STUDIES ON THE HUMAN AND GUINEA PIG SERUM COMPLEMENT SYSTEMS WITH LIPOSOMAL MODEL MEMBRANES Thesis for the Degree of PM D. MICHIGAN STATE UNIVERSITY KRISTINE CALLENBACH KNUDSON; 1971 LIBRARY Michigan Sure UnivefiitY This is to certify that the thesis entitled Studies on the Human and Guina Pig Serum Complement Sys tems with Liposomal Model Membranes ' presented by Kristine Callenbach Knudson has been accepted towards fulfillment of the requirements for _Bh_=D-__degree in msmhialngy and ' Public Health Major professor 5/ zgg:1‘4f3/%7;¥<:ééf::\~7’ I / I Date W 0-7639 III II I IIIIIIIIIIIIIIIIIIIIIIII 3 1293 01004 5270 . . m I .3 I994 ABSTRACT STUDIES ON THE HUMAN AND GUINEA PIG SERUM COMPLEMENT SYSTEMS WITH LIPOSOMAL MODEL MEMBRANES BY Kristine Callenbach Knudson The objective of this study was to investigate the human and guinea pig serum complement systems with liposomal model membranes. Liposomes prepared from sheep erythrocyte membranes at a concentration of 2 pmoles phospholipid/m1 of galactose marker solution were used. Modifications in the procedure for preparation of liposomes, which included varying the phospholipid concentration and prolonged sonic- ation, resulted in preparations of great stability. A simplified assay for the detection of released galactose indicative of liposome lysis was developed based on the oxidation of galactose by galactose dehydrogenase (EC 1.1.1.48) and the corresponding reduction of nico- tinamide adenine dinucleotide (NAD) was monitored spectrophotometrically at 340 nm. Liposomes were lysed by guinea pig serum (as the source of complement) in conjunction with anti-sheep cell antibody and the degree of lysis was dependent on serum concentration. Based on this dependency, the liposome system was developed into a quantitative assay for measurement of complement activity. Electron micrographs Kristine Callenbach Knudson of the serum-lysed liposomes revealed that no discrete lesions were produced during lysis, but rather the liposomes seemed to fragment. Studies on the lysis of liposomes by fresh human serum and on the binding of functionally pure complement components indicated that at least two mechanisms were operating simultaneously to cause lysis of the liposomes. The first mechanism appeared to be the classical complement sequence, requiring all nine complement compon- ents, antibody, Ca++, and Mg++. The second mechanism appeared to require the late-acting components (C3-C9) and Mg++, and the C3 proactivator system was hypothesized to be involved. Using specific antisera against C3 and the C3 proactivator, it was demonstrated that the C3 proactivator system participated in the lysis of liposomes. In addition, a factor, sensitive to diisopropylfluorophosphate and stable to heating at 56°C, was detected in the anti-sheep cell anti- serum and this factor apparently participated in liposome lysis. STUDIES ON THE HUMAN AND GUINEA PIG SERUM COMPLEMENT SYSTEMS WITH LIPOSOMAL MODEL MEMBRANES By Kristine Callenbach Knudson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1971 DEDICATION To my husband, David, who always offered encouragement, understanding, and helpful discussions, and to my parents who instilled in me the value of a good education. ii ACKNOWLEDGEMENTS I would like to express my sincere thanks and appreciation to Dr. David H. Bing for all his help and guidance during my graduate program. His willingness to discuss at any time my progress and problems and to offer his assistance in countless ways is greatly appreciated. Appreciation is also expressed to the other members of my Ph.D. guidance committee, Dr- Leland Velicer, Dr. Robert Moon and Dr. Philip Filner. I would also like to thank Dr. Robert Brubaker for acting as the moderator of my committee. Sincere thanks also go to Laurence Kater, Sandra Spurlock, Bonnie Mernitz and Carlos Sledge for their friendly presence in the laboratory, constructive criticism, and helpful discussions. I would also like to thank the Department of Microbiology and Public Health for its continual financial support during the course of my study. This support was in the form of department assistant- ships, a National Science Foundation Summer Fellowship for Teaching Assistants, and a pre-doctoral fellowship provided by a grant awarded to the department by the National Institutes of Health. iii TABLE OF CONTENTS INTRODUCTION LITERATURE REVIEW Part I. The Complement System Nomenclature of Complement The Complement Sequence Biologically Active By-Products of the Complement System II. Alternative Pathways into the Complement System Cobra Venom Factor Plasmin and Trypsin Endotoxic Lipopolysaccharide The Properdin System III. Liposomal Model Membranes BIBLIOGRAPHY ARTICLE 1 Quantitative Measurement of Guinea Pig Complement with Liposomes. K.C. Knudson, D.H. Bing and L. Kater. 1971. Journal of Immunology. 106: 258. ARTICLE 2 Evidence for Alternate Mechanisms for Immune Lysis of Liposomal Model Membranes. K.C. Knudson and D.H. Bing. Manuscript in preparation. ARTICLE 3 A.Simplified Method for the Centrifugation of Liposomes. K.C. Knudson and D.H. Bing. Manuscript in preparation. iv Page II 11 14 17 20 28 34 6O LIST OF FIGURES Figure Page 1. The Complement Sequence 5 2. Relationship of Alternate Pathways with the Complement System 9 INTRODUCTION The complement system consists of nine components comprised of eleven proteins which interact sequentially with each other to mediate cellular injury and promote the inflammatory response. Druing the past thirty years a model system of sheep erythrocytes, rabbit anti-sheep cell antibody and serum has been used to study the consequences of the activated complement system. Using this model system, a great amount of information on the complement system was obtained. But to study the enzymatic activities of the individual complement components bound to a membrane as in the in vivo situation, an alternative model system was necessary to avoid the contamination of enzymatic reactions by the cellular contents of erythrocytes. The liposomal model membrane system appeared to be the alternative membrane system for these studies. Liposomes are vesicular lipid bilayers, prepared, in this instance, by swelling the phospholipid extract of sheep erythro- cyte membranes in an aqueous galactose solution. The purpose of this study was to use the liposomal model membrane system as a tool for the study of the guinea pig and human serum complement systems. The first component of complement (CI) binds to the cell membrane through an interaction of one of its subunits, CIq, with antibody present on the cell surface. Initial studies on the binding of C1 to liposomes indicated that the C1 was bound non-specifically in the absence of antibody, with little enhancement of binding when antibody was present. Since previously it had been shown that fresh serum (as the source of complement) lysed liposomes, and that the second and eighth components of complement were required for lysis, the roles of other factors in the lytic mechanism.were investigated. This thesis is organized into four sections. The first is a literature review in which information on the complement system, the known alternate pathways into the complement system, and liposomal model membranes is presented. The second section consists of a published manuscript on the application of liposomes for the quanti- tative measurement of guinea pig complement. The third and fourth sections consist of a manuscript and a communication to be submitted for publication and concern the evidence for alternative mechanisms for lysis of liposomes and a procedure for the centrifugation of liposomes. LITERATURE REVIEW Part I The Complement System An extensive review of the complement system is beyond the scope of this survey. Several comprehensive reviews have been written (1,2,3,4,5). The following review will be concerned with the nomen- clature of the complement system, highlights of the complement reaction sequence, and a brief discussion of some of the system's biological implications. Nomenclature of Complement. The term complement is applied to a system of factors occurring in normal serum that are characteris- tically activated by antigen-antibody interaction and subsequently mediate a number of biologically significant consequences. Most of the information of the complement system has been derived from studies with a model system consisting of sheep erythrocytes (E) treated with rabbit antibody (A) which are then lysed upon the addition of fresh normal serum as the source of complement (C)(1,6,7). The present terminology devised for the complement system is based upon this immune lytic sequence. This terminology was agreed upon at a series of discussions arranged by the World Health Organization (8). The nine distinct complement components which interact sequen- tially with each other in immune hemolysis are numbered C1-C9. The first four components are designated C1, C4, C2, and C3 because of long-standing usage, but the five remaining components are numbered sequentially (3:3,, C5, C6, C7, C8, C9). Intermediate complexes are designated by EAC followed by those components which have interacted. The components enumerated after EAC denote a state of reactivity and not necessarily their physical presence (34g., EAC1,4,2,3). Alterna- tively, the example may be shortened to EAC1-3. A bar or rule placed over a complement component is used to indicate that the component is in an active enzymatic or other biological state (e.g., C1 or C5,6,7). The loss of defined activity by a complement component is denoted by the suffix "i". Fragments which result frmm cleavage of complement components during the course of their reaction are suffixed sequen- tially with lower case letters (24g., C3a). The Complement Sequence. A detailed diagram of the complement sequence is shown in Figure 1. The complement system is activated upon interaction of C1 with an antigen-antibody complex. C1 is comprised of three subunits, designated C1q, C1r and C13, which function as a unit and are bound together by Ca++(9). C1q possesses the binding site of C1 for the antibody and in binding to the anti- body undergoes a conformational change (10). C1r is then activated and in turn acts upon C13 converting the inactive proenzyme to its active esterase form, CT; (9,11). CT splits C4 into two fragments, the larger of which is bound to the cell membrane to form the stable complex, EAC1,4b (12,13). C2 is also cleaved by CT and the larger fragment, C2a, forms a cell-bound complex of EAC1,4b,2a in the presence of Mg++ (14,15). A o.m.a.o.nmmmm.a~.as.wvaou may «.30 no emu + emu wuo< mu onano moo u eue< H omouuo>aou Aooo.oqr u 32v thunooum Illllllwnouu>afloaoum mu ++wz ++wz Amoov Houoau ano> aunoo 4 Aooo ow u 32v Aeuesoem mo ooaoavom unwfiofiafioo ecu gums maaanuam ouoauoua< mo magmaofiuofiom N MMDUHm 10 sedimentation coefficient (35). It has a molecular weight of 80,000 (37). Recent studies on this serum factor show that it possesses three different forms (38). One form (A) complexes with CoF, form B cleaves C3 in the absence of CoF, and form C is present in eluates from zymosan previously treated with serum at 17°C. It is hypothesized that the changes in the proactivator proteins are caused by an unknown enzyme in serum (38). The proactivator forms a complex with CoF which has a molecular weight of 220,000 and a 8 value of 98 (36,37). This complex, termed C3 activator, acts enzymatically on C3 cleaving it into two fragments, one of which has anaphylatoxin activity similar to C3a (36,37,38). The formation of the complex between GOP and C3PA is dependent on the presence of Mg++ with the formation being prevented by prior addition of EDTA (39) and upon the temperature (40). It does not react on cell-bound C3 and the activation of fluid-phase C3 by this complex is independent of antibody (40). In in vitro studies with guinea pig serum, CoF was found to inactivate not only C3, but also C5, C6, C7, C8 and C9 (41). CoF had no detectable effect on the early components. The apparent discrepency in the literature con- cerning the consumption of the six terminal components was thought to be due to variation in serum dilution and CoF concentration. The consumption of C5-C9 was dependent on the concentration of the react- ants, whereas the consumption of C3 was not (41). Other studies with guinea pig serum demonstrated that GOP and guinea pig serum would cause hemolysis of unsensitized erythrocytes (42,43). The complex formed would react with C-EDTA to cause depletion of the terminal components. The hemolytic activity of GOP 11 was dependent upon an intact complement system (42). Lysis of unsensitized erythrocytes by CoF and guinea pig serum.was dependent on the formation of a complex between CoF and the proactivator. Hemolysis required the late-acting components. No lysis occurred in genetically C6-deficient rabbit serum or in zymosan-treated serum (39). Plasmin and Trypsin. Incubation of C3 with small amounts of trypsin for short periods of time produces a cleavage product of C3 with identical properties to C3a (30). Plasmin can also cleave a fragment of 6,000 molecular weight from C3 which possesses chemotactic activity for polymorphonuclear leukocytes (44). This chemotactic factor is different from the previously described chemotactic factor derived from.C5:6:7 both in terms of requirements for generation and in phys- ical properties (45). However, the biological properties of the various C3 cleavage products are identical (43). Endotoxic Lipopolysaccharide. The terminal portion of the comple- ment system is a potent source of biologically active by-products. These by-products perform functions which are very similar to those observed in studies on the biological activities mediated by endo- toxins (46). The interaction of serum and endotoxic lipopolysaccharide (LPS) generates the complement-dependent biologically active products possessing neutrophil chemotactic activity and anaphylatoxin (47,48, 49). Following the administration of LPS to animals or man, several physiological changes take place, among which are contraction of smooth muscle and increased vascular permeability. The small amounts of LPS required to bring these alterations about has led to speculation that LPS does not act directly on the tissues, but indirectly through 12 a system of serum.proteins, namely the complement system (46,47,50). Endotoxins when injected into animals consume complement or are anti- complementary (51,52,53,54). Endotoxic LPS has a potent ability to interact with complement during incubation in normal mammalian serum (55,56). Lesions indicative of terminal complement component activ- ation appear on LPS after reactions with fresh serum (55), as well as on the bacterial cell from which the LPS was derived (49,57). A correlation between the endotoxicity of LPS and its ability to conswme complement exists (5). The complement-consuming ability of LPS is lost following several different modifications which result in loss of biological activity. Especially noticeable is the dramatic decrease in consumption of the terminal components during detoxifi- cation (56). The most striking characteristic of LPS interaction with comple- ment is that a preferential consumption of each of the six terminal components occurs (54). These findings support the hypothesis based on the previously described electron microscope data that the lesions observed on LPS are produced by activation of C9 in a manner similar to that required for the production of lesions on the erythrocyte membrane. LPS has little ability to consume substantial amounts of the early-acting components (C1,C4,C2) (51,55,56,58). Because of the marked consumption of the terminal components with little or no consumption of the early-acting components by LPS, it was unclear whether the LPS activated the C3-C9 components via the usual mechanism of antibody-C1, C4, C2, or whether another pathway into the system existed. Studies with immunoglobulin-deficient sera showed 13 that bacterial LPS could induce complement consumption and that large amounts of antibody were not necessary for this interaction to occur (59,60). Extremely small quantities of antibody were hypothesized to suffice for the initiation of the complement sequence (60). LPS was hypothesized to interact with trace amounts of immunoglobulin in serum in a way as to lead to consumption of the terminal components, but to little, if any, consumption of the early-acting components (60). The necessity for additional serum factors for the activation of the six terminal components by LPS was studied. Incubation of puri- fied preparations of the six terminal components with LPS induced no complement consumption (54). Prior incubation of bacterial endotoxin in undiluted normal serum formed a serum-endotoxin intermediate, termed LPS-X, which contained at least six different serum proteins including a ¥'-globulin and C3 (61). LPS-X, but not LPS, destroyed 2 purified C3, C5, C8 and C9. C3 was cleaved by LPS-X into two frag- ments, one of which was indistinguishable from C3a (61). Even though LPS can efficiently activate the terminal components without detectable consumption of C1, C4 or C2 (54), the participation 'of the early-acting components, as well as antibody, cannot be excluded because LPS may promote an extremely efficient Cz:2 convert- ase formation (1). Experiments performed by Jensen using a specific C4 inactivator found in shark serum demonstrated that endotoxin failed to fix the terminal components when incubated with the C4 inactivator (62). These results would indicate that at least small quantities of the early-acting components are required to consume C3-C9. This data would support the hypothesis of an extremely efficient utilization of 14 the early-acting components by LPS. The Properdin System. The properdin system emerged from studies on the mechanism of inactivation of the third component of complement (C3)1 by zymosan, an insoluble carbohydrate derived from yeast cell walls. In 1954 Pillemer and associates isolated properdin and described its properties and role in the immune system (51). The properdin system consisted of a unique serum.euglobulin, termed properdin, Mg++3 and serum factors resembling C1, C2 and C4 (51,63,64). Properdin reacted with zymosan at 17°C to form a complex (PZ) which in turn reacted with and inactivated C3 at 37°C (51). A.method for the assay of properdin was devised based on the requirement of properdin for the inactivation of C3 by zymosan (65).. Properdin could be eluted from the P2 complex (51,65,66) and this was its main means of isolation. The inactivation of C3 by zymosan and properdin occurs in two stages (51). The first stage is the combination of properdin with zymosan at 17°C to form the P2 complex. Two other serum factors are required at this stage for formation of the P2 complex. The first of . these two factors is Factor A, which is hydrazine-sensitive, but has been shown not to be identical with C4 (also hydrazine-sensitive) (67). The second required factor, Factor B, is heat-labile, but could not be identified with either C1 or C2, which are also sensitive to heat (68). Mg++ is also required for the formation of the P2 complex (51). Mg++ can be replaced by Co++ or Mn++, but Mg++ is the most effective cation (69). The second stage is the inactivation of C3 by P2 (51). Mg++ 1C3, as recognized in 1954, was the only complement component known to follow the C1,4,2 sequence. C3, therefore, refers to one or more of the components in the sequence beginning with C3 (C3,C5-C9). 15 is also required for this stage, but Factors A and B are not (51,67,68). The properdin system was implicated in a variety of immunologic reactions. The properdin system was determined to be bacteriocidal against a variety of Gramrnegative microorganisms (70,71). Removal of properdin from the serum also abolished bacteriocial activity. Addition of properdin to properdin-deficient serum restored activity against bacteria. Because of similarities in the requirements for properdin activity and the hemolysis of erythrocytes from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), properdin was determined to be required for the hemolysis of PNH cells (72). Properdin was also postulated to have a role in virus neutralization (73) . The concept of properdin as being a unique serum protein was challenged by several investigators (74,75,76). Nelson suggested that properdin was an antibody or group of antibodies to zymosan, present in small amounts in serum, and fixed complement and destroyed C3 in a manner similar to that of an antigen-antibody complex (74,77). However, several important differences existed between a typical antigen-antibody reaction and the reaction of properdin and zymosan. The formation of P2 was very dependent on environmental conditions, complexing only in a very small termpeature and pH range, requiring Mg++, Factors A and B, and being very dependent on ionic strength (51, 78,79). Also PZ had very little effect on the early-acting cmmponents, whereas antigen-antibody complexes inactivate C1, C2 and C4, with little effect on C3 (79). From these differences properdin was designated as a unique serum protein and not antibody to zymosan. 16 One of the reasons for the controversy over the existence of properdin was the heterogeniety of the properdin preparation (51,65, 66,80). More than a decade after the first description of properdin, a report appeared which discussed the properties of a highly purified properdin (79). This purified properdin consisted of a single protein band in electrophoresis, contained no antigenic determinants to IgM, IgG or IgA, or to any of the complement components, agglutinins to zymosan or antibody to the test bacterial strain. It was thus re- established that properdin was not an antibody. It could be distin- guished from immunoglobulin in both its physiochemical and antigenic properties and by its behavior in an immunologic system. It was re- confirmed that properdin was not a member of the complement system, performing none of the functions of complement in the assay system. The purified properdin had a sedimentation coefficient of 5.28 and a molecular weight of 223,000. Recently the LPS-X factor has been related to properdin (81). Both LPS and properdin interact with the complement system through an intermediate (LPS-X or PZ). At this time, however, the relationship between X and properdin cannot be defined. Also it has been estab- lished thatthe C3 proactivator (C3PA) is Factor B of the properdin system (82). Identity of the other factors of the C3PA alternate pathway with factors of the properdin system have yet to be deter- mined. 17 Part III Liposomal Model Membranes One of the most successful systems for the study of lipid bilayers and their biological importance is the liposomal model membrane (83). To form these model membranes dry lipids are swollen in an aqueous solution to form liquid crystalline structures, which are called liposomes (83). Low molecular weight substances can be trapped in the aqueous interiors of the liposomes (83). With the aid of electron microscopy, the polyene antibiotic filipin was shown to produce pits in erythrocyte membranes and lecithin-cholesterol liposomes similar to the lesions observed in erythrocytes after immune hemolysis with anti- body and complement (84). Based on these observations the liposomes were investigated as a model membrane system with which to study the mechanism of complement action (85). Liposomes prepared from the phospholipid extract of sheep erythrocyte membranes released their trapped marker (glucose) when incubated with both rabbit antiserum prepared against sheep erythrocytes (as the source of antibody) and guinea pig serum (as the source of complement) (85). A spectrophoto- metric assay based on the detection of the released glucose with hexokinase, ATP, glucose-6-phosphate dehydrogenase and NAD was developed (85). Other studies indicated that liposomes prepared from the phospholipid extract could bind (neutralize) antibodies in rabbit hemolysin and fix guinea pig complement (86). The antigen that confers immune sensitivity to the liposomes appears to be the Forssman antigen (87). This antigen is found in 18 sheep erythrocyte membranes and is also present in the phospholipid extract of these membranes. The proposed structure for the Forssman antigen is: N-Ac-Gal(¢.1-3)Gal (fi1-3)Gal (p1-4)-Glc-1-ceramide (87) . Since the Forssman antigen is present both in the intact sheep erythro- cyte and in the phospholipid used to prepare the liposomes, antiserum prepared against the Forssman antigen may be used as the source of antibody for the complement- and antibody-mediated lysis of liposomes. This antiserum is called amboceptor or hemolysin. Modification of the preparation of the liposomes and of the spectrophotometric assay produced more stable liposomes and provided for a less complicated enzymatic assay for the detection of the trapped marker, galactose (88). With these stable liposomes, it was shown that the degree of lysis of the liposomes was dependent on the amount of complement added to the system (88). Electron micrographs revealed the liposomes to consist of vesicles bounded by several bilayers (88). Studies on the mechanism of liposome lysis by complement and antibody indicated that lipids alone may serve as the substrate for complement (87). It did not appear that cell membranes contained any unique and specific receptor sites. Additional studies revealed that no degradation of the phospholipid occurred during liposome lysis (89). The complement-dependent damage to liposomes did not appear to occur by the enzymatic rupture of covalent bonds in the phospholipids. The use of purified human complement components for the lysis of liposomes indicated that loss of the marker was dependent on the presence of C2 19 and C8 (90). C9 did not appear to be an absolute requirement for liposome lysis although its presence enhanced marker release. 10. 11. 12. BIBLIOGRAPHY MUller-Eberhard, H.J. 1968. Chemistry and reaction mechanisms of the complement system. Adv. Immunol. .8: 1. MUller-Eberhard, H.J. 1968. The serum complement system. In. Textbook of Immunopathology, Vol. I. P.A. 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The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. Exp. Med. 127: 371. Ward, P.A., C.G. Cochrane, and H.J. Mallet-Eberhard. 1965. The role of serum complement in chemotaxis of leukocytes in vitro. Jo Exp. Med. 122: 3270 Ward, P.A., C.G. Cochrane, and H.J. MUller-Eberhard. 1966. Further studies on the chemotactic factor of complement and its formation in vivo. Immunology 11: 141. Ward, P.A., and E.L. Becker. 1967. Mechanisms of inhibition of chemotaxis by phosphonate esters. J. Exp. Med. 125: 1001. Klein, P.G., and H.J. Wellensiek. 1965. Multiple nature of the third component of guinea pig complement. 1. Separation and characterization of three factors a,b, and c, essential for hemolysis. Immunology g: 590. Mflller-Eberhard, H.J., U.R. Nilsson, A.P. Dalmasso, M.J. Polley, and M.A. Calcott. 1966. A molecular concept of immune cytolysis. Arch. Path. .QE‘ 205. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 23 Cochrane, C.G., and H.J. Mflller-Eberhard. 1967. Biological effects of C'3 fragmentation. Fed. Proc. '26: 362. Cochrane, C.G., H.J. Mflller-Eberhard, and K. Fjellstrom. 1968. Capacity of a cobra venom protein to inactivate the third component of complement (C'3) and to inhibit immunologic reactions. J. Clin. Invest. .41: 21a. Gfltze, 0., and H.J. MUller-Eberhard. 1971. Isolation of the precursor and the active form of the C3 activator from.hummn serum. Complement Workshop, Jan. 27-29, Baltimore, MD. .Ballow, M., and C.G. Cochrane. 1969. Two anticomplementary factors in cobra venom: Hemolysis of guinea pig erythrocytes by one of them. J. Immunol. 103: 944. Bitter-Suermann, D., M. Dierich, W. Konig, and U. Hadding. 1971. Enzymatic activation of C3 and C5 by a complex derived from a purified cobra venom factor and a serum globulin. Complement Workshop, Jan. 27-29, Baltimore, MD. Shin, H.S., H. Gewurz, and R. Snyderman. 1969. Reaction of a cobra venom factor with guinea pig complement and generation of an activity chemotactic for polymorphonuclear leukocytes. Proc. Soc. Exp. Biol. Med. .131: 203. Pickering, R.J., M.R. Wolfson, R.A. Good, and H. Gewurz. 1969. Hemolysis induced by cobra venom factor activation of terminal complement (C') components in guinea pig serum.(GPS). Fed. Proc. 28; 818. Pickering, R.J., M.R. Wolfson, R.A. Good, and H. Gewurz. 1969. Passive hemolysis by serum and cobra venom factor: a new mechanism inducing membrane damage by complement. Proc. Natl. Acad. Sci. ‘62: 521. Ward, P.A. 1967. A new chemotactic factor released from C'3 by plasmin. Fed. Proc. 22: 244. Ward, P.A. 1967. A plasmin-split fragment of C'3 as a new chemotactic factor. J. Exp. Med. 126: 189. Mergenhagen, S.E., R. Snyderman, H. Gewurz, and H.S. Shin. 1969. Significance of complement to the mechanism of action of endo- toxin. Curr. Topics Microbiol. Immunol. 22: 37. Lichtenstein, L.M., H. Gewurz, N.F. Adkinson, Jr., H.S. Shin, and S.E. Mergenhagen. 1969. Interactions of the complement system with endotoxic lipopolysaccharide: the generation of an anaphy- latoxin. Immunology 16: 327. 24 48. Gewurz, H., H.S. Shin, and S.E. Mergenhagen. .1968. Interactions of the complement system with endotoxic lipopolysaccharide: consumption of each of the six terminal complement components. J. Exp. Med. 128: 1049. 49. Snyderman, R., H. Gewurz, and S.E. Mergenhagen. 1968. Interaction of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polymorphonuclear leuko- cytes. J. Exp. Med. .128: 259. 50. Gewurz, H., H.S. Shin, R.J. Pickering, R. Snyderman, L.M. Licht- enstein, R.A. Good, and S.E. Mergenhagen. 1969. Interactions of the complement system with endotoxic lipopolysaccharides: comple- ment-membrane interactions and endotoxic-induced inflammation. In Cellular Recognition. R.T. Smith and R.A. Good, editors. Appleton-Century-Crofts, New York. 305. 51. Pillemer, L., L. Blum, I.H. Lepow, 0.A. Ross, E.W. Todd, and A.C. Wardlaw. 1954. The properdin system and immunity. I. Demon- stration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 129: 279. 52. Kostka, J., and J. Sterzl. 1963. The action of endotoxin on complement. Folia Micrbiol. Z: 191. 53. Pearlman, D.S., J.B. Sauers, and D.W. Talmadge. 1963. The effect of adjuvant amounts of endotoxins on the serum.hemolytic comple- ment activity in rabbits. J. Immunol. 21: 748. 54. Muschel, L.H., K. Schmoker, and P.M. Webb. 1964. Anticomplemen- tary action of endotoxin. Proc. Soc. Exp. Biol. Med. 117: 639. 55. Bladen, H.A., H. Gewurz, and S.E. Mergenhagen. 1967. Inter- actions of the complement system with the surface and endotoxic lipopolysaccharide of Veillonella alcalescens. J. Exp. Med. 125: 767. . 56. Gewurz, H., S.E. Mergenhagen, A. Nowotny, and J.K. Phillips. 1968. Interactions of the complement system with native and chemically modified endotoxins. J. Bact. ‘25: 397. 57. Bladen, H.A., R.T. Evans, and S.E. Mergenhagen. 1966. Lesions in Escherichia coli membranes after action of antibody and comple- ment. J. Bact. ‘21: 2377. 58. Gewurz, H., R.J. Pickering, G. Naff, R. Snyderman, S.E. Mergen- hagen, and R.A. Good. 1969. Decreased properdin activity in acute glomerulonephritis. Int. Arch. Allergy '36: 592. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 25 Gewurz, H., R. Snyderman, H.S. Shin, L.M. Lichtenstein, and S.E. Mergenhagen. 1968. Complement (C') consumption by endotoxic lipopolysaccharide (LPS) in immunoglobulin-deficient sera. J. Clin. Invest. ‘41: 39a. Gewurz, H., R.J. Pickering, R. Snyderman, L.M. Lichtenstein, R.A. Good, and S.E. Mergenhagen. 1970. Interactions of the comple- ment system with endotoxic lipopolysaccharides in immunoglobulin- deficient sera. J. Exp. Med. ‘121: 817. Shin, H.S., R. Snyderman, E. Frideman, and S.E. Mergenhagen. 1969. Cleavage of guinea pig C'3 by serumrtreated endotoxic lipopolysaccharide. Fed. Proc. ‘28: 485. Jensen, J.A. 1969. A specific inactivator of mammalian C'4 iso- lated from nurse shark (Ginglymostoma cirratum) serum. J. Exp. Med. 130: 217. Pillemer, L., L. Blum, J. Pensky,and I.H. Lepow. 1953. The requirement for magnesium ions in the inactivation of the third component of human complement (C'3) by insoluble residues of yeast cells (zymosan). J. Immunol. 11: 331. Pillemer, L., I.H. Lepow, and L. Blum. 1953. The requirement for a hydrazine-sensitive serum factor and a heat-labile serum factor in the inactivation of human C'3 by zymosan. J. Immunol. ‘11: 339. Pillemer, L., L. Blum, I.H. Lepow, L. Wurz, and B.W. Todd. 1956. The properdin system and immunity: III. The zymosan assay for properdin. J. Exp. Med. 103: 1. Todd, B.W., L. Pillemer, and I.H. Lepow. 1959. The properdin system and immunity: IX. Studies on the purification of human properdin. J. Immunol. 82: 418. Pensky, J., L. WUrz, L. Pillemer, and I.H. Lepow. 1959. The properdin system and immunity. XII. Assay, properties and partial purification of a hydrazine-sensitive serum factor (Factor A) in the prOperdin system. Z. Immunoforschg. .118: 329. Blum, L., L. Pillemer, and I.H. Lepow. 1959. The properdin system and immunity. XIII. Assay and properties of a heat- labile serum factor (Factor B) in the properdin system. Z. Immunoforschg. 118: 349. Wardlaw, A.D., L. Blum, and L. Pillemer. 1956. Replacement of magnesium.by cobalt and manganese in the interactions of the properdin system with bacteria and zymosan. J. Immunol. 81: 43. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 26 Wardlaw, A.D., and L. Pillemer. 1956. The properdin system and immunity. V. The bacteriocidal activity of the properdin system. J. Exp, Med. 103: 553. Wedgewood, R.J. 1960. Antibody requirement for interaction of properdin and bacteria. Fed. Proc. 12: 79. Hinz, C.F.,Jr., W.S. Jordan,Jr., and L. Pillemer. 1956. The properdin system and immunity. IV. The hemolysis of erythro- cytes from patients with paroxysmal nocturnal hemoglobinuria. J. Clin. Invest. 32: 453. Wedgewood, R.J., H.S. Ginsberg, and L. Pillemer. 1956. The properdin system and immunity. VI. The inactivation of Newcastle disease virus by the properdin system. J. Exp. Med. 104: 707. Nelson, R.A. 1958. An alternative mechanism for the properdin system. J. Exp. Med. 108: 514. Mayer, M.M. 1961. Complement and complement-fixation. 12 Experimental Immunochemistry. E.A. Kabat and M.M. Mayer, editors. Charles C Thomas, Springfield. 133. Muschel, L.H. 1961. The antibody-complement system and properdin. A review. Vox. Sang. 6: 385. Blum, L., L. Pillemer, and I.H. Lepow. 1959. The properdin system and immunity. XI. Studies on the interaction of zymosan with the properdin system. Z. Immunoforschg. 118: 313. Lepow, I.H. 1961. The properdin system: A review of current concepts. IE_Immunochemica1 Approaches to Problems in Micro- biology. M. Heidelberger and 0.J. Plescia, editors. Rutgers University Press, New Brunswick. 280. Pensky, J., C.F. Hinz,Jr., B.W. Todd, R.J. Wedgewood, J.T. Boyer, and I.H. Lepow. 1968. Properties of highly purified human properdin. J. Immunol. 100: 142. Lepow, I.H., L. Pillemer, M. Schoenberg, B.W. Todd, and R.J. Wedgewood. 1959. The properdin system and immunity. X. Characterization of partially purified human properdin. J. Immunol. ‘83: 428. Marcus, R.L., H.S. Shin, and M.M. Mayer. 1971. An alternate complement pathway: 03 cleaving ability, not due to C4,2a on endotoxic lipopolysaccharide after treatment with guinea pig serum; releation to properdin. Proc. Natl. Acad. Sci. .§§‘ 1351. 82. 83. 84. 85. 86. 87. 88. 89. 90. 27 Goodkofsky, I., and I.H. Lepow. 1971. Functional relationship of Factor B in the properdin system to C3 proactivator of human serum. J. Immunol. In press. Bangham, A.D., M.M. Standish, and B.C. Watkins. 1965. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13: 238. Kinsky, S.C., S.A. Luse, D. Zopf, L.L.M. vanDeenen, and J.A. Haxby. 1967. Interaction of filipin and derivatives with ery- throcyte membranes and lipid dispersions: Electron microscopic observations. Biochim. Biophys. Acta. 132: 844. Haxby, J.A., C.B. Kinsky, and S.C. Kinsky. 1968. Immune response of a liposomal model membrane. Proc. Natl. Acad. Sci. ‘61: 300. Alving, C.R., S.C. Kinsky, J.A. Haxby, and C.B. Kinsky. 1969. Antibody binding and complement fixation by a liposomal model membrane. Biochemistry 8: 1582. Kinsky, S.C., J.A. Haxby, D.A. Zopf, C.R. Alving, and C.B. Kinsky. 1969. Complement-dependent damage to liposomes prepared from pure lipids and Forssman hapten. Biochemistry 8: 4149. Knudson, K.C., D.H. Bing, and L. Kater. 1971. Quantitative measurement of guinea pig complement with liposomes. J. Immunol. 106: 258. Inoue, K., and S.C. Kinsky. 1970. Fate of phospholipids in liposomal model membranes damaged by antibody and complement. Biochemistry 2: 4767. Haxby, J.A., 0. GBtze, H.J. MUller-Eberhard, and S.C. Kinsky. 1969. Release of trapped marker from liposomes by the action of purified complement components. Proc. Natl. Acad. Sci. '64: 290. ARTICLE 1 Quantitative Measurement of Guinea Pig Complement with Liposomes By K.C. Knudson, D.H. Bing and L. Kater Reprinted from Journal of Immunology, 106: 258, 1971 28 [HE JOURNAL OF IMMUNOLOGY vm. Hm, an. I. Jnuum'y w. Copyright © 1971 by The ‘Villiams & Wilkins Co. Printed in H.S.A. QUANTITATIVE MEASUREMENT OF GUINEA PIG COMPLEMENT WITH LIPOSOMESU" KRISTINE C. KNUDSON,3 DAVID II. BING AND LAURENCE KATER‘ From [ht Dvpurhncnl of .Ilivmbiulog/l (mrl Publir Hmllh, Michigan Sin/e L'nivcrxily. Ens! Lansing, Michigan 4x833 {waived for publication] Mun-h 4, 1970 I'I"|"‘l III ill Tm: JOURNAL or IMMUNOLOGY Copyright © 1971 by The Williams 6: Wilkins Co. -. _—_m. _ .« .._ T-“v~ mew)”; f,, -v Vol. 106, No. 1. January 1971 Printed in U.S.A. QUANTITATIVE MEASUREMENT OF GUINEA PIG COMPLEMENT WITH LIPOSOMES" 2 KRISTINE C. KNUDSON,’ DAVID H. BING AND LAURENCE KATER‘ From. the Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48823 Received for publication March 4, 1970 Liposomes were investigated as an alternative reagent to sheep erythrocytes for quantitative titration of whole complement activity. The liposomes were prepared with various concentrations of phospholipid and were sonicated for more effective dispersion. It was found that reproducible complement titrations could be performed with sonicated liposomes prepared with l or 2 nmol of phospholipid per milliliter of marker solution (0.3 M galactose). Electron micrographs of liposomes treated with complement and hemolysin showed no discrete lesions, but rather aggregates of liposome fragments. Lipid dispersions, termed liposomes, can be used as a model membrane system to study lytic mechanisms. Low molecular weight substances can be trapped in the aqueous regions of the liposomes (1). Using electron microscopy Kinsky et al. (2) showed that the antibiotic filipin pro- duced pits in erythrocyte membranes and lecithin- cholesterol liposomes similar to the holes observed in erythrocytes after immune hemolysis with complement and antiserum. Based on these observations, Haxby et al. (3) investigated the feasibility of using liposomes as an artificial membrane system to study the mechanism of complement action. Liposomal membranes were prepared from sheep erythrocytes, and a spectro- photometric assay was devised for the detection of the trapped marker (glucose) released by the lytic action of complement and antiserum. Further studies by Alving et al. (4) indicated that these liposomes could bind (neutralize) antibodies in rabbit hemolysin as well as fix whole guinea pig complement. 1 This work was supported by Public Health Service Grant l-ROI-AM-B679-Ol ALY from the National Institutes of Health and by the Michi- gan Heart Association. ’ This is Journal Article 5016 from the Michigan Agricultural Experiment Station. 3 Supported by Public Health Service pre-doc- toral training grant GM-01911-02 from the Na- tional Institutes of Health. ‘ Supported by a Medical Student Summer Research Fellowship from the Michigan Heart Association. We were interested as to whether liposomes could be routinely used to quantitatively measure complement activity. Our experiments indicated that stable liposomes could be prepared by soni- cating the lipid derived from sheep erythrocytes in the marker solution. Furthermore, these liposomes could be specifically lysed by comple- ment and hemolysin, and the extent of lysis was dependent on the complement concentration. Examination of complement and antibody- treated liposomes indicated that no discrete holes were produced, but rather the liposomes seemed to fragment during lysis. MATERIALS AND METHODS Complement. Guinea pig blood was obtained by cardiac puncture from retired breeders donated by the Michigan State Department of Health, and the blood was allowed to clot at 0°C over- night. The serum was clarified by centrifugation at 23,500 X G to remove any lipid present. Any natural hemolytic antibody was removed by absorption with washed erythrocytes (5). The serum was then dialyzed overnight against cold 0.1 M Tris-HCl buffer, pH 7.4, containing 0.001 M Mg++ and 0.00015 M Ca“ to remove endog- enous galactose. After dialysis the guinea pig serum (GPS) was titrated for complement (C) activity with 5 X lOs/ml sensitized erythrocytes (5). The titer was 360 CH5.) units /ml. Antisera. Commercial anti-sheep hemolysin (whole hemolysin) prepared against washed whole sheep erythrocytes was obtained from 258 1971] Baltimore Biological Laboratory (Baltimore, Md). Anti-Forssman hemolysin was prepared against boiled sheep erythrocyte stromata as described by Mayer (5). Two hundred milliliters of sera from two rabbits were pooled and the globulins were precipitated by adding 100% saturated (P31102504 to achieve a final concentra- tion of 40% saturation. IgM and IgG immuno- globulins were separated by gel filtration on a Bio-Gel A 1.5 column (4 x 100 cm). Fractions containing the IgM were pooled, concentrated by dialysis against dry sucrose, dialyzed and frozen at —40°C until use. The IgM antiserum was heated at 56°C for 30 min before use. Anti-Foreman antibody concentration. Quanti- tative precipitin assays were carried out with 50 pl of whole hemolysin or IgM with 0.0025 to 7.0 pg N Forssman antigen in a volume of 0.25 ml. The tubes were incubated at 37 °C for 60 min and 7 days at 0°C with daily mixing. The micro- Kjeldahl technique described by Shifi'man at al. (6) was used to determine nitrogen concentration in the washed precipitates. The antibody nitrogen concentrations were 34.4 pg N /ml and 101.8 pg N /ml for whole hemolysin and IgM respectively. Preparations, extinction and fractionation of membranes. Sheep blood was collected in Alsever’s solution and aged at least 3 weeks before use. The erythrocyte membranes were isolated by the method of Dodge at al. (7). The membranes were washed or dialyzed as outlined by Haxby et al. (3) and Kinsky at al. (8). The extraction procedure was that of Bligh and Dyer (9) with experimental details provided by Haxby at al. (3) and Kinsky et al. (8). Organic phosphate was determined by analyst for total phosphorus (P) by acid hydrol- ysis (10). Determination of total phosphorus is a reliable measurement of organic phosphate, be- cause most of the latter is present in the chloro- form soluble phospholipids (3). Liposome preparation. Liposomes were pre- pared as described by Haxby et al. (3). Portions of the chloroform soluble fraction containing 2 pmol phosphorus were taken to dryness by vac- uum evaporation or by a stream of nitrogen gas. All preparations were further dried by evacuation in a desiccator for 1 hr. The phospholipid was rehydrated in 0.3 M galactose in the ratios of 10 pmol P /ml galactose, 2 pmol P/ml galactoseand 1 pmol P/ml galactose. The dried phospholipid residue was dispersed by a glass bead during agitation with a Vortex mixer. Sonication of liposomes. Sonication was carried MEASUREMENT OF COMPLEMENT WITH LIPOSOMES 259 out with modifications of the procedure reported by Huang (11). A Bronwill U-20 Biosonik (Will Corporation, Rochester, N. Y.) was used for all sonication. The sonicating chamber contained 70 ml of the buffer solution used in the spectropho- tometric assay with the addition of 0.3 M galac- tose. The dispersed phospholipid was transferred to a dialysis bag and both the bag and chamber bufl'er were flushed with nitrogen gas. Sonication of the liposomes was carried out for 2.5 hr with circulating ice water (0°C) as a coolant for the sonication chamber. Dialysis of the liposomes was carried out at 4°C againt 1000 ml of isotonic salt solution (0.075 M KCl—0.075 M NaCl) for 5 hr to remove untrapped marker from the liposome preparation. Spectrophotonwtric assay. The assay using lipo. somes wasbasedupon the oxidation of the released galactose with the nicotinamide adenine dinu- cleotide (NAD)-dependent B-galactose dehydro- genase. The reduction of the NAD was followed spectrophotometrically at 340 nm. B-Galactose dehydrogenase suspended in 2.2M (NHJSO. (EC 1.1.1.48) and NAD were ob- tained from Boehringer-Mannheim (New York, N. Y.). The enzyme was diluted in water to 1 M (NHOSO. (2.27 mg/ml) before use. NAD was prepared to a concentration of 10 mg/ml (15 mM). The bufier solution contained 0.1 M Tris- HCl, pH 7.5, 0.058 M NaCl, 0.001 M MgCl: and (MIDI-SM 0801: (II: ='- 0.15). A typical reaction mixture contained 1.2 ml Tris-H01 bufl'er, 0.05 ml NAD and 10 pl B-galac- tose dehydrogenase. Preliminary experiments had shown that these concentrations of enzyme and cofactor were sufficient to oxidize all the galactose released. Appropriate quantities of liposomes (20 pl), CPS and antibody (AS) (20 pl) were added. The GPS and AS were not added until the resid- ual galactose (i.e., that not removed by dialysis) had been oxidized. All assays were performed at 25°C using a Shimadzu multi-convertible double 40 Model S spectrophotometer and a Sargent Model SR recorder. A modification of the spectrophotometric assay was used for some experiments. Liposomes, GPS and AS were incubated at 37°C for 60 min after which time a sample was removed and assayed for the galactose released. A typical reaction mixture consisted of : 30 pl liposomes, 0.1 ml CPS, 0.2 ml IgM anti-Forssman antibody and 0.04 ml Tris-H01 buffer in a total volume of 0.37 ml. After incubation 0.25 ml was transferred into 260 KRISTINE C. KNUDSON, DAVID H. BING AND LAURENCE KATER a cuvette and 0.05 ml N AD, 0.9 m1 Tris-HCl buffer and 10 pl of enzyme were added. The reac- tion was allowed to progress until there was less than a 5% change in absorbance over a period of 1 min. Total galactose. Total galactose was determined by lysis of the liposomes in Triton X-100 (Rohm and Haas Co., Philadelpha, Pa.). To 20 pl of lipo- somes was added 0.1 ml of 10% Triton X-100 and the mixture was incubated at room temperature for 15 min. Tris-HCI bufier (1.1 ml), NAD (0.05 ml) and fi-galactose dehydrogenase (10 pl) were added and the reaction followed spectrophotomet- rically. Trapped galactose. The amount of trapped galactose was defined as the total galactose (as determined by lysis with Triton X-100) minus the free galactose remaining in the liposome prepara- tion after dialysis against isotonic salt solution. The concentration of galactose was calculated using a molar extinction for NADH of 6.22 X 10‘ and assuming that for each mol of NADH formed, 1 mol of galactose was oxidized. Degree of trapped marker released. The degree, Y, of trapped galactose released by the addition of C and AS was determined from the total amount of marker released divided by the amount of trapped marker. A correction was made for the free galactose present in the liposome preparation. Electron microscopy. Untreated liposomes, lipo- somes lysed with Triton X-100, and the spectro- photometric reaction mixtures of liposomes lysed with C and AS were examined in a Philips EM 300 electron microscope operated at 80 kv. A drop of the liposome preparation was placed on a collo- dion-carbon-coated grid and the excess was drained 03 after 20 see. In the case of the lipo- somes lysed with C and AS, the drop remained on the grid for 10 min to insure the deposition of a sufficient number of liposomes to the grid. A drop of phosophotungstic acid, pH 7.0, was added and after 20 see the excess was removed. RESULTS Galactose release. Liposomes containing galac- tose could be made from a chloroform extract of sheep erythrocyte membranes, and the trapped marker could be released by the action of C and AS (Table I). No lysis of the liposomes occurred with GPS that had been heated at 56°C for 30 min. Liposome preparations which were not soni- [VOL. 106 cated were found to be unstable, containing no trapped galactose 24 hr after preparation. The instability caused a greater amount of liposome leakage (galactose released independent of C and AS). This spontaneous leakage of galactose inter- fered with the detection of the complement- mediated lysis of liposomes, and the liposomes could not be used for titration of C for more than 1 day. Sonication of the liposomes, however, proved to be a very effective means of greatly in- creasing the stability of the liposomes. Sonicated liposomes were stable for at least a month with little leakage of galactose during that time. When non-sonicated liposomes prepared with a concentration of 1 pmol P /ml galactose were used, galactose release was dependent on the C con- centration and the response was sigmoidal with the addition of AS (Fig. 1). The lag in release of marker occurred consistently and was similar to the lag observed in the hemolytic assay (5). The galactose release from sonicated liposomes pre- pared in the ratios of 2 mel P /ml galactose and 1 pmol P /ml galactose was also dependent on the C concentration and the response was sigmoidal with the addition of AS (Fig. 2). The lag period observed with these sonicated liposomes was shorter than that seen with the non-sonicated preparations (2 min compared to 6 min for 40 CH” units for sonicated and non-sonicated lipo- somes respectively). » The data shown in Figures 1 and 2 were used to determine the CL” (the number of C units re- quired to release 50% of the trapped marker) and 1 /n (the slope of the plot of log X, pl of GPS, as log Y/(l — Y)) (5) (Table I). The CLso values of the GPS titrated with lipo- somes and either whole hemolysin or IgM anti- Forssman antibody were compared. Since purified IgM released little or no galactose at 25°C over a period of 30 min with 10 CH” to 50 CH” units /20 p1 of liposomes, the liposomes were incubated in the presence of C and AS for 60 min at 37°C. Larger liposome concentrations were used to in- crease the sensitivity. Despite the difierences in conditions and concentrations, if the reaction mixtures were normalized to contain the same concentration of liposomes, the same amount of GPS would cause 50% lysis (Table 1). Electron microscopy. Electron micrographs of the liposomes prepared with the various phospho- lipid concentrations revealed strikingly different arrangements. Liposomes prepared with 10 pmol 1971] MEASUREMENT OF COMPLEMENT WITH LIPOSOMES 261 TABLE I Complement titrations with hemolysin and I all! anti-Forssman antibody“ Antibody Lipsome Preparation cm. Added 95,373,403” or... l/n Whole hemolysin: 1 pmol P/ml of 0.3 M galactose; 50 64.6 11.5 0.647 10 pl - 6.88 pg anti- mechanical mixing;28.78 nmol 40 69.7 body N galactose trapped/10 pl (10 pl 30 54.5 used for assay) 20 43.3 10 0.1 Heated GPS 1.7 20 pl - 13.76 pg anti- 1 pmol P/ml of 0.3 M galactose; 50 72.3 21.3 0.794 body N 22.03 nmol galactose trapped/ 40 70.1 20p]; sonicated (20pl used for 30 assay) 20 40.2 10 25.6 Heated GPS 13.9 Liposomes only 9.5 20 pl - 13.76 pg anti- 2 pmol P/ml of 0.3 M galactose; 100 55.3 11.9 0.931 body N . sonicated; 56.43 nmol galac- 50 56.1 tose trapped/20 pl (Z) pl used 40 51.3 for assay) 30 47.7 20 29.1 10 18.5 Heated GPS 5.7 Liposomes only 5.7 IGM: 0.2 ml = 40.22 1 pmol P/ml of 0.3 M galactose; 16.7 43.1 3.2 0.914 pg antibody N mechanical mixing; 17.12 nmol 13.3 49.3 ' galactose trapped/10 pl (10 pl 10.0 43.3 used for assay) 6.6 25.6 3.3 14.5 0.2 ml -= 40.22 pg anti- 2 pmol P/ml of 0.3 M galactose; 16.7 41.6 2.1 0.704 body N sonicated; 39.55 nmol galac- 13.3 34.0 tose trapped/10 pl (10 'pl used 10.0 20.2 used for assay) 6.6 15.9 3.3 27.7 ' Reactions carried out with whole hemolysin were performed at 25°C for 30 min after the addition of AS; those involving IgM were performed at 37°C for 60 min. The percentage of trapped galactose released by C and AS has been corrected for the nonspecific leakage of galactose from the liposomes. The calculations were made from plots of log X (pl of GPS) vs log Y/ (1-Y). P /ml marker solution without sonication were composed of monolayers with no definite closed structure, i.e., they consisted of sheets of what appeared to be stacked membranes. Most of the stacks were composed of about 16 layers. The liposomes seem to be in the forms Thompson and Henn (12) described for hand shaken lipid disper- sions. Liposomes made with this same ratio of P to marker solution with sonication were still com- posed of many layered sheets frequently in con- voluted formations. These sheets were mostly about 9 layers thick, although they ranged from 8 to 20 layers in thickness. Smaller, more closed structures consisting of five layers were seen with sonicated liposomes prepared with 2 pmol P/ml galactose (Fig. 3). Liposomes lysed with C and AS revealed opaque spherical aggregates com- posed of smaller particles of approximately 188 A in diameter (Fig. 4). In contrast the Triton- lysed liposomes appeared as debris which had agglutinated into spheroid masses (Fig. 5). 262 Ul'm'xibo .< in}: 111111L11111L1|111 0 1O 20 30 minutes Figure 1. Dependence of galactose release on complement concentration. C, 40 CH»; I, 30 CH"; O, 20 CH“; C], 10 CH"; A, heated GPS; and A, liposomes only. Spectrophotometric assay was performed at 25°C for 30 min follow- ing the addition of whole hemolysin. DISCUSSION The purpose of this investigation was to test complement-mediated lysis of liposomes as an alternative system to the classical hemolytic assay for the quantitative measurement of whole guinea pig complement. We have shown that the complement-mediated release of trapped galac- tose from liposomes is similar to the lysis of sheep erythrocytes by complement in the presence of antibody, and have confirmed the results of Haxby et al. (3). From a technical standpoint we found soni- cated liposomes to be a good substitute for eryth- rocytes in the assay of complement. The lipo— somes obtained by sonication of the lipid disper- sion are stable for at least a month, and perhaps longer. Since galactose can be detected with one enzyme and one cofactor, the substitution of gap lactose for glucose has greatly simplified the pre- viously published procedures for detecting the marker (3). Small volumes of serum (30 to 140 pl) and antibody (20 pl) are used, thereby making it possible to conserve valuable reagents. A single assay uses only 20 pl of the liposome preparation. This means that as a minimum it is possible to get 4000 assays from 125 ml of whole sheep blood. Because the extent of lysis is measured using en- zymatic techniques, it might also be possible to automate the whole system for routine measure- ment of complement activity. KRISTINE C. KNUDSON, DAVID H. BING AND LAURENCE KATER [von. 1% I z - , ,. u 0 I ‘— _ 0 e ' . . . r e ' . . v! .0 1 . ‘ e 4', . dd'. - . o 4 A l 1 l i l l l l l l l l 1 l O 10 20 30 ”7 minutes Figure 3. Galactoee release with sonicated liposomes. O, 100 CH”; V, 50 CH“; O, 40 CH“; ., 30 CH"; O, m CH"; U, 10 CH“; ‘, heated GPS; and A, liposomes only. The spectrophoto- metric assay was performed at 25°C for 30 min following the addition of whole hemolysin. We found that the ratio of phospholipid to marker solution during suspension of the lipid dispersion and the method of suspension of the lipid in the marker solution were both extremely critical. By increasing the amount of galactose so there was 1 ml per 1 to 2 pmol of phosphorus (representing phospholipid), and sonicating the lipid and sugar mixture in an inert atmosphere, we were able to reproducibly prepare liposomes which did not spontaneously release galactose, but were sensitive to lysis by complement in the presence of hemolysin. Decreasing the amount of galactose with or without sonication of the lipid solutions resulted in liposomes which were insen- sitive to lysis by complement, or which sponta- neously released galactose so quickly that they were not suitable for assay purposes. A possible explanation for these results is sug- gested by the electron microscopic studies of the liposomes. We noted that the electron micro- graphs of liposomes prepared with 10 pmol of P/ml of marker solution with mechanical agita- tion on a Vortex mixer contained no discrete vesicles but appeared as randomly layered sheets consisting of predominantly 16 layers. The ab- sence of discrete vesicles would explain the con- tinued leakage of “trapped” galactose. Sonication of an identical preparation gave discrete vesicles, predominantly nine layers thick. These prepara- tions, however, were also not lysed by comple. ment and hemolysin. We suspect therefore that q 0‘ 4 ()MPLEMENT W ITH Lll’( )S( ).\ll ‘ \SU R EMENT ()F ( a 4.‘ M l‘ at?» 1. 7 >._o>:§a7.. .E .SSJWE Xv mm .5.“ 13.5:37. 9?. £35.17. SIZE :55 EE: m M: 5:33:33... 6 :: /. 82 H Era—.52 .Ec: utfiEZEEZEA «New Be voyage»; meEcmca: $35.23.; .m. 323k 264 KRISTINE C. KNUl)S()N, DAVII) li. BING AND LAURENCE KATER pa. 4 o .‘a e .4 1‘ IF .. :'._ A .a .> Figure 4. Sonicntcd liposomes prepared with a ratio of 2 pmol 1’ 'ml lll.ll‘l{el‘ solution. The liposomes were treated with (. Negatively stained with 2% phospliotungstic acid. our tcchuicul diflicultics with mcchunicully mixed prcpnrutions wcrc duc to thc guluctosc not. licing completely t-ruppcd. ln thc sonicutcd prcpurations which could not. he lyscd liy cmnplcmcnt but con— tuined trzippcd guluctosc, :ill of thc complcmcnt wus utilizcd lici'orc thc liposomc was lyscd. This hypothcsis is further supportcd in that thc prcp» :irntions of liposomcs which wcrc lyscd with com— plcmont andnntiliody wcrc discrctc vcsiclcs with fivc layers. Liposomes trcutcd with hemolysin wcrc :l. scn» sitive reagent for quznitituti\'cl_\' mcusuring wholc complement. activity. An individual 11>Sll_\' was «lone at 25°C and was complctc in 30 min. \\'c noted that, in no instuncc was it pimililc to cuuw and AS according to the procedures d(‘f\'('rll)(‘(l for the spcctrophotometric assay. Marker = 1000 A (X 218,000). 100',“ rclcnsc of 1110 marker with complement. ’l‘lic most trnppcd galactose which could be re- 1 ‘uscd was lictwccn 50"; and 70‘; (see Fig. 2). If wc call the dcgrcc of lysis at which the system is suturatcd with complement 100’, lysis, normalize thc other points to this vulu * and recalculate the 50'; cndpoint, wc find tlicrc ur‘ approximately 100 to 200 (‘Lw units. ml of scrum, a figure close to thc figure of 360 (‘llw unit's ml determined for this lot of serum. Both of thcsc facts indicate that lllHlN‘HlllL'S can lic substitutcd for crythrorw’tcs in complcmcnt»mcdi:itcd immunc lysis without loss ol sciwitivit)’, and thc vulucs olitaincd for the (‘lm and 1 it probably urc :1 property of this particular i'cnucnt. 'l‘hc suitability of the system [VOL. 106 _..——.___-—— ___—_— —‘_-.__-‘__._—‘— —-‘-——~‘ _ W M2 —1 ' _ Figure 5. Sonicated liposomes prepared with a ratio of 2 pmol P/ml marker solution and lysed with 1.0’; Triton X-lOt). Negatively stained with 2‘}; phosphotungstic acid. Marker = 1000 .°\ (X 218,000). for quantitatively measuring activity of the in- dividual components will need to be tested in further experiments. Complement action on whole erythrocytes results in 85 to 110 A holes (13) and, we were. curious as to the nature of the lesions in lipt.)somes caused by complement and antibody. To examine this question we undertook an electron micro- scopic study of conu)lement-antibody and deter- gent lysed liposomes. The picture seen with com~ plement- and antibody-treated liposome prepara— tions was very difierent than that seen with the Triton-lysed liposomes. There were no regular sized lesions or “holes” in the antibody—comple- ment-treated liposomes. Although the multi- layered structures seen in the untreated liposomes were gone, there still were large globular st ruin tures present which upon close examination seemed to be an association of fragments of the phospholipid layers which were released from the liposome (see arrow, Fig. 4). At this time we hyu pothesize that the large structures are liposome “ghosts” and that complement action destroyed the original liposome layers by releasing by as yet. an unknown chemical process variable size pieces MEASUREMENT OF COMPLEMENT WITII LIPOSOMES 136.3 of the original phospholipid layer. ()nce released, these small pieces tend to mass in solution much in a manner similar to that in which the liposomes were originally formed. .\s the lipid masses are only small segments of what was once a long sheet of phospholipid, they do not form layered struc~ turcs, but they remain associated with the lipo— some "ghost" by virtue of nonspecific hydro- phobic l)onding. The size of the piece of liposome released by complement can apparently vary, as the size of the small globules varied widely in size (92 to 3‘22 A). In any case, we feel that the small globules may represent the product of complement action on antibody