcnkart and Blumenthal (1975) which ucre performed with ultra-thin membranes of oxidized cholesterol. An initial exploration of a lipsomal model system for studying ADCC was made by Juy (‘I (I/. (1977) with mouse spleen cells, anticardiolipin rabbit serum and sphingomyelin, cholesterol liposomca containing cardiolipin. Interpretation of these experiments is handicapped by the susceptibility of the glucose marker to metabolic breakdown and by very large background markerrelease in the various controls. especially that containing antibody. As a consequence, marker release that could be attributed to ADCC was only about l&I Y,, of the total release. With the aid of‘ Dr. Judith B. Willoughby of this laboratory. we haw incestigated another liposomal model system comprising ““Rb marker. dimyristoyl lecithmcholesterol dlcetyl phosphate liposomes containing Forzsman hapten. anti-Forssman I@ antibody and normal mouse spleen cells that have been depleted of adherent cells by passage through a glass bead column. In this system, background release from liposomes treated with spleen cells was quite low, but treatment of the liposomes with antibody still caused undesirably high marker release. as in the experiments ofJuy c’/(I/. (1077). The largest net marker release that could be attributed to ADCC in our experiments has 10”,,. In addition to the difficulty caused by the large background marker release, there is interpretational uncertainty because it is not known u priori whether marker release from the liposomes is due to liposomal membrane damage and, hence, proceeds directly from the liposomcs into the medium. or whether marker is transferred by fusion or ingestion from the liposomes to the spleen cells. followed by secretlon. In order to

Immunologically

u

Mediated

’

5

15

25 Minutes

35

45

Fig. 20. KinettcsofHbRband i’Cr release from liposomes by complement. Liposomes (prepared as described in Fig. 4). containing HbRb and “Cr. were treated with IgM antiForssman Ab (A,) + 5 ~1 GPS as a source of C. The graph displays the percentage of each marker released vs time. (0) n6Rb; (0) 51Cr. (From Shin ct 01.. 1978u.) resolve this issue, we have performed a series of experiments in which 51Cr was introduced as a second marker. The use of 51Cr, in addition to s6Rb, would help to answer this question because 51Cr marker becomes bound to cell protein. As a consequence, in the event of marker transfer from liposomes to spleen cells, appearance of 51Cr in the medium would be retarded relative to that of 86Rb. In order to assess the degree of relative retardation to be expected in this case, a control experiment was performed by measuring spontaneous release of 51Cr and 86Rb from spleen cells pre-labelled with both markers (Fig. 19). It is evident from this experiment that the initial velocity of SLCr release was less than one-fifth that of 86Rb. This gives an indication of the relative retardation of

Membrane

829

Damage

51Cr release to be expected if lymphocyte-mediated marker release from liposomes were to proceed via the spleen cells. On the other hand, in the event of membrane damage to the liposomes and consequent direct release of marker into the medium, as in the case of complement attack. one would expect that markers like SICr and X6Rb would be released more or less simultaneously, provided that the size of the lesion is large relative to the diameters of the markers. Such simultaneous release has been observed in a control experiment in which liposomes containing R6Rb and 51Cr were attacked by IgM antibody and complement (Fig. 20). However, in the case of lymphocytemediated membrane attack, moderate retardation of 51Cr release would be expected due to secondary marker uptake by cells (vi& in&). The results of a double marker ADCC experiment are shown in Fig. 21 which displays the kinetics of release of s6Rb and “‘Cr from liposomes that were incubated alone, or with spleen cells, or with antibody, or with antibody + spleen cells. The kinetics of specific net marker release, calculated by subtraction of the controls, are shown in the inset of Fig. 21. It is evident than there was retardation of 51Cr release, but much less than that to be expected by comparison with Fig. 19. The interpretation of this experiment is complicated by the fact that there is secondary uptake of both markers from the medium by the spleen cells, followed by release. Therefore, as noted already, even if all the marker release proceeded directly and simultaneously from the liposomes into the medium, one would not expect coincidence of the s6Rb and 5*Cr release curves. A control experiment for measuring the magnitude of this effect showed that the percentage decrease of *ORb and 51Cr in the medium 1.5 hr after

wCr fL+sc<

-Rb

(L’SC)

w 2

4

6

8

IO 12 Hours

14

yR4ycr I6 I8

(LI. 20

o 0 l 22

Fig. 2 I. Releu\e of markers from liposomes containing HbRb and “Cr by antibody and spleen cells. The make-up of each reaction mixture is indicated in parentheses behind the marker represented. The control experiments for assessing non-specific release include incubation of liposomes alone(L). ltposomes + spleen cells (L + SC) and liposomes + IgG antibody (L + AG). In the case of liposomes Incubated alone. and with liposomes incubated with antibody, both markers were released virtually at the same rate and. therefore. a single curve has been drawn through both sets of experimental potnts. The curves m the inset display the net release of “6Rb and 51Cr attributable to ADCC. Further correction of these net release data 1s required to take account of secondary marker uptake by the spleen cells, as explained in the text. Correction of HbRb ( x ) and 51Cr (+) release for this effect is shown for the I..5 hr and 4 hr samples. (From Shin PI cl/.. 197X0.)

itself. have the capacity to inhibit ADCC (Fig. 22). The authors interpreted this effect as an indication that ADCC involves phospholipase activity. However, we do not share this interpretation because we believe that the membrane attack in ADCC is mediated by insertion of channel-forming peptides rather than by phospholipasc activity. In line wjith this belief. we interpret Frye and Friou’s results as competition of Rosenthal’s inhibitor or phosphatidyl choline for hydrophobic peptides that thrm channds in lipid bilayers by insertion. Thus, in our view, the phosphatidyl choline inhibition experiments tend to support the BLM studies of Henkart and Blumenthal ( 1975).

As a working hypothesis we have proposed the concept that immunologically specific interaction between lymphocyte and target cell activates putative lymphocyte membrane-associated proteins and that this leads to exposure of hydrophobic peptides which then become inserted in the lipid bilayer of the target cell and form trans.membrane channels (scheme in Fig. 23). At present there are three experimental observations that can be interpreted as being in accord Hypothetic01 Mechanism of Cytolysis

addition OF spleen cells was I2 and 40”,,. respectively: after 4 hr it was 3 I and 50”,,, respectively. When the I .5 and 4 hr data in the inset of Fig. 21 were corrected for the secondary uptake effect by application of the respective decreases to “Cr and X”Rb. the retardation of 5‘Cr relative to S”Rb became negligible. as shown also in Fig. 21. Therefore. the experiment in Fig. 21 indicates that the lymphocyte-marker release was due to liposomal membrane damage and consequent direct release into the medium. However, since the precision of the data is not high due the low extent 01 marker release. and since the correction of secondary uptake represents a crude approximation, we cannot exclude the possibility that a small proportion 01 marker may have been released via the spleen cells.

3. Inhibition

of A DC‘<‘ /I), lecithin

or u lecithin

mulog

It has been reported by Frye and Friou (1975) that Rosenthal’s inhibitor. a synthetic analog of lecithin and an inhibitor ofphospholipase A. ;I> well as lecithin

by Lymphocytes

with this hypothesis. namely: (I) The BLM experiments of Henkart and Blumenthal (1975). (2) the demonstration of marker release from liposomes (Juy of ~1.. 1977; Shin ct ul., 1978~) and (3) the demonstration that lecithin and a lecithin analog inhibit cytolysis by lymphocytes (Frye bt Friou, 1975).

Immunologically

Mediated

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C.

d.,

Fowble J. W. & Joseph K. c‘. (1974) 11, 475. Barfort P.. Arquilla E. R. & Vogelhut P. 0. (196X)S~~rcww 160, 1119. Bhakdi S.. Bjerrum 0. J.. Rother U.. Kniifermann H. & Wallach D. F. H. (1975) Riwhit~7. hi&>,.\. .4( to 406. 21. Dourmashkln R. R. ( 1978) /nnrrurr~~k~,~~~ 35, 205. Eisenberg M.. Hall J. E. & Mead (- A (107.1) J. t?ww Biro/ 14, 143. Fettiplace R.. Andrew D. M. & Haydon D. A. (1971) .I rm’m. Blol. 5, 277. Frye L. D. & Frlou G. J. (1975) ~Vcrlwc, 258, 333. Gatelv M. K.. Mawr M. M. & Hennev C. S. (1976) Cc,// lmrnunol. 27, 82.. Giavedoni E. B. & Dalmasso A. P. (1976) J /wvrm 116, 1163. Goldlust M. B., Shin H. S.. Hammer C. H. & Mayer M. M. (1974) J. Imntun. 113. 99x. Goldman J. N.. Ruddy S. & Austen K. F. (1972) ./ Inrmw~. 109. 357 Goldman J. N. & Austcn K. F. (1974) _I itlfwt ni.5 129. 444. Gordon L. G. M. & Haydon D A. (1971) Bi~d~mr hiop/~~~.\ .‘lctu 2, 1014. Gotre 0. & Miiller-Eberhard H. J. (1970) ./. (‘Y/I .Mczl/ 132, 989. G&e 0. & Miiller-Eberhard H. J. (1971) J (‘x/7. &fr,rl 134, 90s. Green H., Fleischer R. A., Barrou P.&Goldberg B. (1959).1 r.\-p. Mecl. 109, 5 I I. Green H.. Barrow P. & Goldberg B. ( 1959~1)J c’_vp ,Mcd 1 IO. 699. Hammer C. H.. Nicholson A. & Mayer M. M. (1975) Proc. nutn. Ac~ad. SC; l:.S A. 72, 5076. Hammer C. H.. Shin M. L.. AbramowtT A. S. & Mayer M. M. (1977) J. Immw 119, I. Haxby J.. Kinsky C. & Kinsky S. (1968) Proc, wtn .Auul.SC,; C’.S.A. 61, 300 Haydon D. A. & Hladky S. B. (1972) Q. Rrr Bioph,vs 5, 1X7. Henkart P. & Blumenthal R. (1975) Proc. wlrt~ Ac& .%I. C’S A 72, 2789. Hladky S. B. & Haydon D. A. (1972) Biuchm~. h~oph~.r .4ctu 274, 294. Humphrey J. H. & Dourmashkin R. R. (1969) .4d1 Invmw~ol. Immunochemistr~~

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lnoue K. & Nelson R. A.. Jr. (1965) J. lmnnrn 95, 355. lnoue K. & Nelson R. A.. Jr. (1966) J. /nvwr~ 96, 386. lnoue K.. Kinoshita T . Okada M. & Akiyama Y. (1977) J Inwnun. 119, 65. Juliano R. L. (1973) Bioc~him h+/~~.\ Autr -30, 341. Juy D., Billecocq A.. Faure M. & Bona C’. (1977) .C~I~/ J Immutwl. 6, 607. Kinoshita T.. lnoue K.. Okada M. & Akiyama Y. (1977) J /mmun. 119, 73. Kmsky S. C.. Bon\en P P. M.. Kinsky C B.. van Deenen L. L. \d & Rosenthal A. F. (1971 ) B/oc~hcw hioph~~\. .lc,to 233. 8 15.

Mcmhranc

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x31

Klnskq S. C. (1972) Bioc#~wr hw,p/~~5 .4c,rtr 265. I. Kolb W. P.. Ha\b> J. A.. .Arroya\e C. M. & MiillerEbcrhard. H. J. (1973) .I <‘\,I ,Mvd. 138. 42X. Kolb W. P. & Miller-Ebcrhard H. J. (1973)./ <‘yp .wcc/ 138, 43X Lachmann P J.. Munn I;. A. Rr Weissmann G (1970) /wlnflrrl0/0~~~~19. 983. Lachmann P. J. &Thompson R. A. ( 1970) J. c\p .Mcr/ 131, 643. Mart/ t. ( 1977) C’rurtcw,p7~opic~.\ It~~t~~trmh~~/ 7, 301. Maqer M M ( I961 ) In Development of the one-hit theory 01 immune hcmol>\is. /r?r~~,lllloc~llc~fl~~l 11, .4p/J”‘““/r~.~ to Prrh/er~~~ in bficrd~idoy,~(Fdlted by Hcidelberger M. Kr Plescia 0. J.) pp. 26X-179. Rutger\ IJnlwrsity Press. Nw Brunswxk. New Jersey. Mayer M. M. (1972) Prcx wr!~ .4cwc/. .Sc,i C’ .S..4 69, 2954. Maqer M. M. (1977) .I Iwrtrw~ 119, 119.5 Maqcr M. M. ( 197X) //trr~,c’~, 1.~~~ I .%,I. 72. 1966 77. Michaels D. W.. Abramo\lt/ A. S.. Hammer C‘ H Kr Maler M. %I. (1976) f’t.w trc/t/t ,.1cd SC,/L’S.4 73. ‘852. Michaela D. W.. Abramo\ltl A. S.. Hammer C‘. H. & Mayer M. M. (lY7X) .I /wwo~ 120. 17X5. Michaels D. W. & Maqcr M. M. (197X(r) Biop/r~,\. .I 21. I25a. Mueller P. 6i Rudin D. 0. (1970) (‘1o.1. TO/J Bwcv~c~r~ 3, 157. Muller R. U. & Finkelsteln A ( 1972) ./. (rw I’l~~..,io/ 60, 263. Miiller-Eberhard H. J. ( 1975) -lwr Kc,\, Rioc~/wrn 44, 697. Podack E R.. Kolh W. P. & Milllcr-Ehcrhard H J. (1977) /-<&I Proc~ Fdt~ .Am .%I< \ <‘\,I I&I/ 36, 1’00. Podack E. R.. Halverson C’.. b:\wr A. t... Kolh W. P. & Miiller-Eberhard H. J. (197X) .I /wf11w1 120, 1791. Rautcrbcrg E. W. (197X) .I /wvw? 120, 1797. Robinson N. C K: Tanlixd (‘. (1475) Birdrtw~rtr~ 14, 369. Rudd> S.. Glgh I. & Austcn K F. (197’) !I<,II, ll,~,y/ .I .wct/ 287 14X9 . 545. 591. 64’ Shin H. S.. Plckerlng R. J. Xc ‘Llakcr M. M. (lY71) J. /r~wnrrt~ 106, 4X0. Shin M. L.. Paznekaq W. A.. Abramoviv A S. & Mayer M. M. (1977) .I. Iwviwr7 119. 1358. Shin M. L.. Parncka\ W. A & Maher M. M. (lY7X)./ /rwvw 120, 1996. Shin M. L.. Willoughby J. B.. Shin H. S.. Whitlow M. B. & Mayer M. M. (I 9780) ~&I. I’rr~c ~?(/II At11 .Soc.r(,\/~ Hwl. 37, 1272. Tanford C. ( 1973) In 7%w If~~&op/whit U/w/, p. 126. John Wile). New York. Thompson R. 4. & Lachmann P J. (1970) .I (‘I/,