Antigen mobility in membranes and complement-mediated immune

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp , July 1975 Chemistry Antigen mobility in membranes and complement-mediated immune attack (lipid bilayers/phase equilibria/cholesterol/cardiolipin/spin labels) GILLIAN M. KITCH HUMPHRIES AND HARDEN M. MCCONNELL* Stauffer Laboratories for Physical Chemistry, Stanford University, Stanford, California Contributed by Harden M. McConnell, April 15,1975 ABSTRACT The complement fixing activity of liposomes containing cholesterol, dimyristoylphosphatidylcholine (or dipalmitoylphosphatidylcholine), and 3 mol % of cardiolipin has been studied as a function of cholesterol concentration by use of human syphilitic serum containing cardiolipin-specific (Wasserman) antibodies. It is found that complement fixation increases rapidly for cholesterol concentrations above 35 mol %. Spin label studies have been used to study the incorporation of cardiolipin in the relatively rigid phase of binary mixtures of cholesterol and dimyristoylphosphatidylcholine (or dipalmitoylphosphatidylcholine). It is concluded that cardiolipin is included in such a phase of these lipids for cholesterol concentrations above 35 mol %. These results indicate that a relatively rigid lateral distribution of this monovalent antigen in the plane of the membrane facilitates complement fixation and concomitant complement-mediated membrane damage. It is possible that both afferent and efferent immune responses to certain cell surface antigens depend on the lateral mobilities of these antigens in the plane of the membrane. The purpose of the present paper is to provide preliminary evidence for this conjecture for the case of complementmediated immune attack on model membranes (liposomes) containing a monovalent antigen (cardiolipin). We have chosen liposomal bilayer membranes for this investigation since their chemical composition can be precisely defined, and their physical properties (e.g., "fluidity" or antigen mobility) are amenable to quantitative study with a variety of physical techniques. Complement-mediated immune attack on these membranes is the simplest response of the immune system that mimics cytotoxic attack. It is well known from classical immunological literature (1, 2) that complement is fixed by cholesterol-rich lipid carriers containing specific "incomplete" lipid antigens (haptens), and the work of Kinsky and colleagues (3, 4) has given numerous convincing demonstrations that complement-mediated damage to liposomal membranes containing such antigens renders them permeable to a variety of substances. METHODS AND MATERIALS Cardiolipin was purchased from General Biochemicals as a solution in ethanol and chloroform. Thin-layer chromatogra- Abbreviations: DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; TEMPO, 2,2,6,6-tetramethylpiperidine-l-oxyl; VBS, veronal-buffered saline prepared according to Kabat and Mayer, ref. 6; VBS-TEMPO, VBS with 0.46 mm TEMPO and flushed with argon; C', guinea pig complement; X, h, mole fraction of cholesterol. * To whom correspondence should be addressed. phy on Silica Gel G with CHCl3/CH3OH/H2O (65:25:4) as solvent gave a single spot on treatment with H2SO4 and heat. A sample was digested with perchloric acid and assayed for phosphate. Dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) were purchased from Calbiochem. Each phosphatidylcholine was shown to have a sharp melting point at the respective reported value (230 and 410) by the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) solubility method (5). Cholesterol was recrystallized twice from hot ethanol, dried, and shown to have a melting point of (accepted value ). Stock ethanolic solutions of all lipids used were prepared and kept under argon at -20. Antigen preparations contained 3 mol % of cardiolipin and various mole fractions of cholesterol (Xh = 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, and 0.60) with either DMPC or DPPC. A solution containing 1.0 Amol of the required lipid mixture was dried on an argon-filled rotary evaporator under reduced pressure, redissolved in 1 ml of CHC13, and dried again. Complete removal of the solvents was ensured by leaving the flask on the evaporator for 1 hr at 40. A few glass beads and 3.00 ml of veronal-buffered saline (VBS) were introduced, and the lipids were suspended with the aid of a Vortex mixer to give 10 um cardiolipin. Suspensions were prepared on the day of use. Rabbit antiserum against sheep erythrocytes (hemolysin) and guinea pig complement (C') were purchased from Difco, assayed by the methods of Kabat and Mayer (6), and frozen in convenient small volumes. Human syphilitic serum was obtained from the Santa Clara County Health Facility, San Jose, pooled in lots of sera, heated to 560 for 30 min, and tested for reactivity and anticomplementarity. Lots showing the highest reactivity and lowest anticomplementarity were selected and frozen. Normal human serum was obtained from volunteers and similarly treated. C' fixation was accomplished by incubating 100Ml of antigen suspension (1.0 nmol of cardiolipin), 40 Ml of syphilitic serum, and about 0.6 C'H5s units of C' in a total volume of 0.50 ml at 6, 160, 260, and 370 for periods of 19, 17, 5, and 2 (or 3) hr, respectively. VBS was used as diluent throughout; duplicate tubes were always set up; controls were always included containing (a) C' only (hemolytic control), (b) C' and antigen only (antigen controls), and (c) C' and serum only (serum control). On completion of the incubation period, the tubes were chilled on ice, and 0.20 ml of VBS containing 5 X 107 optimally hemolysin-sensitized sheep erythrocytes [prepared by the methods of Kabat and Mayer (6)] were added to each tube. The set of tubes was incubated at 370 for 60 min. Times for complete hemolysis were noted. Any tubes not showing complete homolysis at 60 min were centrifuged and 2483

2 2484 Chemistry: Humphries and McConnell the absorbances were read at 541 nm. Two lots of pooled syphilitic serum were tested both with DMPC- and DPPCcontaining antigens. Control experiments were also performed in which (i) "antigen" preparations containing cholesterol and DMPC or DPPC but no cardiolipin were used with syphilitic serum, (ii) normal serum was used in place of syphilitic serum with cardiolipin-containing antigens, and (Wi) sensitized sheep erythrocytes were incubated for several hours at 370 with antigen preparations. The effects of antigen and antibody concentrations were studied as follows: (a) using the standard volume of syphilitic serum, (40 ua), DPPC/cholesterol/cardiolipin antigens were used at volumes of 200, 100, and 50,d per tube (i.e., 2.0, 1.0, and 0.5 nmol of cardiolipin per tube); and (b) using the standard volume of the full range of antigens, (100 Ml), syphilitic serum was used at volumes of 40 ul and 20,A. For determination of phase transition behavior, lipids were prepared in a manner similar to that used for antigen preparation except that 18.0 MAmol of total lipid was dried down and suspended in 130 ul of VBS-TEMPO. The ratio of lipid/water/tempo was the same as that used by Shimshick and McConnell (5), but used VBS rather than water'as the aqueous phase. For preparations in the critical range, i.e., those for which X&h = 0.35 and 0.45, controls were examined in which (a) cardiolipin was replaced by the appropriate lecithin, and (b) 18 MAmol of lipid was suspended in 54 ml of VBS-TEMPO and centrifuged and the bulk of the supernatant removed. Suspensions were pipetted into argonfilled sample cells consisting of 50-,ul disposable pipettes sealed at one end. After the cell was half filled, more argon was admitted above the liquid and the top was sealed by heating. Cells were placed horizontally in the temperaturecontrolled cavity of a paramagnetic resonance spectrometer. COMPLEMENT FIXATION The method used for the determination of complement fixation is a modified, calibrated version of the Kolmer test from which it was derived (7). It involves two types of measurement: (i) time taken for complete lysis of indicator cells if this is less than 60 min; and (ii) fraction of cells lysed at 60 min if lysis is incomplete at that time. This method was adopted for the following reasons: (a) it allows determination of a fairly wide range of complement activities; and (b) by eliminating the need to centrifuge and spectrophotometrically examine all tubes, it minimizes the risks inherent in handling human serum and allows for far speedier data collection. These advantages are important because each set of experiments used eight different antigen formulae at four different temperatures, included many controls, and used duplicate tubes throughout. For limiting quantities of C', we have shown that partial lysis of 5 X 107 indicator cells in 0.70 ml during 60-min incubation at 370 obeys von Krogh's equation (6), X where y = fraction of cells lysed, n = 0.2, and x is a measure of complement activity proportional to the volume of guinea pig serum supplied. Hence, one unit of C' activity is here defined as that required to lyse 50% of indicator cells under the specified conditions. [One unit of C' in our system is approximately the same as 0.1 C'H50 in the Kabat and Mayer y Proc. Nat. Acad. Sci. USA 72 (1975) 3 4 UNITS COMPLEMENT FIG. 1. Calibration curve for the rate-dependent assay of residual C'. The various symbols represent data from three independent calibrations (see text for further details). (6) schemes for C' titration and fixation tests.] For the C' fixation tests, residual C' activity, sufficient to induce 10-90% lysis under the specified conditions, was calculated by application of von Krogh's equation to experimentally determined values of y, with the exponent set at 0.2. Since values of y between 0.1 and 0.9 only are admissible, the method is limited to determination of complement activities between about 0.60 and 1.55 units. In order to calibrate the rate-dependent method of analysis, 5 X 107 sensitized cells in a total volume of 0.70 ml and supplied with different quantities of C' (between about 0.60 and 6.00 units) were incubated at 370. Some of the tubes showed complete lysis at times less than 60 min. These times were recorded. A-ll tubes not showing complete lysis were centrifuged and spectrophotometrically examined along with controls for total and zero hemolysis. By plotting log (volume of C' solution used) against log [y/(l - y)], it was possible to retitrate the sample of guinea pig serum and hence to calibrate a plot of "minutes for complete hemolysis" against "volume of C' solution used," as shown in Fig. 1. Three separate such experiments were performed and the data combined. The calibration curve was used between the values of 1.70 and 3.00 units. The total range of the two methods was between 0.60 and 3.00 units. Not all points could be measured with the same accuracy, but it is unlikely that for any value in this range it was much worse than unit, and for most values it was better. Depletion of C' activity by the antigen preparations alone was never observed. All samples of human serum used, both syphilitic and normal, were somewhat anticomplementary. Typical C' fixation by DPPC- and DMPC-containing antigens under various conditions of temperature and time is illustrated in Fig. 2. Depletion of C' activity by preparations with Xch = 0.25, 0.30, and 0.35 was invariably constant, usually of about the same level as the serum controls, but occasionally elevated from this. This latter effect was apparently a function of the particular batch of serum used, and significant elevation was only observed at the higher temperatures. The response at Xch = 0.40 was variable, ranging from the same as that observed at Xch = 0.35 to considerably higher. At Xch = 0.45, C' fixation was invariably substantially greater than at 0.35, and it increased linearly in the range Xch = 0.45 to Neither normal serum in the presence of cardiolipin-containing antigens, nor syphilitic serum in the presence of "antigens" lacking cardiolipin, depleted C' activity to a greater extent than the serum controls. In all cases

3 Chemistry: Humphries and McConnell MOLE FRACTION CHOLESTEROL FIG. 2. C' fixation as a function of X:h. The baseline represents the appropriate serum controls in each case. (A) Effect of varying the volume of antigen preparation supplied: A, 50 Isl; 0, 100,gl; 0, 200 #d. For the experiment illustrated, DMPC-containing antigens were used and fixation was for 3 hr at 37. (B) Effect of varying the volume of syphilitic serum supplied: 0, 20 Ml; *, 40 Al. For the experiment illustrated, DPPC-containing antigens were used and fixation was for 19 hr at 60. these controls were at a level measurable with an accuracy of 0.15 unit. Antigen preparations did not destabilize antibody-sensitized sheep cells. The level of C' fixation increased with increasing antibody concentration (Fig. 2B), but at around the level used in the standard assay it decreased with increasing antigen concentration (Fig. 2A). Clearly, C' fixing activity is biphasic with respect to antigen concentration. Similar experiments not reported here in detail have shown that fixation by antigen preparations with Xch = can be inhibited by 35% cholesterol/62% DMPC/3% cardiolipin, but not by 35% cholesterol/65% DMPC. This indicates that antigen preparations having low cholesterol content bind antibody although they show little C' fixing activity. Preliminary experiments, using a previously reported method (8), show that immune lysis of spin label loaded antigen preparations varies as a function of cholesterol content in much the same way as complement fixation. Freeze-fracture electron microscopy showed no detectable change in liposome structure for Xch = Antigen preparations with other compositions have not been examined in this way OU120 LdlO or100 g60 k MOLE FRACTION CHOLESTEROL FIG. 3. Incomplete phase diagram for DMPC-cholesterol in excess water [Shimshick and McConnell (9)], with the addition of upper and lower transition temperatures (0) for the system in which 3 mol % of DMPC is replaced by 3 mol % of cardiolipin at all Xch studied. For clarity, limits of error have been omitted from the new data. Error in composition is the same as that shown in the original diagram; transition temperatures shown are all 12O or less. At Xch = 0.40, two upper transitions are indicated. Proc. Nat. Acad. Sci. USA 72 (1975) 2485 LECITHIN-CHOLESTEROL PHASE DIAGRAM Fig. 3 gives the DMPC-cholesterol phase diagram reported by Shimshick and McConnell (9). Points on the fluidus and solidus curves in this diagram were obtained by studies of the binding of the spin label TEMPO to binary mixtures of these lipids, as a function of temperature. The paramagnetic resonance spectrum of TEMPO in an aqueous dispersion of lipids shows two signals, one due to TEMPO in the aqueous phase, and one due to TEMPO in the fluid hydrophobic phase of the bilayer. The fraction of TEMPO in the hydrophobic phase is measured (approximately) by the TEMPO spectral parameter, f, discussed earlier (5). Typically, regions of lateral phase separations of phospholipids into relatively fluid and relatively rigid domains are recognized by a particularly strong temperature dependence of,f; high-temperature discontinuities in df/dt have been used to identify points on the fluidus curve, and low-temperature discontinuities in df/dt have been used to identify points on the solidus curve. This method of establishing phase diagrams has been verified for certain binary mixtures of phospholipids by nuclear magnetic resonance (Brulet and McConnell, unpublished) and freeze-fracture electron microscopy (10, 11). Nuclear magnetic resonance can be used to study the composition of the fluid and solid domains, using '3C-labeled phospholipids. With freeze-fracture electron microscopy, the solid phase lipids can be identified by characteristic band patterns, and fluid phase lipids can be identified by the absence of band patterns, as well as by the inclusion of small concentrations of membrane proteins that are preferentially soluble in the fluid phase. We have thus far been unable to detect coexisting fluid and solid domains in the temperature-composition region denoted F+S in Fig. 3, using electron microscopy. The lateral phase separation implied by the horizontal line at the melting point of the pure phosphatidylcholine has been observed at temperatures below this melting point. The absence of distinguishable domains in the F+S region may be due simply to the fact that the boundaries between fluid and solid domains have a low free energy. Fortunately, our interpretation of the effect of cardiolipin on TEMPO binding does not depend on whether distinct domains are present, or whether the F+S region represents a continuous phase transition. Fig. 3 shows that' the high-temperature "fluidus" break in df/dt is strongly affected by about 3 mol % of cardiolipin. This can arise from three distinct effects: (a) cardiolipin acts as an impurity," and has a melting point-depressing effect; (b) cardiolipin, bearing a large negative charge, causes the bilayer membranes to repel one another, increasing their hydration and fluidity; and (c) cardiolipin increases the surface charge of the membrane, producing a lateral expansion, leading to a reduction in transition temperature (13)'. In any case, cardiolipin must be incorporated in the "fluid" lipid phase. A striking result, however, is the absence of any effect of cardiolipin on the "solidus" curve, below Xh = We therefore conclude that for Xch S 0.35, the cardiolipin is gradually excluded from the cholesterol-lecithin region as the temperature is lowered, and is totally excluded at temperatures below the solidus curve. The cardiolipin is probably present in the membranes in the form of concentrated patches of pure cardiolipin; these are doubtless fluid patches under the conditions of our C' fixation experiments since the chain melting phase transition of cardiolipin is well below 6. At Xbh > 0.35, the cardiolipin obviously has a profound effect on the lower temperature break in df/dt; i.e., on the

4 2486 Chemistry: Humphries and McConnell "solidus curve." There are only two evident interpretations of this result. For Xh > 0.35 the cardiolipin is either (a) homogeneously incorporated in the solid phase of the membrane or (b) forms a separate phase rich in cholesterol and cardiolipin and thus reduces the concentration of cholesterol in the lecithin-cholesterol phase. In either case, the temperature for complete solidification is expected to be substantially reduced. Also, in either case, the cardiolipin must become relatively strongly immobilized (14-19). There is additional evidence for the above conclusions. Let fi be the absolute value of f corresponding to the lower temperature break points in plots of f against temperature. A plot of fi against Xch (not given here) shows a pronounced maximum at Xch For Xch < 0.35, there is - an enhanced increase in fi with increasing Xch because of the increasing solubility of TEMPO in the pure cardiolipin phase with increasing temperature. For Xch > 0.35, fi decreases with increasing Xch because of the inclusion of cardiolipin in the rigid cholesterol-rich phase, in spite of the fact that the corresponding temperatures are also increasing. No maximum of this type is observed in the absence of cardiolipin. Control experiments indicate that the results shown in Fig. 3 are not caused by the use of VBS rather than water as the aqueous phase, and that these results are also unchanged when the ratio (Mg++ + Ca++)/(cardiolipin) at the time of liposome generation is varied from that used in the TEMPO solubility assay to that used in the standard method for antigen preparation. An equivalent perturbation of the cholesterol-dppc phase diagram has been observed but the data are not presented here. DISCUSSION We have provided evidence that C'-mediated immune attack on bilayer membranes of lecithin and cholesterol containing a low concentration of the monovalent antigen cardiolipin is related to the lateral mobility of this antigen in the plane of the membrane. A significant level of C' fixation is only observed when the cholesterol concentration is increased to Xch > 0.35, where the spin-label data indicate that cardiolipin is included homogeneously in a relatively rigid bilayer membrane, or forms a separate rigid phase containing a high concentration of cholesterol. In either case, extensive studies of the effects of cholesterol on molecular motion in membranes (14-19) leave little doubt that all molecular motions (including lateral mobility) of cardiolipin are significantly restricted under these conditions. We interpret the precipitous increase of complement fixation for Xch > 0.35 as due to the abrupt incorporation of this antigen in a rigid membrane. This concentration of cholesterol is of particular interest in view of the calorimetric studies of Hinz and Sturtevant (20) and the x-ray diffraction studies of Engelman and Rothman (17). Both groups have proposed that a 2:1 lecithin-cholesterol mole ratio represents a special stoichiometry where the lecithin-cholesterol interaction is maximal; Engelman and Rothman (17) have shown by model building that at this stoichiometry a partially ordered structure can be formed in which cholesterol-cholesterol contacts are avoided, and each phospholipid molecule interacts with two cholesterol molecules. The spin-label data of Marsh and Smith (14) also indicate that the "condensing effect" of cholesterol is especially strong for Xch > From these studies it is reasonable to conclude that the thermodynamic activity of cholesterol increases above Xch Proc. Nat. Acad. Sci. USA 72 (1975) 0.35 so that excess cholesterol molecules are free to interact with cardiolipin, either by homogeneous inclusion of cardiolipin in the solid phase, or by the formation of concentrated patches of cardiolipin and cholesterol. We suggest that a relatively rigid lateral distribution facilitates C' attack by increasing the effective valency of the antigen. Water-soluble monovalent antigens (haptens) free in aqueous solution do not fix C' (6), so it is reasonable to suppose that lipid-soluble monovalent antigens free in two-dimensional highly fluid bilayers behave likewise in not fixing complement. It has been suggested that distortion of IgM by its binding to critically spaced repeating antigenic determinants on a large molecule or a cell surface is a necessary prerequisite for C' fixation (21-23). [Syphilitic antibodies of the Wassermann type are principally IgM (24).] In a relatively rigid membrane, some distortion of IgM could arise in order for several sites to bind to antigen. The lateral mobilities of the membrane antigens need not be zero, but rather, only need be low enough that the conformational change in IgM is triggered, and persists long enough to permit the first step in C' fixation. Similarly, an increase in the effective valency of the antigens should potentiate C' fixation by specific IgG. It is probable that phase separations of the type suggested result in changes in lateral distribution of cardiolipin in addition to changes in mobility. It is quite possible that these distribution effects also contribute to the shape of the profile of C' fixation versus cholesterol content. We believe the significance of studies such as the one described here extends beyond C'-mediated attack on cell membranes. Certainly in some instances the afferent and efferent immune responses to cell surface antigens involve entirely different processes; for example, lymphocyte stimulation to produce specific antibodies, followed by C'-mediated cytotoxic attack. Logic suggests that these processes follow the same selection rules. Thus, conditions of monovalent hapten mobility in membranes required for C'-mediated cytotoxic attack on these membranes are likely to be the same as required for direct lymphocyte stimulation by the same membranes. There are many studies with water-soluble haptens that suggest this reciprocal relationship. Haptens free in solution normally do not produce an afferent immune stimulation, whereas haptens covalently linked to a carrier (e.g., a protein) do (25). Haptens free in solution do not normally fix C', but haptens covalently linked to a carrier do (26). It is entirely plausible that the lateral distribution and mobilities of cell surface components are important in other biological processes, such as specific cell-cell recognition. In cells, the mobilities and distributions of some membrane components are of course not controlled solely by lipid composition, but involve submembranous structures as well (27-29). We are very grateful to Ms. Harada of the Santa Clara County Health Facility, San Jose, Calif. for obtaining syphilitic sera. This work was supported by the National Science Foundation, Grant no. BMS , and has benefited from facilities made available by the Advanced Research Projects Agency through the Center for Materials Research at Stanford University. 1. Kolmer, J. A. (1928) in Serum Diagnosis by Complement Fixation (Lea and Febiger, Philadelphia), pp Rapport, M. M. (1970) in Handbook of Neurochemistry (Plenum Press, New York, London), Vol. III, pp Uemura, K. & Kinsky, S. C. (1972) Biochemistry 11, Six, H. R., Uemura, K. & Kinsky, S. C. (1973) Biochemistry 12, Shimshick, E. J. & McConnell, H. M. (1973) Biochemistry 12,

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