166
Eur. J. Immunol. 1975.5: 166-170
M. Inbar and M. Shinitzky
15 David, C.S. and Reisfeld, R.A., Biochemistry 1974. 13: 1014. 16 Haglund, H., in Click, D. (Ed.) Methods of Biochemical Analysis, vol. 19, Interscience Press, New York 1971, p. 1 . 17 Rodbard, D. and Chrambach, A., Anal. Biochem. 1971.40: 95. 18 Weber, K. and Osborn, M., J. Biol. Chem 1969.244: 4406.
20 Nakamuro, K., Tanigaki, N. and Pressman, D., Proc. Nut. Acud Sci. US 1973. 70: 2863. 21 Billing, R.J., Mittal, K.K. and Terasaki, P.I., Tissue Antigens 1973.3: 251. 22 Reisfeld, R.A., Pellegrino, M.A. and Kahan, B.D., Science 1971. 172: 1134.
19 Miyakawa, Y.,Tanigaki, N., Kreiter, V.P., Moore, G.E. and Pressman, D., TYansplantation 1973.15: 312.
23 Reisfeld, R.A. and Kahan, B.D., in Inman, F.P. (Ed.) Contemporary Topics in Immunochemisfry, Vol. 1 , Plenum Press, New York 1972, p. 51.
M. Inbar and M. Shinitzky Laboratory of Membranes and Bioregulation, The Weizmann Institute of Science, Rehovot
Decrease in microviscosity of lymphocyte surface membrane associated with stimulation induced by concanavalin A Microviscosity in the surface membrane lipid core of rat lymph node lymphocytes was determined b y fluorescence polarization analysis of the fluorescent hydrocarbon probe 1,6-diphenyl-l,3,5-hexatriene when embedded in the membrane of intact cells, in relation t o in virro stimulation induced by concanavalin A. The apparent microviscosity in the stimulated cell population was found to be distinctively lower than in the nonstimulated cell population, based on which the membrane microviscosity of the blast cells was estimated. For each of the six experiments which were carried out the estimated microviscosity in the blast cell membranes falls between the upper and the lower limits represented b y freshly drawn intact lymphocytes and leukemic cells, respectively. In accord with this observation it is suggested that a controlled reduction in the microviscosity of the surface membrane is one of the processes which take place during the stimulation of lymphocytes b y antigens in vivo.
1. Introduction The dynamics of cell surface membrane constituents may play a major role in regulation of processes which are associated with cell growth, differentiation and immune response [ 1-61. In principle, the mobility of functional membrane receptors is controlled b y the local microviscosity and t h e gross plasticity properties of the membrane which are determined to a major extent by the composition of the cell surface lipid components and their distribution pattern [7]. Recently, with the aid of a sensitive technique based on fluorescence polarization analysis of the fluorescent hydrcl carbon probe 1,6-dipheny1-1,3,5-hexatriene (DPH) embedded in the surface membrane lipid core of intact cells, we have shown that the microviscosity in the surface membrane of normal lymphocytes from rats o r mice is almost twice that of malignant lymphoma cells from an ascites form of a Moloney virus-induced lymphoma in mice [8]. This difference in membrane microviscosity originates from a cholesterol deficiency, of about half the normal amount, in the surface membrane of the lymphoma cells [8]. Restoration of cholesterol in these cells is accompanied by a marked reduction in their tumorigenicity [ 9 ] . An analogous difference in micro-
[I 8821 Correspondence: Michael Inbar, Laboratory of Membranes and Biore-
gulation, The Weizmann Institute of Science, Rehovot, Israel Abbreviations: Con A: Concanavalin A DPH: 1,6-Diphenyl-1,3,5hexatriene EM: Eagle’s medium YAC: An ascites form of a
Moloney virus-induced lymphoma &MM: a-Methyl-D-mannopyranoside C/PL: Molar ratio of cholesterol-to-phospholipids [3H]dThd: Tritiated thymidine
viscosity was also observed in our laboratory between human lymphocytes obtained from peripheral blood of normal donors and from chronic lymphatic leukemic patients (Inbar, Shinitzky and Ben-Bassat, submitted for publication). These findings have led us to conclude that microviscosity in the surface membrane o f normal and abnormal leukocytes may possess a major regulatory task in various biological functions of these cells in vivo [ 101. T h e present study was undertaken t o determine the membrane microviscosity of lymphocytes stimulated in virro by the mitogen concanavalin A (Con A) as a model system for possible changes in membrane microviscosity which are associated with immune response processes in vivo.
2. Materials and methods 2.1. Cells
The normal lymphocytes were from lymph nodes of 4-5week-old CR/RAR rats. Lymphocytes were collected b y teasing the tissue apart and allowing t h e tissue pieces to sediment [3]. The malignant lymphoma cells used were from an ascites form of a Moloney virus-induced lymphoma (YAC) [ 111. These cells were grown in A strain mice b y intraperitoneal inoculation of l o 5 cells per animal, and were collected for the experiments 10-1 5 days afterwards. The normal lymphocytes and the YAC cells were collected in 0.15 M KCl and washed three times with 0.15 M KCI before use 8 . Con A (Miles-Yeda, Rehovot, Israel) was stored a t 4 C in a phosphate buffer solution, pH 7.2, containing 1 M NaCl.
u
Em. J. ImmunoL 1975.5: 166-170
Membrane microviscosity and lymphocyte stimulation
167
2.2. Stimulation of lymphocytes Lymphocyte stimulation by Con A [ 12, 131 was evaluated by both the induction of thymidine incorporation and the formation of blast cells. Lymphocytes (5 x 1O6 cells) in 0.5 ml Eagle’s medium (EM) were mixed with 0.5 ml EM containing 2 pg Con A in a 35 mm petri dish; 1 ml EM with fetal calf serum was then added to yield a final concentration of 1 pg Con A/ml and 10 % serum. The cells were incubated in a COz incubator at 37 “C up to 72 h. In the control cultures, 0.1 M &-methyl-D-mannopyranoside (a-MM) was added in order to saturate all the specific carbohydrate binding sites of the mitogen before incubation with lymphocytes. Cells were labeled with [3H]thymidine ([3H]dThd, The Radiochemical Centre, Amersham, England) by adding 0.2 pCi in 0.1 ml EM to each plate for 24 h. The incorporation of [ 3H]thymidine was measured after precipitation with 10 % cold trichloroacetic acid and filtration over 0.45 p Millipore filters. Radioactivity counts were measured with a toluene scintillation fluid. The percent of blast cells was determined by a microscope view after staining the cells with May-Grihwald-Giemsa [ 131.
2.3. Fluorescence polarization and membrane microviscosity The fluorescent hydrocarbon 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as a probe for monitoring the microviscosity in the cell surface membrane lipid core [8]. For labeling, 2 x 10-3 M DPH (Aldrich, puriss) in tetrahydrofuran was first diluted 1 000-fold with vigorously stirred aqueous 0.15 M KC1. Stirring was continued for 15 min at 25 “C and a clear and stable aqueous dispersion of 2 x 10“ M DPH, which is practically void of fluorescence, was obtained [8]. One volume of cell suspension (5 x lo6 cells/ml) in 0.15 M KCl was mixed with one volume of the DPH dispersion and incubated at 25 “C for 60 min. The labeled cells were then washed and resuspended in 9.15 M KC1 and immediately used for fluorescence measurements [8]. Under these conditions the fluorescent probe labeled only the cell surface membrane (see discussion) to a level of about 1 probe molecule per 1000 lipid molecules. The DPH-labeled lymphocytes were fully viable as inferred from Con A stimulation experiments analogous to those carried out with the unlabeled lymphocytes. Suspensions of unlabeled cells of the same concentrations were used as reference samples. All fluorescence measurements were carried out at 25 “C. Fluorescence polarizations and intensity were measured as previously described [8]. For excitation a 366 nm band generated from a 500 W mercury arc, which was passed through a Glan-air polarizer, was used. The fluorescence light was detected in 2 independent cross-polarized channels, equipped with Glan-Thompson polarizers, after passing through an aqueous solution of 2 M NaN02 used as a cut-off filter for wave lengths below 390 nm. Fluorescence polarization and intensity were obtained by simultaneous measurement of I,,/I-, and Illwhere I,, and I, are the fluorescence intensities polarized parallel and perpendicular to the direction of polarization of the excitation beam, respectively. These values relate to the degree of fluorescence polarization, ‘Pto the fluorescence anisotropy, ‘r’, and to the total fluorescence intensity, “F”, by the following equations:
+
F = Ill 21, =
11(111/11
+ 2)
Correction for background light was made with the reference cell sample as previously described [ 141. The method used in the present study for evaluation of microviscosity has been outlined previously in conjunction with studies on the hydrocarbon region of synthetic micelles [ 141, liposomes [ 15, 161, biological membranes [ 17, 181, and intact cells [ 8, 181. The method is based on the fluorescence polarization properties of the fluorescent hydrocarbon probe as described by the Perrin equation for rotational depolarization of a nonspherical fluorophore: TO
r
= 1
T*T
+ c(r)77
where ‘r’ and ‘r,,’ are the measured and the limiting fluorescence anisotropies, ‘T’ is the absolute temperature, ‘7’is the excited state lifetime and ‘77’ is the viscosity of the medium. C(r) is a parameter which relates to the molecular shape of the fluorophore, and has a specific value for each r value [ 141. For DPH excited at 366 nm ro = 0.362 and the relation given in Eq. (11) is close to linear with C r) values of (8.6 f 0.4) x lo5 poise x deg-’ x sec-’ [8, 16\. With the aid of C(r) and the determined r, T and T , the microviscosity was evaluated [8].
3. Results Six identical but separate experiments were carried out. In each of the experiments rat lymph node lymphocytes were incubated in EM containing 10 % fetal calf serum in the presence of 1 pg/ml Con A with o r without 0.1 M a-MM. The degree of fluorescence polarization of the treated cells labeled with DPH was determined every 24 h up t o 7 2 h after incubation in relation to the induction of thymidine incorporation (see Fig. 1 and Table 1). In all of the six experiments the degree of fluorescence polarization of the incubated lymphocytes was found to increase with time which reflected an increase in their membrane microviscosity. However, a considerably smaller increase in the degree of fluorescence polarization of DPH was observed in the stimulated cultures, which was correlated with the induction of thymidine incorporation and the formation of blast cells (see Table 1). In all the experiments after 72 h the Con A stimulation was concomitant with 20-30 % blast cell formation. Assuming that the remaining 70-80 % cells can be presented by the cells in the nonstimulated control cultures, the degree of fluorescence polarization of DPH in the surface membrane of the blast cells (Pb)could be estimated from the recorded values in the stimulated and the control cultures ( P and P c , respectively) using the addition law of fluorescence polarization [ 191. For the above specific case this law is expressed by the following equation:
where ‘f,,’ and ‘fC( are the fractions of the fluorescence signals emitted by the blast cells and by the nonstimulated cells, respectively. Since the volume of a blast cell is about three
Eur. J. lmmunol. 1975.5: 166-170
M. Inbar and M. Shinitzky
168
times greater than that of a normal lymphocyte, which corresponds to about twice the surface area, we assumed that the average fluorescence intensity generated by a blast cell is double that of a nonstimulated cell. According t o this assumption in cell cultures containing 20 % blast cells, one gets f, = 1 / 3 and f, = 2/3 and in cell cultures containing 30 % blast cells f, = 6 / 1 3 and f, = 7/13. With the aid of these figures and the recorded values of P, and P we have evaluated the P, values for cultures containing 2 0 % and 30 % blast cells (see Table 2). The range of microviscosity of the blast cells was estimated b y assuming that the values of DPH are the same in all cell types measured. This assumption was based on a previous study [8] where we found that at 25 OC r of DPH-labeled normal lymphocytes and leukemic cells differ by only about 5 %, though these two cell types display an almost 2-fold difference in membrane microviscosity. With the aid of t h s assumption, and the value of 2.8 poise recorded for the microviscosity at 25 O C in freshly drawn rat lymphocytes [8], the microviscosity in the surface membrane o f the blast cells could be deduced. The re-
/
sults, which are displayed graphically in Fig. 2, indicate that the membrane microviscosity of t h e blast cells possesses an intermediate value between t h e upper limit represented by the noncultured nonstimulated lymphocytes and the lower limit represented by t h e noncultured malignant lymphoma cells [8]. Table 1. Thymidine incorporation and the degree of fluorescence polarization (P)of DPH embedded in the surface membrane lipid core of stimulated and nonstimulated lymphocytes
Exp. no.
Treatmend
1
A
B 7
A
B 3
A
B 4
A
5
B A B
6
A
B
[3H]dThd incorporated (cpmlplate) 11195 104
0.284 0.319
8621 100
0.313 0.338
8864 70
0.322 0.370
8664 122
0.315 0.360
11030 60
0.308 0.360
8720 95
0.310 0.360
a) A lymphocytes were incubated for 72 h with 1 pg/ml Con A. B lymphocytes were incubated with the same concentration of Con A in the presence of 0.1 M 0r-m
Table 2. Estimated range for the degree of fluorescence polarization (Pd of blast cells stimulated with Con A and labeled with DPHd
Exp. no. 0.271
0
I
2L 18 Time of incubation(h)
I
72
Figure 1. Changes in the degree of fluorescence polarization of DPH at 25 OC (P)and the rate of thymidine incorporation in stimulated ( 0 ) and nonstimulated ( 0 ) lymphocytes. For experimental details see text.
s 0.275
1 2 3 4 5 6
- 0.23 - 0.28 - 0.26 - 0.26 - 0.21 0.70- 0.25
0.20 0.26 0.22 0.22 0.15
a) Lymphocytes were stimulated with 1 pg/ml Con A and the estimated values were derived from fluorescence polarization data taken after 72 h of incubation. The lower and the upper limits represent 20 % and 30 % blast cells in the stimulated cultures, respectively .
L
0
4. Discussion
IQ200L
11*221
Figure 2. Degree of fluorescence polarization of DPH (P)(25 "C) and membrane microviscosity (T) at 25 "C o f A - nonstimulated lymphocytes [ 81, B - blast cells and C - malignant lymphoma cells [ 81. The bars represent the range experimental results.
Fluorescence polarization provides a sensitive technique for evaluation of dynamic properties of lipid regions in biological membranes [8, 17, 181. This technique was applied in this study for monitoring changes i n microviscosity of lymphocyte surface membranes which follow stimulation induced b y the mitogen Con A. As a fluorescence probe we have used DPH which was shown in previous studies to be highly efficient in measurements of microviscosities and fusion activation energies in lymphocyte membranes [8, 91 as well as in other systems [ 16, 181. The fluorescence microscope view obtained with the DPHlabeled cells is of a glowing ring around t h e cell periphery, which suggests that DPH is predominantly located in the cell surface membranes. This assignment is supported by
Membrane microviscosity and lymphocyte stimulation
Eur. J. ImmunoL 1975.5: 166-170
a series of observations which are given below, all strongly indicating that the detected DPH emission is almost exclusively generated from the surface membranes. The apparent degree of fluorescence polarization, P, relates directly to the microviscosity of t h e labeled region and in biological membranes it is determined to a great extent b y the molar ratio of cholesterol to phospholipids, C/PL [lo]. Since free cholesterol is almost exclusively located in the cell surface membrane, t h e C/PL levels there are substantially greater than in all inner membranes [20]. However, in intact cells the C/PL level in the surface membrane can be increased or decreased b y cholesterol partitioning with external lipid dispersions like liposomes [ 81. Throughout t h e acquisition of DPH by rat lymphocytes or mice lymphoma cells, P remains virtually constant [8], and n o changes in P are detectable upon incubation of t h e DPH-labeled cellsat 25 OC for 60 min in the absence or presence of u p t o 1 0-2 M glucose, or sodium azide. These observations could b e accounted for either b y a rapid and passive partitioning of DPH between the surface membrane and the cell inner membranes, or b y an almost exclusive inclusion of DPH in the cell surface membrane. The latter possibility, however, is much more favorable since b y increasing or reducing t h e C/PL level in the surface membrane of rat normal lymphocytes or mice lymphoma cells, the accompanying changes in P a r e as expected from a n isolated membrane [8].In fact, b y monitoring the degree of fluorescence polarization of DPH-labeled lymphocytes a good estimate for t h e C/PL level in t h e cell surface membrane can be obtained (in preparation).
In all six experiments which were carried out, it was observed that the degree of fluorescence polarization, P, of DPH in the cultured lymphocytes increases with time of incubation, which reflected an overall increase in microviscosity of t h e cell surface membranes. However, in t h e cell samples where stimulation b y Con A was induced, the increase in P was found t o be considerably smaller than in t h e nonstimulated cell samples (Table 1). This difference was attributed t o t h e contribution of t h e blast cells to t h e P value recorded for the stimulated cell samples. This contribution could be estimated from the fluorescence polarization and the blast formation data obtained with t h e stimulated and t h e nonstimulated cultures. For all six experiments performed, the estimated range of P values in t h e blast cells corresponds to microviscosities below that which was recorded in the surface membrane of freshly drawn rat lymph node lymphocytes, but above that recorded for mice lymphoma cells (see Fig. 2). Thus, in contrast to the nonstimulated cultured lymphocytes, where the microviscosity increased with time of incubation, t h e formation of blast cells is concomitant with a reduction of t h e microviscosity in t h e cell surface membrane. Microviscosity in t h e lipid core of biological membranes is predominantly determined b y its molar ratio of cholesterol t o phospholipids C/PL. For DPH-labeled systems the derived microviscosity relates t o C/PL by t h e approximate empirical dependence l o g 7 = 0.17
'v
+ 0.6 C/PL
(IV)
where is the microviscosity at 25 OC given in poise [ lo]. Taking the average 7 values of 2.8 and 1.9 for intact rat lymph node lymphocytes and for the blast cells (see Fig. 2), the C/PL value estimated from Eq. (11) for t h e blast cells is smaller by a factor of about 2.5 from that estimated f o r t h e rat lympho-
169
cytes. This marked decrease in C/PL of t h e blast cell surface membranes undoubtedly originates from the enhanced cellular biosynthesis of phospholipids which is triggered by the binding of Con A [ 2 1, 221. Moreover, the estimated 2.5-fold decrease in C/PL and t h e 2-fold increase in the surface area of the blast cells indicate that the total amount of membrane cholesterol remains approximately constant during the formation o f blast cells which was induced b y Con A. The P value of the nonstimulated lymphocytes was found t o increase from 0.273 before incubation t o an average value of 0.351 after 72 h of incubation (see Table l ) , which correspond t o 7 of 2.8 and 6.0 poise, respectively. This marked increase in microviscosity presumably originates from a 2.2fold increase in C/PL (see Eq. IV) in the surface membrane of the cultured lymphocytes. An analogous increase of a similar magnitude in the molar ratio of total cell cholesterol t o phospholipids was also observed in cultured human lymphoblasts [ 231. The increase in cholesterol level in the cultured lymphocytes could be the result of influx of unesterified cholesterol from the 10 % fetal calf serum present in the culture medium or less likely from an enhanced cellular biosynthesis of cholesterol. In all of our experiments the detectable changes in P o r 7 could be observed only 20-24 h after the addition of Con A (see Fig. 1). These results are in contrast t o the findings of Ferber e t al. [24] and Bamett et al. [25] where increase in membrane fluidity was observed 4 h [24] and 15 min [25] after the addition of the mitogen. Whatever the reason for this discrepancy may be, the decrease in membrane microviscosity (increase in membrane fluidity) which was determined here with DPH is of the same time scale as the increase in the thymidine incorporation and the formation of blast cells in the stimulated cultures. Microviscosity in biological membranes is the major physical parameter which controls lateral and rotational mobilities of the membrane proteins. The observed decrease in microvis. cosity in the stimulated lymphocytes may cause considerable increase in the mobilities of specific receptors or enzymes which can thus turn o n processes like that of DNA synthesis in the blast cells. Since the C/PL is the dominant factor which determines the microviscosity in the cell surface membrane it may play a major role in determining various cellular activities. W e have recently suggested a general working hypothesis which describes the role of the surface membrane cholesterol and the microviscosity in regulation of growth processes in leukocytes [ l o ] . I t was proposed that in leukocytes the upper limit of membrane microviscosity is found in normal nonstimulated cells, whereas the lower limit is found in malignant leukemic cells. Based on this model, and the results which were presented in this study, it is suggested that a controlled reduction of C/PL and microviscosity in the surface membrane of lymphocytes in vivo, t o levels which are still above the threshold beyond which the development of leukemia can occur, may induce the triggering signal for immune response processes. Received August 13, 1974, in revised form October 29, 1974. 5. References 1 Taylor, R.B., Duffus, W.P.H., Raff, M.C. and de k t r i s , S., NatureNew Biol. 1971.233: 225.
2 Singer, S.J. and Nicolson, G.L., Science 1972. 175: 720. 3 Inbar, M. and Sachs, L., FEBS Lett. 1973.32: 124.
170
S.J. Black
Eur. J. ImmunoL 1975.5: 170-175
4 Inbar, M., Ben-Bassat, H., Fibach, E. and Sachs, L., Proc. Nut. Acad. Sci. US 1973. 70: 2577. 5 Shinitzky, M., Inbar, M. and Sachs, L., FEBS Lett. 1973.34: 247. 6 Inbar, M,Shinitzky, M. and Sachs, L., J. Mol. Biol. 1973.81: 245. 7 Gitler, C.,Annu. Rev. Biophys. Bioeng. 1972. 1 : 51. 8 Shinitzky, M. and Inbar, M., J. Mol. Biol. 1974. 85: 603. 9 Inbar, M. and Shinitzky, M., Proc. Nut. Acad. Sci. US 1974. 71: 21 28. 10 Inbar, M and Shinitzky, M., Proc. Nut. Acad. Sci US 1974. 71: 4229. 11 Klein, E. and Klein, G., J. Nut. Cancer Inst. 1964.32:547. 12 Powell, A.E. and Leon, M.A., Exp. Cell Res. 1970.62: 315. 13 Inbar, M., Ben-Bassat, H. and Sachs, L., Exp. Cell Res. 1973. 76. . - . -141 .-.
14 Shinitzky, M., Dianoux, A.C, Gitler, C. and Weber, G., Biochemistry 1971.10: 2106. 15 Cogan, U.,Shinitzky, M., Weber, G. and Nishida, T., Biochemistry 1973.12:521.
16 Shinitzky, M. and Barenholz, Y., J. Biol. Chem. 1974.249: 2652.
S.J. Black* Department of Zoology, University of Edinburgh, Edinburgh
17 Rudy, B. and Gitler, C., Biochim. Biophys. Acta 1972.288: 231. 18 Aloni, B., Shinitzky, M. and Lime, A., Biochim BiOPhYS. Acts 1974.348:438. 19 Weber, G., Biochem. J. 1952.51: 145. 20 Martonosi, A., in Manson, L.A. (Ed.) “Biomembranes” Vol. 1, Plenum Press, New York-London 1971,p. 223. 21 Fisher, D.B. and Mueller, G.C., Biochim. Biophys. Acta 1969. 176: 316. 22 Resch, K. and Ferber, E., Eur. J. Biochem. 1972.27: 153. 23 Gottfried, E.L., in Nelson, G.J. (Ed.) “Blood Lipids and Lipoproteins, Quantitation, Composition and Metabolism”, Wiley Interscience Press, New York 1972,p. 400. 24 Ferber, E., Reilly, C.E., de Pasquale, G. and Resch, K., in Lindhal-Kissling,K. and Osoba, D. (Eds.) Lymphocyte Recognition and Effector Mechanisms, Academic Press, New York 1974,p 529. 25 Barnett, R., Scott, R.E., Furcht, L.T. and Kersey, J.H., Nature 1974.249: 465.
Antigen-induced changes in lymphocyte circulatory patterns The effect of a splenic o r lymph node anti-sheep red blood cell response o n lymphocyte migration patterns in mice was studied. It was found that trapping of lymphocytes in these stimulated organs was indiscriminate and was followed by a period of restricted cell entry or localization; furthermore, reduced cell localization in the spleen, during the splenic response, was accompanied b y a reduction in the number of cells localizing in unstimulated brachial and axillary lymph nodes. These results were taken t o indicate that major changes occur in lymphocyte circulation during strong splenic immune responses.
1. Introduction Migration of lymphocytes into lymphoid tissues seems to be of great importance in determining t h e ability of that tissue t o give rise t o a humoral immune response [ 1-31. This is probably associated with selection, b y antigen, of specifically reactive lymphocytes from t h e recirculating cell pool [4, 51 which in turn may be aided by a temporary hold u p in lymphocyte traffic at the site of antigenic challenge [6-81. Trapping of lymphoid cells has been documented for several species and is known t o follow the presentation of many different antigens and adjuvants [6, 9-1 11. It is also considered t o be indiscriminate [9- 131, although this has never been clearly demonstrated. [I 8781
* Work conducted while a member of the Dept. of Zoology, University of Edinburgh. Correspondence: Samuel J. Black, Institut fur Genetik der Universitat zu Koh, D-5 K o h 41,Weyertal 121,Federal Republic of Germany Abbreviations: SRBC: Sheep erythrocytes CRBC Chicken erythrocytes C P Cyclophosphamide monohydrate i.p.: Intraperitoneally i.v.: Intravenously s.c.: Subcutaneously PFC Plaque-forming cell(s) [ ‘2sI)dUrd: 5-[ ‘2s1)odo-2deoxyuridine FdUrd 5-flUOrOdeoxyuridine GvH: Graft-versus-host TCA: Trichloroacetic acid [ 3H]Leu: ~-[4,5-~H]leucinecpm: Counts per minute
Substantial indiscriminate trapping of circulating lymphocytes in an antigen-stimulated lymphoid organ would almost certainly affect the patterns of lymphocyte migration through the complete animal and hence would be expected to influence the immune potential of uninvolved lymphoid tissues. It was thus the purpose of this investigation t o establish: 1) the specificity of lymphocyte arrest in stimulated lymphoid tissues, 2) the effect of splenic or lymph node immune responses o n the passage of lymphocytes into other lymphoid organs.
2. Materials and methods 2.1. Animals
Two-to-four months-old CBA/H and CBA/H-T6T6 mice from inbred stocks maintained in t h e Department of Zoology, University of Edinburgh, were used throughout. Mice of the same strain, age and sex were chosen for each experiment.
2.2. Antigens Sheep erythrocytes (SRBC) and chicken erythrocytes (CRBC) were obtained in Alsever’s solution from Tissue Culture Services, Slough, England. They were washed 4 times in sterile 0.85 % saline and diluted in saline prior to injection. Doses and routes o f immunization are described under experimental protocols.
Eur. J. Immunol. 1975.5: 166-170
M. Inbar and M. Shinitzky
15 David, C.S. and Reisfeld, R.A., Biochemistry 1974. 13: 1014. 16 Haglund, H., in Click, D. (Ed.) Methods of Biochemical Analysis, vol. 19, Interscience Press, New York 1971, p. 1 . 17 Rodbard, D. and Chrambach, A., Anal. Biochem. 1971.40: 95. 18 Weber, K. and Osborn, M., J. Biol. Chem 1969.244: 4406.
20 Nakamuro, K., Tanigaki, N. and Pressman, D., Proc. Nut. Acud Sci. US 1973. 70: 2863. 21 Billing, R.J., Mittal, K.K. and Terasaki, P.I., Tissue Antigens 1973.3: 251. 22 Reisfeld, R.A., Pellegrino, M.A. and Kahan, B.D., Science 1971. 172: 1134.
19 Miyakawa, Y.,Tanigaki, N., Kreiter, V.P., Moore, G.E. and Pressman, D., TYansplantation 1973.15: 312.
23 Reisfeld, R.A. and Kahan, B.D., in Inman, F.P. (Ed.) Contemporary Topics in Immunochemisfry, Vol. 1 , Plenum Press, New York 1972, p. 51.
M. Inbar and M. Shinitzky Laboratory of Membranes and Bioregulation, The Weizmann Institute of Science, Rehovot
Decrease in microviscosity of lymphocyte surface membrane associated with stimulation induced by concanavalin A Microviscosity in the surface membrane lipid core of rat lymph node lymphocytes was determined b y fluorescence polarization analysis of the fluorescent hydrocarbon probe 1,6-diphenyl-l,3,5-hexatriene when embedded in the membrane of intact cells, in relation t o in virro stimulation induced by concanavalin A. The apparent microviscosity in the stimulated cell population was found to be distinctively lower than in the nonstimulated cell population, based on which the membrane microviscosity of the blast cells was estimated. For each of the six experiments which were carried out the estimated microviscosity in the blast cell membranes falls between the upper and the lower limits represented b y freshly drawn intact lymphocytes and leukemic cells, respectively. In accord with this observation it is suggested that a controlled reduction in the microviscosity of the surface membrane is one of the processes which take place during the stimulation of lymphocytes b y antigens in vivo.
1. Introduction The dynamics of cell surface membrane constituents may play a major role in regulation of processes which are associated with cell growth, differentiation and immune response [ 1-61. In principle, the mobility of functional membrane receptors is controlled b y the local microviscosity and t h e gross plasticity properties of the membrane which are determined to a major extent by the composition of the cell surface lipid components and their distribution pattern [7]. Recently, with the aid of a sensitive technique based on fluorescence polarization analysis of the fluorescent hydrcl carbon probe 1,6-dipheny1-1,3,5-hexatriene (DPH) embedded in the surface membrane lipid core of intact cells, we have shown that the microviscosity in the surface membrane of normal lymphocytes from rats o r mice is almost twice that of malignant lymphoma cells from an ascites form of a Moloney virus-induced lymphoma in mice [8]. This difference in membrane microviscosity originates from a cholesterol deficiency, of about half the normal amount, in the surface membrane of the lymphoma cells [8]. Restoration of cholesterol in these cells is accompanied by a marked reduction in their tumorigenicity [ 9 ] . An analogous difference in micro-
[I 8821 Correspondence: Michael Inbar, Laboratory of Membranes and Biore-
gulation, The Weizmann Institute of Science, Rehovot, Israel Abbreviations: Con A: Concanavalin A DPH: 1,6-Diphenyl-1,3,5hexatriene EM: Eagle’s medium YAC: An ascites form of a
Moloney virus-induced lymphoma &MM: a-Methyl-D-mannopyranoside C/PL: Molar ratio of cholesterol-to-phospholipids [3H]dThd: Tritiated thymidine
viscosity was also observed in our laboratory between human lymphocytes obtained from peripheral blood of normal donors and from chronic lymphatic leukemic patients (Inbar, Shinitzky and Ben-Bassat, submitted for publication). These findings have led us to conclude that microviscosity in the surface membrane o f normal and abnormal leukocytes may possess a major regulatory task in various biological functions of these cells in vivo [ 101. T h e present study was undertaken t o determine the membrane microviscosity of lymphocytes stimulated in virro by the mitogen concanavalin A (Con A) as a model system for possible changes in membrane microviscosity which are associated with immune response processes in vivo.
2. Materials and methods 2.1. Cells
The normal lymphocytes were from lymph nodes of 4-5week-old CR/RAR rats. Lymphocytes were collected b y teasing the tissue apart and allowing t h e tissue pieces to sediment [3]. The malignant lymphoma cells used were from an ascites form of a Moloney virus-induced lymphoma (YAC) [ 111. These cells were grown in A strain mice b y intraperitoneal inoculation of l o 5 cells per animal, and were collected for the experiments 10-1 5 days afterwards. The normal lymphocytes and the YAC cells were collected in 0.15 M KCl and washed three times with 0.15 M KCI before use 8 . Con A (Miles-Yeda, Rehovot, Israel) was stored a t 4 C in a phosphate buffer solution, pH 7.2, containing 1 M NaCl.
u
Em. J. ImmunoL 1975.5: 166-170
Membrane microviscosity and lymphocyte stimulation
167
2.2. Stimulation of lymphocytes Lymphocyte stimulation by Con A [ 12, 131 was evaluated by both the induction of thymidine incorporation and the formation of blast cells. Lymphocytes (5 x 1O6 cells) in 0.5 ml Eagle’s medium (EM) were mixed with 0.5 ml EM containing 2 pg Con A in a 35 mm petri dish; 1 ml EM with fetal calf serum was then added to yield a final concentration of 1 pg Con A/ml and 10 % serum. The cells were incubated in a COz incubator at 37 “C up to 72 h. In the control cultures, 0.1 M &-methyl-D-mannopyranoside (a-MM) was added in order to saturate all the specific carbohydrate binding sites of the mitogen before incubation with lymphocytes. Cells were labeled with [3H]thymidine ([3H]dThd, The Radiochemical Centre, Amersham, England) by adding 0.2 pCi in 0.1 ml EM to each plate for 24 h. The incorporation of [ 3H]thymidine was measured after precipitation with 10 % cold trichloroacetic acid and filtration over 0.45 p Millipore filters. Radioactivity counts were measured with a toluene scintillation fluid. The percent of blast cells was determined by a microscope view after staining the cells with May-Grihwald-Giemsa [ 131.
2.3. Fluorescence polarization and membrane microviscosity The fluorescent hydrocarbon 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as a probe for monitoring the microviscosity in the cell surface membrane lipid core [8]. For labeling, 2 x 10-3 M DPH (Aldrich, puriss) in tetrahydrofuran was first diluted 1 000-fold with vigorously stirred aqueous 0.15 M KC1. Stirring was continued for 15 min at 25 “C and a clear and stable aqueous dispersion of 2 x 10“ M DPH, which is practically void of fluorescence, was obtained [8]. One volume of cell suspension (5 x lo6 cells/ml) in 0.15 M KCl was mixed with one volume of the DPH dispersion and incubated at 25 “C for 60 min. The labeled cells were then washed and resuspended in 9.15 M KC1 and immediately used for fluorescence measurements [8]. Under these conditions the fluorescent probe labeled only the cell surface membrane (see discussion) to a level of about 1 probe molecule per 1000 lipid molecules. The DPH-labeled lymphocytes were fully viable as inferred from Con A stimulation experiments analogous to those carried out with the unlabeled lymphocytes. Suspensions of unlabeled cells of the same concentrations were used as reference samples. All fluorescence measurements were carried out at 25 “C. Fluorescence polarizations and intensity were measured as previously described [8]. For excitation a 366 nm band generated from a 500 W mercury arc, which was passed through a Glan-air polarizer, was used. The fluorescence light was detected in 2 independent cross-polarized channels, equipped with Glan-Thompson polarizers, after passing through an aqueous solution of 2 M NaN02 used as a cut-off filter for wave lengths below 390 nm. Fluorescence polarization and intensity were obtained by simultaneous measurement of I,,/I-, and Illwhere I,, and I, are the fluorescence intensities polarized parallel and perpendicular to the direction of polarization of the excitation beam, respectively. These values relate to the degree of fluorescence polarization, ‘Pto the fluorescence anisotropy, ‘r’, and to the total fluorescence intensity, “F”, by the following equations:
+
F = Ill 21, =
11(111/11
+ 2)
Correction for background light was made with the reference cell sample as previously described [ 141. The method used in the present study for evaluation of microviscosity has been outlined previously in conjunction with studies on the hydrocarbon region of synthetic micelles [ 141, liposomes [ 15, 161, biological membranes [ 17, 181, and intact cells [ 8, 181. The method is based on the fluorescence polarization properties of the fluorescent hydrocarbon probe as described by the Perrin equation for rotational depolarization of a nonspherical fluorophore: TO
r
= 1
T*T
+ c(r)77
where ‘r’ and ‘r,,’ are the measured and the limiting fluorescence anisotropies, ‘T’ is the absolute temperature, ‘7’is the excited state lifetime and ‘77’ is the viscosity of the medium. C(r) is a parameter which relates to the molecular shape of the fluorophore, and has a specific value for each r value [ 141. For DPH excited at 366 nm ro = 0.362 and the relation given in Eq. (11) is close to linear with C r) values of (8.6 f 0.4) x lo5 poise x deg-’ x sec-’ [8, 16\. With the aid of C(r) and the determined r, T and T , the microviscosity was evaluated [8].
3. Results Six identical but separate experiments were carried out. In each of the experiments rat lymph node lymphocytes were incubated in EM containing 10 % fetal calf serum in the presence of 1 pg/ml Con A with o r without 0.1 M a-MM. The degree of fluorescence polarization of the treated cells labeled with DPH was determined every 24 h up t o 7 2 h after incubation in relation to the induction of thymidine incorporation (see Fig. 1 and Table 1). In all of the six experiments the degree of fluorescence polarization of the incubated lymphocytes was found to increase with time which reflected an increase in their membrane microviscosity. However, a considerably smaller increase in the degree of fluorescence polarization of DPH was observed in the stimulated cultures, which was correlated with the induction of thymidine incorporation and the formation of blast cells (see Table 1). In all the experiments after 72 h the Con A stimulation was concomitant with 20-30 % blast cell formation. Assuming that the remaining 70-80 % cells can be presented by the cells in the nonstimulated control cultures, the degree of fluorescence polarization of DPH in the surface membrane of the blast cells (Pb)could be estimated from the recorded values in the stimulated and the control cultures ( P and P c , respectively) using the addition law of fluorescence polarization [ 191. For the above specific case this law is expressed by the following equation:
where ‘f,,’ and ‘fC( are the fractions of the fluorescence signals emitted by the blast cells and by the nonstimulated cells, respectively. Since the volume of a blast cell is about three
Eur. J. lmmunol. 1975.5: 166-170
M. Inbar and M. Shinitzky
168
times greater than that of a normal lymphocyte, which corresponds to about twice the surface area, we assumed that the average fluorescence intensity generated by a blast cell is double that of a nonstimulated cell. According t o this assumption in cell cultures containing 20 % blast cells, one gets f, = 1 / 3 and f, = 2/3 and in cell cultures containing 30 % blast cells f, = 6 / 1 3 and f, = 7/13. With the aid of these figures and the recorded values of P, and P we have evaluated the P, values for cultures containing 2 0 % and 30 % blast cells (see Table 2). The range of microviscosity of the blast cells was estimated b y assuming that the values of DPH are the same in all cell types measured. This assumption was based on a previous study [8] where we found that at 25 OC r of DPH-labeled normal lymphocytes and leukemic cells differ by only about 5 %, though these two cell types display an almost 2-fold difference in membrane microviscosity. With the aid of t h s assumption, and the value of 2.8 poise recorded for the microviscosity at 25 O C in freshly drawn rat lymphocytes [8], the microviscosity in the surface membrane o f the blast cells could be deduced. The re-
/
sults, which are displayed graphically in Fig. 2, indicate that the membrane microviscosity of t h e blast cells possesses an intermediate value between t h e upper limit represented by the noncultured nonstimulated lymphocytes and the lower limit represented by t h e noncultured malignant lymphoma cells [8]. Table 1. Thymidine incorporation and the degree of fluorescence polarization (P)of DPH embedded in the surface membrane lipid core of stimulated and nonstimulated lymphocytes
Exp. no.
Treatmend
1
A
B 7
A
B 3
A
B 4
A
5
B A B
6
A
B
[3H]dThd incorporated (cpmlplate) 11195 104
0.284 0.319
8621 100
0.313 0.338
8864 70
0.322 0.370
8664 122
0.315 0.360
11030 60
0.308 0.360
8720 95
0.310 0.360
a) A lymphocytes were incubated for 72 h with 1 pg/ml Con A. B lymphocytes were incubated with the same concentration of Con A in the presence of 0.1 M 0r-m
Table 2. Estimated range for the degree of fluorescence polarization (Pd of blast cells stimulated with Con A and labeled with DPHd
Exp. no. 0.271
0
I
2L 18 Time of incubation(h)
I
72
Figure 1. Changes in the degree of fluorescence polarization of DPH at 25 OC (P)and the rate of thymidine incorporation in stimulated ( 0 ) and nonstimulated ( 0 ) lymphocytes. For experimental details see text.
s 0.275
1 2 3 4 5 6
- 0.23 - 0.28 - 0.26 - 0.26 - 0.21 0.70- 0.25
0.20 0.26 0.22 0.22 0.15
a) Lymphocytes were stimulated with 1 pg/ml Con A and the estimated values were derived from fluorescence polarization data taken after 72 h of incubation. The lower and the upper limits represent 20 % and 30 % blast cells in the stimulated cultures, respectively .
L
0
4. Discussion
IQ200L
11*221
Figure 2. Degree of fluorescence polarization of DPH (P)(25 "C) and membrane microviscosity (T) at 25 "C o f A - nonstimulated lymphocytes [ 81, B - blast cells and C - malignant lymphoma cells [ 81. The bars represent the range experimental results.
Fluorescence polarization provides a sensitive technique for evaluation of dynamic properties of lipid regions in biological membranes [8, 17, 181. This technique was applied in this study for monitoring changes i n microviscosity of lymphocyte surface membranes which follow stimulation induced b y the mitogen Con A. As a fluorescence probe we have used DPH which was shown in previous studies to be highly efficient in measurements of microviscosities and fusion activation energies in lymphocyte membranes [8, 91 as well as in other systems [ 16, 181. The fluorescence microscope view obtained with the DPHlabeled cells is of a glowing ring around t h e cell periphery, which suggests that DPH is predominantly located in the cell surface membranes. This assignment is supported by
Membrane microviscosity and lymphocyte stimulation
Eur. J. ImmunoL 1975.5: 166-170
a series of observations which are given below, all strongly indicating that the detected DPH emission is almost exclusively generated from the surface membranes. The apparent degree of fluorescence polarization, P, relates directly to the microviscosity of t h e labeled region and in biological membranes it is determined to a great extent b y the molar ratio of cholesterol to phospholipids, C/PL [lo]. Since free cholesterol is almost exclusively located in the cell surface membrane, t h e C/PL levels there are substantially greater than in all inner membranes [20]. However, in intact cells the C/PL level in the surface membrane can be increased or decreased b y cholesterol partitioning with external lipid dispersions like liposomes [ 81. Throughout t h e acquisition of DPH by rat lymphocytes or mice lymphoma cells, P remains virtually constant [8], and n o changes in P are detectable upon incubation of t h e DPH-labeled cellsat 25 OC for 60 min in the absence or presence of u p t o 1 0-2 M glucose, or sodium azide. These observations could b e accounted for either b y a rapid and passive partitioning of DPH between the surface membrane and the cell inner membranes, or b y an almost exclusive inclusion of DPH in the cell surface membrane. The latter possibility, however, is much more favorable since b y increasing or reducing t h e C/PL level in the surface membrane of rat normal lymphocytes or mice lymphoma cells, the accompanying changes in P a r e as expected from a n isolated membrane [8].In fact, b y monitoring the degree of fluorescence polarization of DPH-labeled lymphocytes a good estimate for t h e C/PL level in t h e cell surface membrane can be obtained (in preparation).
In all six experiments which were carried out, it was observed that the degree of fluorescence polarization, P, of DPH in the cultured lymphocytes increases with time of incubation, which reflected an overall increase in microviscosity of t h e cell surface membranes. However, in t h e cell samples where stimulation b y Con A was induced, the increase in P was found t o be considerably smaller than in t h e nonstimulated cell samples (Table 1). This difference was attributed t o t h e contribution of t h e blast cells to t h e P value recorded for the stimulated cell samples. This contribution could be estimated from the fluorescence polarization and the blast formation data obtained with t h e stimulated and t h e nonstimulated cultures. For all six experiments performed, the estimated range of P values in t h e blast cells corresponds to microviscosities below that which was recorded in the surface membrane of freshly drawn rat lymph node lymphocytes, but above that recorded for mice lymphoma cells (see Fig. 2). Thus, in contrast to the nonstimulated cultured lymphocytes, where the microviscosity increased with time of incubation, t h e formation of blast cells is concomitant with a reduction of t h e microviscosity in t h e cell surface membrane. Microviscosity in t h e lipid core of biological membranes is predominantly determined b y its molar ratio of cholesterol t o phospholipids C/PL. For DPH-labeled systems the derived microviscosity relates t o C/PL by t h e approximate empirical dependence l o g 7 = 0.17
'v
+ 0.6 C/PL
(IV)
where is the microviscosity at 25 OC given in poise [ lo]. Taking the average 7 values of 2.8 and 1.9 for intact rat lymph node lymphocytes and for the blast cells (see Fig. 2), the C/PL value estimated from Eq. (11) for t h e blast cells is smaller by a factor of about 2.5 from that estimated f o r t h e rat lympho-
169
cytes. This marked decrease in C/PL of t h e blast cell surface membranes undoubtedly originates from the enhanced cellular biosynthesis of phospholipids which is triggered by the binding of Con A [ 2 1, 221. Moreover, the estimated 2.5-fold decrease in C/PL and t h e 2-fold increase in the surface area of the blast cells indicate that the total amount of membrane cholesterol remains approximately constant during the formation o f blast cells which was induced b y Con A. The P value of the nonstimulated lymphocytes was found t o increase from 0.273 before incubation t o an average value of 0.351 after 72 h of incubation (see Table l ) , which correspond t o 7 of 2.8 and 6.0 poise, respectively. This marked increase in microviscosity presumably originates from a 2.2fold increase in C/PL (see Eq. IV) in the surface membrane of the cultured lymphocytes. An analogous increase of a similar magnitude in the molar ratio of total cell cholesterol t o phospholipids was also observed in cultured human lymphoblasts [ 231. The increase in cholesterol level in the cultured lymphocytes could be the result of influx of unesterified cholesterol from the 10 % fetal calf serum present in the culture medium or less likely from an enhanced cellular biosynthesis of cholesterol. In all of our experiments the detectable changes in P o r 7 could be observed only 20-24 h after the addition of Con A (see Fig. 1). These results are in contrast t o the findings of Ferber e t al. [24] and Bamett et al. [25] where increase in membrane fluidity was observed 4 h [24] and 15 min [25] after the addition of the mitogen. Whatever the reason for this discrepancy may be, the decrease in membrane microviscosity (increase in membrane fluidity) which was determined here with DPH is of the same time scale as the increase in the thymidine incorporation and the formation of blast cells in the stimulated cultures. Microviscosity in biological membranes is the major physical parameter which controls lateral and rotational mobilities of the membrane proteins. The observed decrease in microvis. cosity in the stimulated lymphocytes may cause considerable increase in the mobilities of specific receptors or enzymes which can thus turn o n processes like that of DNA synthesis in the blast cells. Since the C/PL is the dominant factor which determines the microviscosity in the cell surface membrane it may play a major role in determining various cellular activities. W e have recently suggested a general working hypothesis which describes the role of the surface membrane cholesterol and the microviscosity in regulation of growth processes in leukocytes [ l o ] . I t was proposed that in leukocytes the upper limit of membrane microviscosity is found in normal nonstimulated cells, whereas the lower limit is found in malignant leukemic cells. Based on this model, and the results which were presented in this study, it is suggested that a controlled reduction of C/PL and microviscosity in the surface membrane of lymphocytes in vivo, t o levels which are still above the threshold beyond which the development of leukemia can occur, may induce the triggering signal for immune response processes. Received August 13, 1974, in revised form October 29, 1974. 5. References 1 Taylor, R.B., Duffus, W.P.H., Raff, M.C. and de k t r i s , S., NatureNew Biol. 1971.233: 225.
2 Singer, S.J. and Nicolson, G.L., Science 1972. 175: 720. 3 Inbar, M. and Sachs, L., FEBS Lett. 1973.32: 124.
170
S.J. Black
Eur. J. ImmunoL 1975.5: 170-175
4 Inbar, M., Ben-Bassat, H., Fibach, E. and Sachs, L., Proc. Nut. Acad. Sci. US 1973. 70: 2577. 5 Shinitzky, M., Inbar, M. and Sachs, L., FEBS Lett. 1973.34: 247. 6 Inbar, M,Shinitzky, M. and Sachs, L., J. Mol. Biol. 1973.81: 245. 7 Gitler, C.,Annu. Rev. Biophys. Bioeng. 1972. 1 : 51. 8 Shinitzky, M. and Inbar, M., J. Mol. Biol. 1974. 85: 603. 9 Inbar, M. and Shinitzky, M., Proc. Nut. Acad. Sci. US 1974. 71: 21 28. 10 Inbar, M and Shinitzky, M., Proc. Nut. Acad. Sci US 1974. 71: 4229. 11 Klein, E. and Klein, G., J. Nut. Cancer Inst. 1964.32:547. 12 Powell, A.E. and Leon, M.A., Exp. Cell Res. 1970.62: 315. 13 Inbar, M., Ben-Bassat, H. and Sachs, L., Exp. Cell Res. 1973. 76. . - . -141 .-.
14 Shinitzky, M., Dianoux, A.C, Gitler, C. and Weber, G., Biochemistry 1971.10: 2106. 15 Cogan, U.,Shinitzky, M., Weber, G. and Nishida, T., Biochemistry 1973.12:521.
16 Shinitzky, M. and Barenholz, Y., J. Biol. Chem. 1974.249: 2652.
S.J. Black* Department of Zoology, University of Edinburgh, Edinburgh
17 Rudy, B. and Gitler, C., Biochim. Biophys. Acta 1972.288: 231. 18 Aloni, B., Shinitzky, M. and Lime, A., Biochim BiOPhYS. Acts 1974.348:438. 19 Weber, G., Biochem. J. 1952.51: 145. 20 Martonosi, A., in Manson, L.A. (Ed.) “Biomembranes” Vol. 1, Plenum Press, New York-London 1971,p. 223. 21 Fisher, D.B. and Mueller, G.C., Biochim. Biophys. Acta 1969. 176: 316. 22 Resch, K. and Ferber, E., Eur. J. Biochem. 1972.27: 153. 23 Gottfried, E.L., in Nelson, G.J. (Ed.) “Blood Lipids and Lipoproteins, Quantitation, Composition and Metabolism”, Wiley Interscience Press, New York 1972,p. 400. 24 Ferber, E., Reilly, C.E., de Pasquale, G. and Resch, K., in Lindhal-Kissling,K. and Osoba, D. (Eds.) Lymphocyte Recognition and Effector Mechanisms, Academic Press, New York 1974,p 529. 25 Barnett, R., Scott, R.E., Furcht, L.T. and Kersey, J.H., Nature 1974.249: 465.
Antigen-induced changes in lymphocyte circulatory patterns The effect of a splenic o r lymph node anti-sheep red blood cell response o n lymphocyte migration patterns in mice was studied. It was found that trapping of lymphocytes in these stimulated organs was indiscriminate and was followed by a period of restricted cell entry or localization; furthermore, reduced cell localization in the spleen, during the splenic response, was accompanied b y a reduction in the number of cells localizing in unstimulated brachial and axillary lymph nodes. These results were taken t o indicate that major changes occur in lymphocyte circulation during strong splenic immune responses.
1. Introduction Migration of lymphocytes into lymphoid tissues seems to be of great importance in determining t h e ability of that tissue t o give rise t o a humoral immune response [ 1-31. This is probably associated with selection, b y antigen, of specifically reactive lymphocytes from t h e recirculating cell pool [4, 51 which in turn may be aided by a temporary hold u p in lymphocyte traffic at the site of antigenic challenge [6-81. Trapping of lymphoid cells has been documented for several species and is known t o follow the presentation of many different antigens and adjuvants [6, 9-1 11. It is also considered t o be indiscriminate [9- 131, although this has never been clearly demonstrated. [I 8781
* Work conducted while a member of the Dept. of Zoology, University of Edinburgh. Correspondence: Samuel J. Black, Institut fur Genetik der Universitat zu Koh, D-5 K o h 41,Weyertal 121,Federal Republic of Germany Abbreviations: SRBC: Sheep erythrocytes CRBC Chicken erythrocytes C P Cyclophosphamide monohydrate i.p.: Intraperitoneally i.v.: Intravenously s.c.: Subcutaneously PFC Plaque-forming cell(s) [ ‘2sI)dUrd: 5-[ ‘2s1)odo-2deoxyuridine FdUrd 5-flUOrOdeoxyuridine GvH: Graft-versus-host TCA: Trichloroacetic acid [ 3H]Leu: ~-[4,5-~H]leucinecpm: Counts per minute
Substantial indiscriminate trapping of circulating lymphocytes in an antigen-stimulated lymphoid organ would almost certainly affect the patterns of lymphocyte migration through the complete animal and hence would be expected to influence the immune potential of uninvolved lymphoid tissues. It was thus the purpose of this investigation t o establish: 1) the specificity of lymphocyte arrest in stimulated lymphoid tissues, 2) the effect of splenic or lymph node immune responses o n the passage of lymphocytes into other lymphoid organs.
2. Materials and methods 2.1. Animals
Two-to-four months-old CBA/H and CBA/H-T6T6 mice from inbred stocks maintained in t h e Department of Zoology, University of Edinburgh, were used throughout. Mice of the same strain, age and sex were chosen for each experiment.
2.2. Antigens Sheep erythrocytes (SRBC) and chicken erythrocytes (CRBC) were obtained in Alsever’s solution from Tissue Culture Services, Slough, England. They were washed 4 times in sterile 0.85 % saline and diluted in saline prior to injection. Doses and routes o f immunization are described under experimental protocols.
