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Interactions of lyophilised and rehydrated mouse spleen lymphocytes with phytohaemagglutinin (PHA) and concanavalin A (Con A)
Interactions of Lyophilised and Rehydrated Mouse Spleen Lymphocytes with Phytohaemagglutinin (PHA) and Concanavalin A (Con A) D. THOhfAS
Ah-D
H. ZOLA
I~epclrtnLents of Biological Chemistry and Experimental Immunobiology, Wcllcome Research Laboratories, Langley Court, Bcckenham, Kent, BR3 3BS
Recent studies from these laboratories have shown that a proportion of splenic lymphocytes fr’om CBA/lac mice immuniscd with sheep red blood cells (SRBC) were capable of producing haemolytic antibody ( IgM) plaques when reconstituted after freezing or freeze-drying under controlled conditions in the presence of polyvinylpyrrolidone (PVP) and foetal calf serum (FCS) (5). H owevcr, the ability of splenic cells to form E rosettes with SRBC or to bind to lectin-derivatised Sepharose beads was impaired, particularly by freezedrying (16) and the treated cells were unable to proliferate in response to mitogenic stimuli ( 6). One interpretation of these observations was that a degree of organisation had survived in the cytosol of the spleen cells capable of producing the haemolytic antibodies, whilst certain membrane binding sites had become coated with PVP during the process of freeze-dry& 0 or had been differcntially denatured and were therefore functionally impaired. In order to identify the nature of the damage at the membrane level more preciscly we have examined the interaction of frozen and freeze-dried cells with the lectins phytohaemagglutinin (PHA) and Concanavalin A (Con A) using radioisotope and immunofluorescent techniques. Received Janunry 13, 1976.
Copyright All rights
@ 1977 by Academic Press, Inc. 01 reproduction in any iorm reserved.
XI.4TERIALS
AND METHODS
Concanavalin A (Con A) and Sepharose 4B beads were obtained from Pharmacia, Uppsala, Sweden. Purified phytohaemagglutinin (PHA-P) and sheep ,antirabbit immunoglobulin labelled with fluorescein isothiocyanate were obtained from Wellcome Reagents Ltd., Beckenham, Kent, U.K. The rabbit anti-Con A and rabbit antiPHA sera used in this study were prepared by Dr. D. C. Edwards of these laboratories. Female Californian rabbits weighing approximately 7 kg were injected in the footpads with 1 mg of the lectin emulsified in Freund’s Complete Adjuvant ( FCA ). Approximately 50 days later the rabbits were injected intramuscularly with a similar emulsion and bled out 4 weeks later. The blood was allowed to coagulate and the serum stored at -20°C until required. Preparation
of Spleen Cells (SC)
CBA/lac mice approximately 14 weeks old were sacrified and their spleens removed. Single cell suspensions were prepared in Hanks balanced salt solution (HBSS) and washed in HBSS. The erythrocytes were lysed using Geys haemolytic buffer; lymphocytes were isolated on Ficoll/Triosil (8) and washed three times in HBSS by centrifugation at 200g for 10 min.
ISSN
0011-2240
4G
THOMAS
AND
ZOLA
Cell Corctrts md Ccl1 Vitil~ilily Total cell counts were carried out using conventional haematological techniques (2). Cell viability was measured by the trypan blue dye exclusion technique (3). Binding of [12nI] PHA and [la”Z] Con A to Treated and Untreated Cells
FIG. 1. Absci.sxz: counts of [YJCon A added (cpm X 10d); ordinate: counts of [‘7]Con A bound by 1.25 X 10’ cells (cpm X lo+). The binding of [7]Con A to freshly prepared M---B (SC), frozen/thawed O-O (FTC), and freeze-dried/rehydrated spleen cells 0-O (FDRC).
The iodination of Con A and PHA was carried out at room temperature and pH 7.4 by the chloramine-T procedure described by Hunter (9). Binding experiments were carried out by mixing 10 pl aliquots of dilutions of the labelled lectin with 50 ~1 aliquots of the cells (treated and untreated) containing 1.25 x lo6 cells. The cell suspensions were incubated at 37°C for 30 min, after which time the cells were washed twice in 2 ml of buffer and transferred to clean tubes for radioactivity counts. The reaction tubes were pretreated to reduce the absorption of the lectin by soaking in a solution of 5% KI and 0.5% bovine seruni albumin in phosphate buf-
Preparation of Frozen/Thawed (FTC) and Freeze-DriedjRehydrated Cells (FDRC) SC prepared as described above were suspended in 1470 PVP in HBSS supplemented with FCS and adjusted to 1 x 10’ cells/ml. The cells were then frozen and thawed or freeze-dried and rehydrated by the techniques previously described in detail elsewhere ( 16). The rate of cooling ing was approximately 4”C/min down to -40°C on the precooled shelves of an EF-6 freeze-drier (Edwards High Vacuum Ltd., U.K. ). Approximately half of the total number of cells were held at this temperature for 16-17 hr and then thawed. The remaining cells, having been cooled down to -40°C were then dried by sublimation of ice in vucuo for 16-17 hr. Following freeze-drying the cells were rehydrated slowly ( 16). The percentage recovery of freeze-dried spleen cells was approximately 70%, of which > 95% were viable as judged by the trypan blue dye exclusion test (3).
a 7. 6 5 4 3 2
0
4
a
12
16
20
24
FIG. 2. Abscissa: counts of [%I]PHA added (cpm X lo-‘); ordinate: counts of [‘?]PHA bound by 1.25 X 10’ cells (cpm X 10m4). The binding of [9]PHA to freshly prepared n -W (SC), frozen/thawed O-O (FTC), and freeze-dried/ (FDRC). rehydrated spleen cells 0 -0
XfOUSE
I,ectiu
Bindirlg
SC
+ +
con s
it
to the Sllrfltce (FI)RC)
h 1:
+
Ig
II
:I c:on
SPLEES
+
con
A,L
(FITC)”
A
+
II
47
of Xormal (SC), Frozen (FTC), and Freeze-Dried 11e:tsrued by Imm~moflrlores~ellce
SC + PHA + 1:. a I’HAc + S it II Ig (FIX)
FTC
LYMl’HOCYTES
a con
A
+ s :I, 11 Ig (FITC)
FTC + I’IIA + 1: n 1’H.k + s a I‘l Tg (FITC)
FI)I:c + con .4 + II a rm + S a It Ig (FIX)
x
I;l)l:c + PIIA + I’L a I’HA + S it IL Ig (FITC) l1 It tl Co11 A---rabbit xrlti-Con~snRvalin A serum. b S it 1:. Ig (FITC)---sheep antirabbit inlnlrulogloblllitl r I< :I PHA-rabbit :~ntiphytohtternagglrltinill ,ser,un.
fered saline (PBS) for 2 hr at RT. The tubes were then rinsed three times with distilled water and allowed to drain dry. PH,4 expcrimcnts were carried out using PBS, whilst the Con ,4 experiments were performed in HBSS containing R4nC12 ( 10e3sf) to stabilisc the Con A and 0.25% BSA to reduce nonspecific binding.
Binding of PHA and Con A to Treated and Untreated Cells Using an lmmunofluorescent Technique Binding of Con A and PHA was measured by an immunofluorescence technique described by Thomas et al. (IS). Briefly, 10 ~1 smears of SC, FTC, and FDRC at a concentration of 1 X lo6 cells/ml were allowed to dry in air on glass slides and fixed in alcohol. The fixed cells were reacted with
24.3
5
2.:::
23.5
17 I.5 ‘r
233 218 2ii-4
17 32 12 I
218 2.X) 2:3X
32 -
2.3) 233 223
-
235 213
15 37
220 2.50
30 -
Cells
03
!I2
94
12 I
17 23 I
94
90 > 04 >
-__
ld~ellecl with fl~loresc~ein isothiocyanate.
similar volumes of Con A (10 pg/ml) or PHA (5 pg/ml) for 20 min at RT, after which the unbound lectin was removed by washing three times in PBS. The cells were then reacted with the appropriate rabbit anti-Con A or anti-PHA, diluted 1: 100 with PBS, for a similar period of time and the unbound antibody removed by repeated washing and then reacted with anti-rabbit immunoglobulin labelled with fluorescein isothiocyanate. Finally the cells were washed free of excess %uorescein labelled antibody and the slides viewed for the uptake of the fluorescent dye. The numbers of cells fluorescing using FTC and FDRC preparations were compared with the cells %uorcscing using SC preparations. Controls without lectin, without antiserum to the lectins, or without fluorescein labelled antibody were always included.
4s
THOMAS TABLE
2
A Comparison of White Cell Agglutination of Normal (SC), Frozen (FTC), and Freeze-1)ried Cells (FDRC) Using Con A or PHA Agglutination titre (reciprord of dilution)
Quantit) of lectill required for agglutination (&ml)
SC
200 200
FTC FTC
Con A PHA
200 200
.i
FDRC FDRC -_.-
Con A PHA
20 20
.io 30
AggluGnution of Treated and Cells Using Con A and PHA
numbers of cells taking up the fluorescent dye, note being taken as to whether or not this fluorescence was ringlike, in patches or spots or capped. The experiments were carried out at +4”C and at 37°C with or without sodium azide, a metabolic inhibitor.
5 5
5
Untreated
Measurements of white cell agglutination were carried out using a microscale modification of standard procedures [see for example Thomas et al. ( 17)] at lectin concentrations from 1 to 10e7 “g/ml. Capping of PHA by Antibody Treated and Untreated Cells
ZOLA
RESULTS
Con A PHA
SC
AND
Using
Aliquots ‘of 1.0 ml of SC, FTC and FDRC suspensions at a concentration of 5 X lo6 cells/ml were placed in plastic tubes and centrifuged at 200g for 10 min. The supernatant was removed by decantation and 1.0 ml of the PHA solution at a concentration of 5 pg/ml was added. The suspension was agitated gently and incubated for 20 min at 37”C, after which the cells were harvested and washed by centrifugation. The resulting cell pellet was resuspended in 1.0 ml of a 1:lOO dilution of the rabbit anti-PHA serum and incubated for a further 20 min at 37°C. The supernatant was removed and the cells reacted with antirabbit immunoglobulin labelled with fluorescein isothiocyanate. The suspension was incubated for 20 min at 37”C, washed exhaustively, and examined by fluorescence. The total numbers of cells per field were counted in white light, together with the
The amount of iodinated Con A ( [1”“1] Con A) and PHA ([““IIPHA) bound by spleen cells was not significantly affected by the freeze-thaw or freeze-drying/rehydration procedures (Figs. 1 and 2) although the uptake of [Y]Con A by the frozen cell preparations was slightly increased. This increase was observed repeatedly with Con A but not with PHA. The total number of cells binding Con A and PHA was measured by a double sandwich immunofluorescence technique. The results (Table 1) clearly showed that the number of cells binding the lectins was unaltered by either the freezing or the lyophilisation procedures. Approximately 94% of all cells showed uptake of fluorescent dye. The agglutination of spleen cells by PHA and Con A was not affected by the freezethaw procedure but was reduced by freezedrying (Table 2). Normal and frozen cells were agglutinated by lectin concentrations down to 5 pg/ml whilst 50 /*g/ml of the lectin was required to agglutinate the lyophiliscd cells. The ability of the treated cells to cap PHA was examined using fluorescent antibody. The results, set out in Table 3, showed that normal spleen cell cap formation was optimal, 76% cap formation, when carried out at 37°C and minimal, 13% cap formation, when performed at +4”C. The addition of sodium azide at a final concentration of 10-’ M in the culture medium inhibited cap formation at both 37°C and +4”C. Similar results were obtained when using frozen-thawed cells under similar experimental conditions. Approximately 64% of the fluorescing cells formed caps at optimal
conditions whilst no caps were seen at +4”C. The addition of sodium azide, as expected, completely inhibited cap formation. In contrast to these results, cap formation of PHA by freeze-dried/rehydrated cells was dramatically reduced at optimum culture conditions, producing only 6%) capped cells out of the total number of fluorescing cells. No cells were seen to cap at t4”C and the sodium azide completely inhibited what little cap formation there 1ras at 37°C. DISCUSSION
The preservation of living cells for long periods of time is normally carried out by freezing the cells in the prescncc of a suitable cryoprotectant and then maintaining the cells at the temperature of liquid nitrogen. Earlier stud& had shown that reconstituted mouse spleen cells freeze-dried in the presence of PVP and FCS, were able to bind antilymphocyte serum and to secrete antibody, an energy requiring proccss. However, some membrane alterations wcrc apparent, as indicated by impaired ability of these cells to form E rosettes and to bind to immobilised PHA and Con A ( 16). In addition, such cells were unable to prolifcratc in response to initogcnic stimulus (6). These results were originally intcrprcted on the basis of masking of membranc sites or clifferential denaturation of the sites which react with the lectins Con A and PHA. Ho~cvcr, the present results show quite clearly that the quantity of Con A and PHA bound was not significantly affected by freezing or freeze-drying in the prcscnce of PVP supplemented with FCS. The decreased bincling of freeze-dried ~11s to Icctin/Sepharosc beads may bc esplaincd if the lateral diffusion of macroinoleculcs is impairecl, since the formation of :I stable association between a cell and a derivatiscd Scpharose particle probably rcquircs multiple bonding (1, 7). Evidence for tbr lack of lateral diffusion of membranc proteins was obtained in the Auorcs-
” I’h~toh:telnag-glutillill (pluified --Wellcome Xeagents Ltd., Beckenham). b NnN2 -~sotlirun asride (RI)11 T,ttl., U.K.).
cent studies where it was shown that capping of the bound lectins by antibody was greatly reduced after the cells had been freeze-dried. The impairment of agglutination by lectins after freeze-drying reported in this study could also be due to decreased membrane fluidity, since clustering of reccptors has been observed in Con A agglutination of cells ( 12). However, it would seem that clustering of the receptors is not au absolute requirement for agglutination, ancl becomes unnecessary at high lectin concentrations. Cap formation has been shown to be inhibitecl by the antimctabolite, sodium azide ( 15) and by certain antimitotic drugs such as cytochalasin B and vinblastine (14). Vinblastine is a known inhibitor of microtubule function and cytochalasin B has been reported to be similarly involved with microfilament function (I9), although this is by no means proved. The failure of the cells studied here to cap co&1 possibly be cxplained in terms of irrcvcrsiblc damage occurring during the freczc-drying stage to
50
THOMAS
the metabolic function of the cells. Equally, as microtubule and microfilament functions appear to play an active and synergic role in the formation of caps by the cell (13), damage to these structures is also a strong possibility. Furthermore, these findings could also help to explain earlier results (16) where it was shown that rosette formation was impaired following freeze-drying as normally functioning microfilaments appear to be necessary for the formation of rosettes (1% 4). The exact mechanisms involved in the early stages of mitogenesis by plant lectins are not known, although capping and endocytosis are thought not to be involved, whilst the lateral diffusion of the membrane macromolecules is probably required in order to form the necessary ion channels. Thus the absence of a mitogenic response after freeze-drying and reconstitution could be partly due to the impaired membrane fluidity, Equally it could be accounted for by damage occurring to the mitotic apparatus of the cell or by damage to, or blocking of, the functional lectin receptor. It is known that lectins will bind to any macromolecule containing the appropriate conformation of sugar but only a small proportion of these sites are receptors in the functional sense. This distinction is illustrated by the observations that PHA binds in equal proportions to both T-cells and B-cells ( ll), but it is only the T-cell that responds to the lectin mitotically. In the present study, although the results indicate that the total quantity of bound lectin is not noticeably decreased, the possibility remains that the functional receptors are either masked, possibly by the cryoprotectant PVP, or differentially denatured, thus becoming functionally impaired. In conclusion, it should be emphasised that the results presented in this and earlier studies (6, 16) suggest that freeze-dried/ rehydrated cells have little functional integrity as measured by the response to
AXD
ZOLA
mitogens and by the capping of bound lectins by antibody despite the fact that such cells retain their ability to produce IgM antibodies (5). These studies indicate that attempts to improve the functional integrity of the recovered cells following freezedrying should use a test to assess the membrane fluidity, e.g., capping of lectins by antibody, as a major criterion of success. SUMMARY
Rehydrated lymphocytes freeze-dried in of polyvinyl-pyrrolidone the presence (PVP) and foetal calf serum (FCS) have been shown to retain plaque forming activity (5) but were unable to respond to plant mitogens (6) or to bind to immobilised phytohaemagglutinin (PHA) or Concanavalin A (Con A) ( 16). Studies with fluorescein conjugated and radio iodinated antibody to PHA and Con A showed that the ability of the cells to bind the lectins was undiminished. However, agglutination by lectin and capping of lectinanti-lectin were impaired by freeze-drying, suggesting a defect in membrane fluidity, perhaps resulting from damage to the microfilament and microtubule apparatus of the cell. ACKNOWLEDGMENTS The authors wish to express their gratitude to hlrs. S. Barnes, Mrs. S. Graves, Mrs. A. Niblock, and Mr. N. Kent for invaluable technical assistance. REFERENCES 1. Anderson, J., Edelman, G. hl., Miiller, G., and Sjaberg, 0. Activation of B lymphocytes by locally concentrated Concanavalin A. Eur. J. Immunol. 2, 233 ( 1972). 2. Baker, F. J., Silverton, R. E., and Luckcock, E. D. In “Introduction to Medical Laboratory Technology” (F. J. Baker, R. E. Silverton, and E. D. Luckcock, Eds.), 4th ed., pp. 506-521. Butterworths, London, 1966. 3. Boyse, E. A., Old, L. J., and Chourolinkov, I. Cytotoxic test for demonstration of mouse antibody. In “Methods of Medical Research” (H. N. Eisen, Ed.), Vol. 10. Yearbook Medical Publishers, Chicago, 1964.
4. Cohnen, G., Fischer, K. and Brittinger, G. Human T-lymphocyte rosette formation: inhibition by cytochalasin B. Irnmru~!ogr~ 29, 337 ( 1975). 5. Damjanovic, I’., Edwards, D. C., and Thomas, D. Recovery of hacmolytic placlue-fonnillg ~11s after freeze-drying. Xcltrrrc’ (London) 253, llG (1975). 6. Damjanovic, V., and Thomas, 1). The 11%~of polyvinylpyrrolidone as a cryoprotectnnt in the freezing of human lymphocytes and in preliminary stllclies on freeze-drying. I’roc. C:ech. had. Sci., in press (1976). 7. Greavrs, 1~1. F., and Baumingrr, S. .I\ctivatio:1 of T and B lymphocytes by insoluble phytomitogens. Nuture (London) 235, G7 ( 1972). 8. Harris, R., and likaejiofa, E. 0. Rapid preparation of lymphocytes for ti\suc t),pin:g. Lancct 2, 237 ( 19G9). 9. Hunter, W. bl. In “Handbook of 1Ssperimcntal Immunology” (1~. hl. Weir, Ed.), 2nd cd.. Chap. 17. Black\\~ells, Oxford, 1973. 10. Kersey, J. H., IIom, 1). J., and Buttrick, 1’. IIuman T lymphocyte receptors for sheep crythrocytes: Conditions for binding including inhibition by cytochnlasin B. J. Inrn~~lr~oZ. 112, 862 (1974). 11. Loor, F. Binding and redistribution of lectins on lymphocyte membranes. Eur. J. Imm~uwl. 4, 210 (1974). 12. Nicolson, G. L. Topography of mcmbranc Concnnavalin A sites modified by proteolysis. Nature (I,ontlon) 239, 193 (1972).
13. Pctris de, S. Concannvalin A receptors, immunoglobulins and B antigens of the lymphocyte surface: interactions with Concnvalin A and with cytoplasmic structures. J. Cdl Biol. 65, 123 (1975). 14. Pctris de, S. Inhibition and reversal of capping I)\- cl-tochalasin B, viilblahtine and colchicinc. Sntrrrc (Lontlon) 250, 54 (1974). 15. Ta)-lor, R. B., Dullus, \V. I?. IL, Raff, 31. C., and de Petris, S. Rcdistriblltion and pinoc>tosis of lynlphocyte surface immnnoglobuinduced 1)) anti-immunolin 11&c&.? ~lol~~ili~i antibody. NNtrrrc ( I,ontlon) 233, 225 (1971). 16. Thomas, D., Ed\~ards, D. C., and Damjano\ic, 1’. The surface properties of spleen cells from CB.%/lnc mice following freezing nnd freeze-dq ing using polyvinyIpyrrolidoi~e. Cr&iolv~!/ 13, 191 ( 1976). 17. Thomas, I)., ~Ioccdalc, B., Rahr, I~., Epps, II. B. G., and Ecl\vards, I). C. Sates on the large scale preparation and propertics of anti-lymphocyte serum for itse in mice. C/in. Eql. Immwd. 7, 769 ( 1970). 18. Thomas, D., hlosedale, B., and Zola, I-I. The use of the indirect fluorescent antibody tcchnique in assessing the activity of antilymphocytic sern and antilymphocytic globulins, Clin. EXJI. Immrmol. 8, 987 (1971). 19. \Vcssels, N. K., Spooner, B. S., Ash, J. IT., Bradley, hl. O., Lnduenn, >I. A., Taylor, B. L., \Vrenn, J. T., and Yamada, K;. hI. SlicroBlanir:nts in cellular and developmental processes. Sciulrr 171, 135 ( 1971).
Ah-D
H. ZOLA
I~epclrtnLents of Biological Chemistry and Experimental Immunobiology, Wcllcome Research Laboratories, Langley Court, Bcckenham, Kent, BR3 3BS
Recent studies from these laboratories have shown that a proportion of splenic lymphocytes fr’om CBA/lac mice immuniscd with sheep red blood cells (SRBC) were capable of producing haemolytic antibody ( IgM) plaques when reconstituted after freezing or freeze-drying under controlled conditions in the presence of polyvinylpyrrolidone (PVP) and foetal calf serum (FCS) (5). H owevcr, the ability of splenic cells to form E rosettes with SRBC or to bind to lectin-derivatised Sepharose beads was impaired, particularly by freezedrying (16) and the treated cells were unable to proliferate in response to mitogenic stimuli ( 6). One interpretation of these observations was that a degree of organisation had survived in the cytosol of the spleen cells capable of producing the haemolytic antibodies, whilst certain membrane binding sites had become coated with PVP during the process of freeze-dry& 0 or had been differcntially denatured and were therefore functionally impaired. In order to identify the nature of the damage at the membrane level more preciscly we have examined the interaction of frozen and freeze-dried cells with the lectins phytohaemagglutinin (PHA) and Concanavalin A (Con A) using radioisotope and immunofluorescent techniques. Received Janunry 13, 1976.
Copyright All rights
@ 1977 by Academic Press, Inc. 01 reproduction in any iorm reserved.
XI.4TERIALS
AND METHODS
Concanavalin A (Con A) and Sepharose 4B beads were obtained from Pharmacia, Uppsala, Sweden. Purified phytohaemagglutinin (PHA-P) and sheep ,antirabbit immunoglobulin labelled with fluorescein isothiocyanate were obtained from Wellcome Reagents Ltd., Beckenham, Kent, U.K. The rabbit anti-Con A and rabbit antiPHA sera used in this study were prepared by Dr. D. C. Edwards of these laboratories. Female Californian rabbits weighing approximately 7 kg were injected in the footpads with 1 mg of the lectin emulsified in Freund’s Complete Adjuvant ( FCA ). Approximately 50 days later the rabbits were injected intramuscularly with a similar emulsion and bled out 4 weeks later. The blood was allowed to coagulate and the serum stored at -20°C until required. Preparation
of Spleen Cells (SC)
CBA/lac mice approximately 14 weeks old were sacrified and their spleens removed. Single cell suspensions were prepared in Hanks balanced salt solution (HBSS) and washed in HBSS. The erythrocytes were lysed using Geys haemolytic buffer; lymphocytes were isolated on Ficoll/Triosil (8) and washed three times in HBSS by centrifugation at 200g for 10 min.
ISSN
0011-2240
4G
THOMAS
AND
ZOLA
Cell Corctrts md Ccl1 Vitil~ilily Total cell counts were carried out using conventional haematological techniques (2). Cell viability was measured by the trypan blue dye exclusion technique (3). Binding of [12nI] PHA and [la”Z] Con A to Treated and Untreated Cells
FIG. 1. Absci.sxz: counts of [YJCon A added (cpm X 10d); ordinate: counts of [‘7]Con A bound by 1.25 X 10’ cells (cpm X lo+). The binding of [7]Con A to freshly prepared M---B (SC), frozen/thawed O-O (FTC), and freeze-dried/rehydrated spleen cells 0-O (FDRC).
The iodination of Con A and PHA was carried out at room temperature and pH 7.4 by the chloramine-T procedure described by Hunter (9). Binding experiments were carried out by mixing 10 pl aliquots of dilutions of the labelled lectin with 50 ~1 aliquots of the cells (treated and untreated) containing 1.25 x lo6 cells. The cell suspensions were incubated at 37°C for 30 min, after which time the cells were washed twice in 2 ml of buffer and transferred to clean tubes for radioactivity counts. The reaction tubes were pretreated to reduce the absorption of the lectin by soaking in a solution of 5% KI and 0.5% bovine seruni albumin in phosphate buf-
Preparation of Frozen/Thawed (FTC) and Freeze-DriedjRehydrated Cells (FDRC) SC prepared as described above were suspended in 1470 PVP in HBSS supplemented with FCS and adjusted to 1 x 10’ cells/ml. The cells were then frozen and thawed or freeze-dried and rehydrated by the techniques previously described in detail elsewhere ( 16). The rate of cooling ing was approximately 4”C/min down to -40°C on the precooled shelves of an EF-6 freeze-drier (Edwards High Vacuum Ltd., U.K. ). Approximately half of the total number of cells were held at this temperature for 16-17 hr and then thawed. The remaining cells, having been cooled down to -40°C were then dried by sublimation of ice in vucuo for 16-17 hr. Following freeze-drying the cells were rehydrated slowly ( 16). The percentage recovery of freeze-dried spleen cells was approximately 70%, of which > 95% were viable as judged by the trypan blue dye exclusion test (3).
a 7. 6 5 4 3 2
0
4
a
12
16
20
24
FIG. 2. Abscissa: counts of [%I]PHA added (cpm X lo-‘); ordinate: counts of [‘?]PHA bound by 1.25 X 10’ cells (cpm X 10m4). The binding of [9]PHA to freshly prepared n -W (SC), frozen/thawed O-O (FTC), and freeze-dried/ (FDRC). rehydrated spleen cells 0 -0
XfOUSE
I,ectiu
Bindirlg
SC
+ +
con s
it
to the Sllrfltce (FI)RC)
h 1:
+
Ig
II
:I c:on
SPLEES
+
con
A,L
(FITC)”
A
+
II
47
of Xormal (SC), Frozen (FTC), and Freeze-Dried 11e:tsrued by Imm~moflrlores~ellce
SC + PHA + 1:. a I’HAc + S it II Ig (FIX)
FTC
LYMl’HOCYTES
a con
A
+ s :I, 11 Ig (FITC)
FTC + I’IIA + 1: n 1’H.k + s a I‘l Tg (FITC)
FI)I:c + con .4 + II a rm + S a It Ig (FIX)
x
I;l)l:c + PIIA + I’L a I’HA + S it IL Ig (FITC) l1 It tl Co11 A---rabbit xrlti-Con~snRvalin A serum. b S it 1:. Ig (FITC)---sheep antirabbit inlnlrulogloblllitl r I< :I PHA-rabbit :~ntiphytohtternagglrltinill ,ser,un.
fered saline (PBS) for 2 hr at RT. The tubes were then rinsed three times with distilled water and allowed to drain dry. PH,4 expcrimcnts were carried out using PBS, whilst the Con ,4 experiments were performed in HBSS containing R4nC12 ( 10e3sf) to stabilisc the Con A and 0.25% BSA to reduce nonspecific binding.
Binding of PHA and Con A to Treated and Untreated Cells Using an lmmunofluorescent Technique Binding of Con A and PHA was measured by an immunofluorescence technique described by Thomas et al. (IS). Briefly, 10 ~1 smears of SC, FTC, and FDRC at a concentration of 1 X lo6 cells/ml were allowed to dry in air on glass slides and fixed in alcohol. The fixed cells were reacted with
24.3
5
2.:::
23.5
17 I.5 ‘r
233 218 2ii-4
17 32 12 I
218 2.X) 2:3X
32 -
2.3) 233 223
-
235 213
15 37
220 2.50
30 -
Cells
03
!I2
94
12 I
17 23 I
94
90 > 04 >
-__
ld~ellecl with fl~loresc~ein isothiocyanate.
similar volumes of Con A (10 pg/ml) or PHA (5 pg/ml) for 20 min at RT, after which the unbound lectin was removed by washing three times in PBS. The cells were then reacted with the appropriate rabbit anti-Con A or anti-PHA, diluted 1: 100 with PBS, for a similar period of time and the unbound antibody removed by repeated washing and then reacted with anti-rabbit immunoglobulin labelled with fluorescein isothiocyanate. Finally the cells were washed free of excess %uorescein labelled antibody and the slides viewed for the uptake of the fluorescent dye. The numbers of cells fluorescing using FTC and FDRC preparations were compared with the cells %uorcscing using SC preparations. Controls without lectin, without antiserum to the lectins, or without fluorescein labelled antibody were always included.
4s
THOMAS TABLE
2
A Comparison of White Cell Agglutination of Normal (SC), Frozen (FTC), and Freeze-1)ried Cells (FDRC) Using Con A or PHA Agglutination titre (reciprord of dilution)
Quantit) of lectill required for agglutination (&ml)
SC
200 200
FTC FTC
Con A PHA
200 200
.i
FDRC FDRC -_.-
Con A PHA
20 20
.io 30
AggluGnution of Treated and Cells Using Con A and PHA
numbers of cells taking up the fluorescent dye, note being taken as to whether or not this fluorescence was ringlike, in patches or spots or capped. The experiments were carried out at +4”C and at 37°C with or without sodium azide, a metabolic inhibitor.
5 5
5
Untreated
Measurements of white cell agglutination were carried out using a microscale modification of standard procedures [see for example Thomas et al. ( 17)] at lectin concentrations from 1 to 10e7 “g/ml. Capping of PHA by Antibody Treated and Untreated Cells
ZOLA
RESULTS
Con A PHA
SC
AND
Using
Aliquots ‘of 1.0 ml of SC, FTC and FDRC suspensions at a concentration of 5 X lo6 cells/ml were placed in plastic tubes and centrifuged at 200g for 10 min. The supernatant was removed by decantation and 1.0 ml of the PHA solution at a concentration of 5 pg/ml was added. The suspension was agitated gently and incubated for 20 min at 37”C, after which the cells were harvested and washed by centrifugation. The resulting cell pellet was resuspended in 1.0 ml of a 1:lOO dilution of the rabbit anti-PHA serum and incubated for a further 20 min at 37°C. The supernatant was removed and the cells reacted with antirabbit immunoglobulin labelled with fluorescein isothiocyanate. The suspension was incubated for 20 min at 37”C, washed exhaustively, and examined by fluorescence. The total numbers of cells per field were counted in white light, together with the
The amount of iodinated Con A ( [1”“1] Con A) and PHA ([““IIPHA) bound by spleen cells was not significantly affected by the freeze-thaw or freeze-drying/rehydration procedures (Figs. 1 and 2) although the uptake of [Y]Con A by the frozen cell preparations was slightly increased. This increase was observed repeatedly with Con A but not with PHA. The total number of cells binding Con A and PHA was measured by a double sandwich immunofluorescence technique. The results (Table 1) clearly showed that the number of cells binding the lectins was unaltered by either the freezing or the lyophilisation procedures. Approximately 94% of all cells showed uptake of fluorescent dye. The agglutination of spleen cells by PHA and Con A was not affected by the freezethaw procedure but was reduced by freezedrying (Table 2). Normal and frozen cells were agglutinated by lectin concentrations down to 5 pg/ml whilst 50 /*g/ml of the lectin was required to agglutinate the lyophiliscd cells. The ability of the treated cells to cap PHA was examined using fluorescent antibody. The results, set out in Table 3, showed that normal spleen cell cap formation was optimal, 76% cap formation, when carried out at 37°C and minimal, 13% cap formation, when performed at +4”C. The addition of sodium azide at a final concentration of 10-’ M in the culture medium inhibited cap formation at both 37°C and +4”C. Similar results were obtained when using frozen-thawed cells under similar experimental conditions. Approximately 64% of the fluorescing cells formed caps at optimal
conditions whilst no caps were seen at +4”C. The addition of sodium azide, as expected, completely inhibited cap formation. In contrast to these results, cap formation of PHA by freeze-dried/rehydrated cells was dramatically reduced at optimum culture conditions, producing only 6%) capped cells out of the total number of fluorescing cells. No cells were seen to cap at t4”C and the sodium azide completely inhibited what little cap formation there 1ras at 37°C. DISCUSSION
The preservation of living cells for long periods of time is normally carried out by freezing the cells in the prescncc of a suitable cryoprotectant and then maintaining the cells at the temperature of liquid nitrogen. Earlier stud& had shown that reconstituted mouse spleen cells freeze-dried in the presence of PVP and FCS, were able to bind antilymphocyte serum and to secrete antibody, an energy requiring proccss. However, some membrane alterations wcrc apparent, as indicated by impaired ability of these cells to form E rosettes and to bind to immobilised PHA and Con A ( 16). In addition, such cells were unable to prolifcratc in response to initogcnic stimulus (6). These results were originally intcrprcted on the basis of masking of membranc sites or clifferential denaturation of the sites which react with the lectins Con A and PHA. Ho~cvcr, the present results show quite clearly that the quantity of Con A and PHA bound was not significantly affected by freezing or freeze-drying in the prcscnce of PVP supplemented with FCS. The decreased bincling of freeze-dried ~11s to Icctin/Sepharosc beads may bc esplaincd if the lateral diffusion of macroinoleculcs is impairecl, since the formation of :I stable association between a cell and a derivatiscd Scpharose particle probably rcquircs multiple bonding (1, 7). Evidence for tbr lack of lateral diffusion of membranc proteins was obtained in the Auorcs-
” I’h~toh:telnag-glutillill (pluified --Wellcome Xeagents Ltd., Beckenham). b NnN2 -~sotlirun asride (RI)11 T,ttl., U.K.).
cent studies where it was shown that capping of the bound lectins by antibody was greatly reduced after the cells had been freeze-dried. The impairment of agglutination by lectins after freeze-drying reported in this study could also be due to decreased membrane fluidity, since clustering of reccptors has been observed in Con A agglutination of cells ( 12). However, it would seem that clustering of the receptors is not au absolute requirement for agglutination, ancl becomes unnecessary at high lectin concentrations. Cap formation has been shown to be inhibitecl by the antimctabolite, sodium azide ( 15) and by certain antimitotic drugs such as cytochalasin B and vinblastine (14). Vinblastine is a known inhibitor of microtubule function and cytochalasin B has been reported to be similarly involved with microfilament function (I9), although this is by no means proved. The failure of the cells studied here to cap co&1 possibly be cxplained in terms of irrcvcrsiblc damage occurring during the freczc-drying stage to
50
THOMAS
the metabolic function of the cells. Equally, as microtubule and microfilament functions appear to play an active and synergic role in the formation of caps by the cell (13), damage to these structures is also a strong possibility. Furthermore, these findings could also help to explain earlier results (16) where it was shown that rosette formation was impaired following freeze-drying as normally functioning microfilaments appear to be necessary for the formation of rosettes (1% 4). The exact mechanisms involved in the early stages of mitogenesis by plant lectins are not known, although capping and endocytosis are thought not to be involved, whilst the lateral diffusion of the membrane macromolecules is probably required in order to form the necessary ion channels. Thus the absence of a mitogenic response after freeze-drying and reconstitution could be partly due to the impaired membrane fluidity, Equally it could be accounted for by damage occurring to the mitotic apparatus of the cell or by damage to, or blocking of, the functional lectin receptor. It is known that lectins will bind to any macromolecule containing the appropriate conformation of sugar but only a small proportion of these sites are receptors in the functional sense. This distinction is illustrated by the observations that PHA binds in equal proportions to both T-cells and B-cells ( ll), but it is only the T-cell that responds to the lectin mitotically. In the present study, although the results indicate that the total quantity of bound lectin is not noticeably decreased, the possibility remains that the functional receptors are either masked, possibly by the cryoprotectant PVP, or differentially denatured, thus becoming functionally impaired. In conclusion, it should be emphasised that the results presented in this and earlier studies (6, 16) suggest that freeze-dried/ rehydrated cells have little functional integrity as measured by the response to
AXD
ZOLA
mitogens and by the capping of bound lectins by antibody despite the fact that such cells retain their ability to produce IgM antibodies (5). These studies indicate that attempts to improve the functional integrity of the recovered cells following freezedrying should use a test to assess the membrane fluidity, e.g., capping of lectins by antibody, as a major criterion of success. SUMMARY
Rehydrated lymphocytes freeze-dried in of polyvinyl-pyrrolidone the presence (PVP) and foetal calf serum (FCS) have been shown to retain plaque forming activity (5) but were unable to respond to plant mitogens (6) or to bind to immobilised phytohaemagglutinin (PHA) or Concanavalin A (Con A) ( 16). Studies with fluorescein conjugated and radio iodinated antibody to PHA and Con A showed that the ability of the cells to bind the lectins was undiminished. However, agglutination by lectin and capping of lectinanti-lectin were impaired by freeze-drying, suggesting a defect in membrane fluidity, perhaps resulting from damage to the microfilament and microtubule apparatus of the cell. ACKNOWLEDGMENTS The authors wish to express their gratitude to hlrs. S. Barnes, Mrs. S. Graves, Mrs. A. Niblock, and Mr. N. Kent for invaluable technical assistance. REFERENCES 1. Anderson, J., Edelman, G. hl., Miiller, G., and Sjaberg, 0. Activation of B lymphocytes by locally concentrated Concanavalin A. Eur. J. Immunol. 2, 233 ( 1972). 2. Baker, F. J., Silverton, R. E., and Luckcock, E. D. In “Introduction to Medical Laboratory Technology” (F. J. Baker, R. E. Silverton, and E. D. Luckcock, Eds.), 4th ed., pp. 506-521. Butterworths, London, 1966. 3. Boyse, E. A., Old, L. J., and Chourolinkov, I. Cytotoxic test for demonstration of mouse antibody. In “Methods of Medical Research” (H. N. Eisen, Ed.), Vol. 10. Yearbook Medical Publishers, Chicago, 1964.
4. Cohnen, G., Fischer, K. and Brittinger, G. Human T-lymphocyte rosette formation: inhibition by cytochalasin B. Irnmru~!ogr~ 29, 337 ( 1975). 5. Damjanovic, I’., Edwards, D. C., and Thomas, D. Recovery of hacmolytic placlue-fonnillg ~11s after freeze-drying. Xcltrrrc’ (London) 253, llG (1975). 6. Damjanovic, V., and Thomas, 1). The 11%~of polyvinylpyrrolidone as a cryoprotectnnt in the freezing of human lymphocytes and in preliminary stllclies on freeze-drying. I’roc. C:ech. had. Sci., in press (1976). 7. Greavrs, 1~1. F., and Baumingrr, S. .I\ctivatio:1 of T and B lymphocytes by insoluble phytomitogens. Nuture (London) 235, G7 ( 1972). 8. Harris, R., and likaejiofa, E. 0. Rapid preparation of lymphocytes for ti\suc t),pin:g. Lancct 2, 237 ( 19G9). 9. Hunter, W. bl. In “Handbook of 1Ssperimcntal Immunology” (1~. hl. Weir, Ed.), 2nd cd.. Chap. 17. Black\\~ells, Oxford, 1973. 10. Kersey, J. H., IIom, 1). J., and Buttrick, 1’. IIuman T lymphocyte receptors for sheep crythrocytes: Conditions for binding including inhibition by cytochnlasin B. J. Inrn~~lr~oZ. 112, 862 (1974). 11. Loor, F. Binding and redistribution of lectins on lymphocyte membranes. Eur. J. Imm~uwl. 4, 210 (1974). 12. Nicolson, G. L. Topography of mcmbranc Concnnavalin A sites modified by proteolysis. Nature (I,ontlon) 239, 193 (1972).
13. Pctris de, S. Concannvalin A receptors, immunoglobulins and B antigens of the lymphocyte surface: interactions with Concnvalin A and with cytoplasmic structures. J. Cdl Biol. 65, 123 (1975). 14. Pctris de, S. Inhibition and reversal of capping I)\- cl-tochalasin B, viilblahtine and colchicinc. Sntrrrc (Lontlon) 250, 54 (1974). 15. Ta)-lor, R. B., Dullus, \V. I?. IL, Raff, 31. C., and de Petris, S. Rcdistriblltion and pinoc>tosis of lynlphocyte surface immnnoglobuinduced 1)) anti-immunolin 11&c&.? ~lol~~ili~i antibody. NNtrrrc ( I,ontlon) 233, 225 (1971). 16. Thomas, D., Ed\~ards, D. C., and Damjano\ic, 1’. The surface properties of spleen cells from CB.%/lnc mice following freezing nnd freeze-dq ing using polyvinyIpyrrolidoi~e. Cr&iolv~!/ 13, 191 ( 1976). 17. Thomas, I)., ~Ioccdalc, B., Rahr, I~., Epps, II. B. G., and Ecl\vards, I). C. Sates on the large scale preparation and propertics of anti-lymphocyte serum for itse in mice. C/in. Eql. Immwd. 7, 769 ( 1970). 18. Thomas, D., hlosedale, B., and Zola, I-I. The use of the indirect fluorescent antibody tcchnique in assessing the activity of antilymphocytic sern and antilymphocytic globulins, Clin. EXJI. Immrmol. 8, 987 (1971). 19. \Vcssels, N. K., Spooner, B. S., Ash, J. IT., Bradley, hl. O., Lnduenn, >I. A., Taylor, B. L., \Vrenn, J. T., and Yamada, K;. hI. SlicroBlanir:nts in cellular and developmental processes. Sciulrr 171, 135 ( 1971).