Changes in membrane fluidity associated with lymphocyte stimulation by succinyl-concanavalin A

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Changes in membrane fluidity associated with lymphocyte stimulation by succinyl-concanavalin A
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  170 Biochimica et Biophysica A cta. 686 (1982) 170-176 Elsevier Biomedical Press BBA71105 CHANGES IN MEMBRANE FLUIDITY ASSOCIATED WITH LYMPHOCYTE STIMULATION BY SUCCINYL-CONCANA V ALIN A S,N. CHERENKEVICH a, J.M. VANDERKOOI b., and C. DEUTSCHc Department of Biophysics, Byelorussian State University, Minsl< (U.S.S.R.) and b Department of Biochemist~ and Biophysics, Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 (U.S.A.) (Received September 1st, 1981) Key words: Lymphocyte stimulation; Membrane luidity; Succinyl-concanavalin A; Fluorescence anisotropy; (Human) Time-resolved fluorescence anisotropy studies of human peripheral blood lymphocytes labeled with 1 6- diphenyl-l 3 5-hexatriene were carried out at temperatures between 4 and 38°C. For unstimulated freshly- isolated lymphocytes the calculated order parameters were found to be 0.62 at 4°C and 0.44 at 37°C. Mitogen-induced alterations in the order parameter were evident within minutes after addition of succinyl- concanavalin A to the cells increasing to a value of 0.56 after 2 h at 37°C. Both stimulated and unstimulated cells show a decrease in fluorescence anisotropy over the next 2-3 days of culture and after the third day the time-resolved fluorescence anisotropy decay profiles of the two populations of cells were indistinguishable. Our results indicate that there are both short- and long-term changes in the membranes of the cell upon stimulation by mitogen. Introduction Resting (Go) human peripheral blood lympho- cytes may be stimulated to undergo proliferation in vitro with a variety of plant lectins [1]. Within minutes after mitogen addition increased fluxes of K, Na, and Ca occur [2], and within the first few hours there is receptor aggregation and removal. Other initial events include increased transport ot sugars, nucleosides, and elevation in the levels of cellular cyclic nucleotides [ 1,3]. The mitogens need not enter the lymphocyte in order to initiate pro- liferation [4] and therefore, the critical early changes may indeed be membrane events. Our studies are an attempt to obtain informa- tion regarding the role of the lymphocyte mem- brane lipids in mitogen-induced alterations in the cell metabolism and function. 1,6-Diphenyl-l,3,5- hexatriene was used as a probe of the hydrophobic * To whom correspondence hould be addressed. 0005-2736/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press region of the membrane. This probe, developed by Shinitzky and Inbar [5] has two features which make it a useful tool to study membranes. The long axis of the molecule and the absorption and emission dipole coincide, thereby simplifying anal- ysis of anisotropy [6-8]. In addition, the motion of diphenylhexatriene is very sensitive to its lipid milieu, since it preferentially aligns parallel to the fatty acid side chains of the phospholipids [9]. Succinyl-concanavalin A was used in these studies because it produces little or no agglutination or cap formation [10]. Therefore, any effect of mito- gen on diphenylhexatriene anisotropy can be inter- preted without regard to any such effects. Methods and Materials Chemicals. Hank's balanced salt solution, (Ca 2+ , Mg2+)-free, was obtained from Grand Is- land Biological Company. Modified Eagle medium with Earle's salts was made up from its individual  171 components. Normal, unsubstituted Earle's medium contained 116 mM NaC1, 5.2 mM KC1, 0.8 mM MgSO 4, 1.8 mM CaC12, 1 mM NaH2PO 4, 6 mM glucose and 26 mM NaHCO 3. Hypaque (Sodium salt, 50% solution) was obtained from Winthrop Laboratories and succinyl-concanavalin A was obtained from Polysciences. [3H]Thymidine was obtained from New England Nuclear Co. Sigma Chemical Co. provided Ficoll and diphenyl- hexatriene. Lymphocyte Preparation. Heparinized human venous blood was collected from healthy adults. The lymphocytes were separated by gradient centrifugation using a modified Ficoll-Hypaque technique as previously described [11]. The result- ing lymphocyte preparation generally contained less than 10-20% monocytes, as determined by the nonspecific esterase stain [12]. Viability of freshly prepared lymphocytes, as tested by Trypan blue exclusion, was greater than 98%. Cultures were prepared by adding 5.105 cells to a 1-ml solution containing minimum Eagle's medium plus 10% of either pooled human AB serum or fetal calf serum, 1% L-glutamine and 1% penicillin-streptomycin. These were incubated in 5% CO 2 in humidified air at 37°C. Each day, 30 1-ml control cultures and 30 1-ml stimulated cul- tures (50/~g succinyl-concanavalin A per ml) were collected separately, centrifuged at 600 × g for 3min, and resuspended in (Ca2+,Mg2+)-free Hank's balanced salt solution to final concentra- tions of (2-8). 106 cells/cm 3. This cell suspension was then used for the preparation of the samples for the fluorescence time-resolved anisotropy. DNA synthesis in the cell cultures in the ab- sence and presence of succinyl-concanavalin A (50 /~g/ml) was assayed by addition of 0.25 /~Ci of [3H]thymidine 8h before the cultures were col- lected, and washed using a Cell Harvester (Model M12 from Brandel). The incorporation of [3H]thymidine was determined using scintillation counting. Fluorescence anisotropy measurements. The in- strument used to measure fluorescence anisotropy was a single-photon-counting (Ortec, Inc.) nanosecond lifetime instrument under computer control. An air spark-gap was used for excitation. A 344 nm UV interference filter was used to isolate the exciting wavelengths, and a Schott GG 420 filter used for emission. Fluorescence anisot- ropy, r(t) is defined as I~l t) --Ii t) r( t ) = i--:vx-7--4-7-77 lltt)_r~zJ_tt (1) where 1, is the intensity of the vertical component and 1± is the intensity of the horizontal compo- nent of the emitted light. The denominator is proportional to the total fluorescence emitted and is therefore independent of anisotropy. The fluorescence anisotropy, r, of a chromo- phore solubilized in systems with anisotropic ordering is a complex function containing a time- dependent part, r(t), and a time-independent part, r. Nordio and Bresolin [13] and Luckhurst et al. [14] predicted that the time-dependent part of r(t) is composed of an infinite number of exponentials. However, in our study, following Kawato et al. [7] and Lakowicz et al. [8] we have used the phenome- nological approach that r(t) at short times (in the nanosecond range) can be fit by a single- exponential curve. This approach gives useful in- formation about the rotational motion of the fluo- rescence probe in anisotropic systems such as lipo- somes and intact cell membranes [15-17]. In this case, r(t) can be written as: r(t) = (r o - r~o)e-'/* + r~ 2) where ~ characterizes the relaxation time of the fluorescence probe that undergoes anisotropic ro- tational motion. The value of r 0 in this equation refers to the anisotropy of the probe with fully restricted motion; r~ is the value of anisotropy at long times after the flash. The ratio r~/r o was shown to be related to the order parameter S v of a chromophore solubilized in a spherical aggregate of an isotropic solution [7,15,16,18]. In the case of diphenylhexatriene, for which the absorption and emission transition dipole moments coincide with the long axis of the molecule, the following equa- tion can be written [7] for the order parameter, Sv: r 2 -=s; 3) ro We have used the order parameter, S v, to char- acterize the lipid order in cultured lymphocytes  1 /2 which have been stimulated with succinyl- concanavalin A. The value of roo was determined from r(t). Estimates of the limiting anisotropy, r0, given in the literature are divergent, but close to the theoretical value of 0.4 [6-8]. Therefore we used the value of r 0 -- 0.4 in this work. Labelling the cells with diphenylhexatriene. Di- phenylhexatriene (Sigma Chemical Co.) was solubilized in tetrahydrofuran at l0- 3 M and then diluted 500-fold in modified Hank's balanced salt solution, Ca2+-free, followed by vigorous stirring for 1 h at 20 C. Longer incubation times, up to 2 h, had no effect on the value of anisotropy. The final concentration of diphenylhexatriene in cell suspensions (5.105 cells/ml) was 5. 10 -7 M. Under these conditions, the effect of light scattering on the observed r(t) values was negligi- ble. No change in the r(t) profile could be de- tected during the time needed to accumulate a decay curve (around 3 min). After selected experi- ments viability of the cells was measured by Trypan blue experiments and was greater than 90%. Results The fluorescence decay of diphenylhexatriene in human peripheral blood lymphocytes is com- posed of more than one component as illustrated by the time-dependence of the total fluorescence intensity (Iii+ 21.) at 7°C (Fig. 1). In this case the decay curve was fit by two exponentials with lifetimes of 3 and l0 ns with 30 and 70 percent contribution, respectively. The decay profile is typical for that found in biological and synthetic membranes, and is a consequence of heterogene- ity of sites and/or excited-state reactions [9,19,20]. There is a significant difference in the decay profiles for the parallel and perpendicular compo- nents of light as shown in the decay profile of the difference emission (III--IL) (Fig. 1). Using Eqn. 1, we calculated fluorescence anisotropy, r(t); typical anisotropy profiles are shown in Fig. 2 for two temperatures, 7 and 35°C. The diphenyl- hexatriene fluorescence anisotropy decayed rapidly within the first 15-20 ns after excitation to a constant value, roo. The non-zero value of r=, which indicates an apparent infinite rotational relaxation time, is a function of the degree of restriction on molecular rotational motion of the diphenyl- ~> 104 L:...~ ~I, + 2 T L '~ ~., .-~ 10 - ~ ~d@lamp .,4 .... ~?~":':';';/"' .i:i~." 'i 70 6'o io Time, ns Fig. I. Fluorescence decay profile of diphenylhexatriene in human lymphocytes. Total fluorescence decay profile (Iip + 21L), difference (lit- l_L) and lamp function are indicated on the figure. Conditions are given in Materials and Methods. Temperature: 7°C. Dotted line represents the computer best fit to III +21-L curve with a double exponential decay function with lifetimes of 3 and l0 ns. hexatriene imposed by the membrane interior. The value of r~ decreased from 0.14 to 0.08 as the temperature was increased from 7 to 35°C, indi- cating increased disorder. Using Eqn. 2, the order parameter is calculated to be 0.55 and 0.31, respec- tively, for these temperatures. The addition of succinyl-concanavalin A alters the anisotropy de- cay profile at 7 and 35°C by changing both ro~ and the rate of decay of r(t). Fig. 2 shows that at 7°C the mitogen decreased the rate of decay of anisot- ropy such that r(t) reached a plateau about 10-12 ns later than in untreated cells. In addition, the values of r= increased to 0.17 and 0.11 at 7 and 35°C, respectively, upon stimulation. In order to correlate the change in the mem- brane structure with other physiological changes 04 t ___ 7oc o.i1 -- 35°c , i i o 20 30 40 Time ns Fig. 2. Fluorescence anisotropy profile of diphenylhexatriene in human lymphocytes. The conditions are given in Materials and Methods and the temperatures are indicated on the figure. --, no additions; -- -- --, 50/~g succinyl-concanavalin A per ml; Measurements were made 20 rain after addition.  0.4- 0.3- o. o o.2- o (¢1 0.1 0.56 0.52 0.48 0.44 0.40 & 180 • 27 i i i i i ~ i i i io zo 5o 60 Time, nsec B ' 2b ' 4b ' go' 8o' 16o' ~o' // '1~o' Time, rain Fig. 3. Anisotropy decay profiles and order parameter of di- phenylhexatriene in human lymphocytes after addition of suc- cinyl-concanavalin A. Temperature: 37°C. (A) Decay profiles after addition of 50 #g succinyl-concanavalin A per ml. Each decay curve was obtained at the time (in rain) specified in the figure. (B) Order parameter as a function of time after addition of succinyl-concanavalin A. induced by succinyl-concanavalin A, the time- dependence of the response was examined. The effect of mitogen on diphenylhexatriene anisot- ropy occurred within minutes of mitogen addition, as shown in Fig 3. The order parameter, Sv, was calculated from the decay curves and is plotted as a function of incubation time with mitogen (Fig. 3b). It can be seen that the change was complete within 20-30 min after succinyl-concanavalin A addition. Because the lipid order parameter, S~, increases during the process of the succinyl-concanavalin A stimulation of lymphocytes, it was of interest to see whether S~ is also a function of culture time; we therefore determined the value of S~ throughout a 4-day culture period for untreated and stimu- lated lymphocytes (Fig. 4). During the first two days of cultivation the order parameter for di- phenylhexatriene in lymphocytes decreased both 173 Sv 0.6~ 0.51~ 0 4t~ 0.3 02 0 I 2 3 4 DAYS Fig. 4. Change in the order parameter, Sv, of diphenyl- hexatriene in lymphocytes as a function of cell cycle. Condi- tions are given in Materials and Methods. Temperature: 37°C. O, no addition; [~, 50/xg succinyl-concanavalin A per ml. for control cells and stimulated cells. By the time of the third and fourth days of growth the values of S v were the same for control and stimulated cells (Fig4). In order to show that succinyl- concanavalin A stimulated the cells under culture conditions, DNA synthesis was assayed; the cells which were treated with concanavalin A showed 25 ¸ b 2O x i -6 IO- 0 5- T , , I 2 3 4 DAYS Fig. 5. [3H]Thymidine incorporation in succinyl-concanavalin A-treated lymphoeytes. Cells were cultured at 5. lO s cells/ml in minimum essential medium in the presence (O) and absence (0) of succinyl-concanavalin A (50 #g/ml). 10% mixed human serum was used in all cultures and triplicate cultures were used for each data point.  174 enhanced incorporation of [3H]thymidine (Fig. 5), indicating that stimulation had indeed occurred. The values of S v for diphenylhexatriene in lymphocytes were derived from the temperature dependence of r(t) inthe range of 4-40°C (Fig. 6). The dependence of S v upon the temperature is a complex function for all conditions tested. For freshly prepared unstimulated lymphocytes the temperature profile is biphasic; at temperatures below 15°C the order parameter is high, 0.5 to 0.6, indicating restricted motion for diphenyl- hexatriene, while the low value for the order parameter above 32°C indicates that the mem- brane imposes little constraint on the rotation of diphenylhexatriene, and that diphenylhexatriene no longer has a preferential orientation in the membrane. The change in slope of the temperature profile in the range of 15-40°C is similar to that observed in lipid dispersions undergoing 'phase transition' in which the sharp transition tempera- ture is broadened by the presence of a mixture of phospholipids or by the addition of protein [ 15,16]. The temperature profile of S v for diphenyl- hexatriene in 4-day cultured cells was also com- plex, and there appeared to be a broadening of the temperature range over which the slope changes occur. The value of Sv increased after succinyl- concanavalin A addition to freshly isolated cells and to the cells cultured for four days over the entire temperature range. Sip 0.6- 0.5 0.4- 0.3. 0.2- O~I 6 16 2 4 3 2 40 Temperoture *C Fig. 6. The temperature dependence of the order parameter for diphenylhexatriene in lymphocytes. Conditions are given in Materials and Methods. Open and closed symbols refer to day zero and day four of cultivation, respectively. O, O, no addi- tions; [2, I, 50/~g succinyl-concanavalin A per ml. Discussion Peripheral blood lymphocytes are a cell popula- tion that is uniformly in the resting state and that can be activated by the addition of a mitogen such as concanavalin A. This synchrony provides an opportunity to monitor the immediate metabolic and morphologic changes that accompany activa- tion, and the longer-lasting changes occurring over subsequent days of culture. The former category includes increased fluxes of ions and small mole- cules and patching/capping, while the latter includes blast formation, increased RNA synthesis and DNA synthesis. The cell membrane is implicated in the process of stimulation by mitogen. First, it contains the receptors for mitogen. Second, the alterations in transport properties appear to be a primary effect of the lymphocyte stimulant, since increased rates of entry of small molecules have been demon- strated within minutes of the addition of the stimulant and often occur independently of pro- tein and RNA synthesis. In this regard, it is especially interesting to observe that after succinyl-concanavahn A was added, the order parameter for diphenylhexatriene anisotropy increased (from 0.44 to 0.56), indicat- ing that the motion of diphenylhexatriene became more restricted (Fig. 3). More than 50 percent of the change occurred within 6 min after con- canavahn A addition; therefore the changes in the order parameter correlate with the observed rapid mitogen-induced changes in transport and metabolism. The significance of the magnitude of the change in order parameter upon succinyl-concanavalin A addition is hard to evaluate. The fluorescence de- cay of diphenylhexatriene is nonsingle-exponential, a reflection of the heterogeneity of diphenyl- hexatriene location. Since the plasma membrane is less than 20 percent of the total membranes in lymphocytes [21], the changes in the diphenyl- hexatriene order parameter are dramatic indeed and could be indicative of generalized membrane alterations. The functional consequence of mito- gen stimulation might arise from changes in mem- brane phospholipid metabolism. While the relative amounts of the different types of phospholipids in membranes appears to be unaffected by mitogen,
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