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Alteration of rat splenic lymphocyte migration in vitro by the state of microtubule integrity
CELLULAR
IJIiWJKOLOGY
Alteration
DAVID Departments
M.
39, 325-335
(1978)
of Rat Splenic Lymphocyte by the State of Microtubule CENTER,~
of Medicine, of the AfFliuted
I.
STFTIIEN
Haward Hospitals
AND
WASSERMAN,~
Medical Cestrr, Rccei7d
Migration Integrity1 K.
in Vitro
FRANK
AUSTEN
School and the Xobcrt B. Brigham Inc., Boston, d~as.nacl~~~sctts 02120 April
Division
6, 19i8
Rat splenic lymphocytes exhibit a positive chemokinetic response to colchicine and vinblastine. Both agents elicit a dose-dependent increase in chemokinesis with their peak effect at 2 to 4 X IO-’ ill being 3.5 times baseline random migration. The distance traveled by the leading front and the total movement of rat splenic lymphocytes is maximal in the absence of a gradient at all effective concentrations of colchicine or vinblastine. Checkerboard analysis established this response as entirely chemokinetic without any chemotactic component. That this chemokinetic response was due to a shift in the dynamic state of microtubules toward disassembly was supported by the inactivity of lumicolchicine and the capacity of heavy water to reverse the effect in a dose-response fashion. Cytochalasin B suppressed baseline random migration and reversed the chemokinetic response of the rat splenic lymphocytes to 4 X lo-’ M colchicine. The chemokinetic motility of rat splenic lymphocytes may depend not only on microtubule disassembly hat also on the contractile activity of microfilaments.
INTRODUCTION The delayed type hypersensitivity skin reaction is characterized by a lymphocytic infiltrate composed predominantly of cells which are not specifically sensitized for the antigen eliciting the reaction ( 1) . Recent studies with rodent lymphocytes have revealed a capacity of T cells to migrate in response to specific antigen (2) and to lymphocyte products (3), and of B cells to be attracted to anti-Ig 4 (3). In the initial studies, mouse splenic B cells incubated with anti-Ig exhibited increased numbers of motile forms as assessed by direct microscopic observation (4, 5). This increase in numbers of motile forms required a metabolically active cell and was prevented by cytochalasin B even though surface capping of anti-IgG-IgG complexes was not diminished (4). Subsequent studies demonstrated that the motile responses to anti-Ig could he assessed by the Royden chamber technique and was 1 Supported by Grants AI-07722, AI-10356 and RR-OS669 from the National Institutes of Health. 2 Dr. Center is a Fellow of the American Lung Association. 3 Dr. Wasserman is a recipient of an Allergic Diseases Academic Award (AI-00254) from the National Institutes of Health. 4 Abbreviations used in this paper: BS.4, bovine serum albumin; cyclic AMP, cyclic 3’,5’adenosine monophosphate; hpf, high power field; Ig, immunoglobulin; IgG, immunoglobulin G; M, molar; M199, medium 199; and M199-BS.4, Ml99 containing 0.4%, l,ovillc s~-um albumin. 325 000%8749/78/0392-0325$02.00/O All
Copyright 0 1978 rights of reproduction
by
Academic Press, Inc. in any form reserved.
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WASSERMAN,
AND
AUSTEN
chemokinetic and not chemotactic because a gradient of anti-Ig was not necessary (6). However, the response of rat splenic cells to low concentrations of anti-Ig in Boyden chambers was shown to be chemotactic while that to high concentrations was again chemokinetic (3). Further, human B lymphoblasts from cell lines maintained in continuous culture showed a chemotactic response to endotoxin-activated human plasma (7). Human peripheral blood T lymphocytes activated with phytohemaglutinin also responded chemotactically to endotoxin-activated human plasma (8). Mouse T lymphocytes taken from lymph nodes draining sites of challenge with specific antigenic proteins responded chemotactically to a gradient of the same antigen in a modified Boyden chamber assay (2). I n contrast, mouse lymphocytes taken from draining lymph nodes after contact sensitization with oxalazone responded chemokinetically to endotoxin-activated plasma (7). Finally, rat splenic T lymphocytes, but not B lymphocytes, manifested a chemotactic response to T cell lymphokines derived from mixed lymphocyte cultures (3). Thus, chemokinetic and chemotactic motile responses have been documented by in vitro studies with both B and T cells from several species. Colchicine, a microtubule depolymerizing agent, failed to inhibit the induction of motile forms in mouse splenic B cells by anti-Ig (5). However, pretreatment with colchicine did prevent the inhibition of the motile reaction observed with agents that elevated intracellular levels of cyclic 3’,5’-adenosine monophosphate (cyclic AMP) (5), suggesting that cyclic AMP-mediated microtubule assembly (9, 10) prevented lymphocyte motility. Colchicine treatment of cultured human B lymphoblasts prevented their chemotactic response to endotoxin-activated human plasma ; this effect was attributed to interference with the orientation required for directed locomotion, but not with the locomotor mechanism since chemokinesis was enhanced (7). It was subsequently shown that colchicine, vinblastine, and vincristine enhanced the motility .of normal and leukemic human lymphocytes, whereas cytochalasin B inhibited their migration ( 11). Colchicine concentrations in a dose range of 1O-4 M to 10m6M were also shown to increase the number of motile forms of mouse splenic T and B cells in CBA but not ,4 strain mice (12). The present studies demonstrate that colchicine alone induces a chemokinetic response in rat splenic lymphocytes. The optimal concentrations for eliciting the chemokinetic response are in the range of 2 x lo-7 M to 1 x 10m6ill, while concentrations in the range of 10m4 M to 1O-3 M do not affect migration. The action of colchicine is attributable to disassembly of microtubules in that it is not observed with the inactive form, lumicolchicine and is reversed in a dose-response fashion by the introduction of the microtubule stabilizing agent, heavy water. MATERIALS
AND
METHODS
Colchicine, vinblastine (Eli Lilly & Co., Indianapolis, Indiana), cytochalasin B, medium 199 (M199) heavy water (Sigma Chemical Co., St. Louis, Missouri), (Microbiological Associates, Bethesda, Maryland), bovine serum albumin (Miles Laboratories, Inc., Elkhart, Indiana), Sartorius nitrocellulose micropore filters (Beckman Instruments, Mountainside, New Jersey), polystyrene disposable chemotactic chambers (Adaps Corporation, Bedford, Massachusetts), Ficoll-paque@ (Pharmacia Fine Chemicals, Piscataway, New Jersey), and Sprague-Dawley rats (Charles River Breeders, Boston, Massachusetts) were obtained from the manufacturers and suppliers.
DEPEKDENCE
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Lymphocyte migration. The migration of rat splenic lymphocytes was assessed by a further modification of a Boyden chamber technique (3, 13). Sprague-Dawley rats weighing 250 to 300 g were sacrificed by COz intoxication and their spleens were removed. One to two spleens were placed in 100 ml of Ml99 and teased apart with surgical forceps. The resulting cell suspension was freed of cellular clumps and tissue debris by sieving through bolting cloth. The cell suspension was distributed in 10 ml volumes onto ten 10 ml Hypaque-Ficoll cushions (14) and centrifuged for 45 min at 4009 at 25°C. The bands of mononuclear cells at the interface were aspirated, washed twice in Ml99 and incubated in 20 ml of Ml99 containing 0.4% bovine serum albumin (M199-USA) and with carbonyl iron (15) in a shaking water bath for 30 min at 37°C. The cells were then layered on two additional 10 ml Hypaque-Ficoll cushions and were centrifuged for 45 min at 4009 at 25°C. The iron laden monocytes, contaminating polymorphonuclear leukocytes, and free iron particles were pelleted through the cushion. The cells at the Hypaque-Ficoll-Ml99 interface were routinely 95% lymphocytes (SD * 4% in 27 experiments as assessed by differential counts of 200 cells on smears stained with Giemsa or Wright’s stains). Four X lo6 lymphocytes in 1 ml of M199-BSA were placed in polystyrene disposable chemotactic chambers which utilized 8 pm nitrocellulose micropore filters to separate the cells from 1 ml of M199-BSA in a non-disposable lower compartment. For assessment of the chemokinetic response of lymphocytes, the stimulus was introduced into both compartments, whereas for chemotaxis it was placed in the lower compartment only. Migration experiments were carried out for 3 hr at 37°C in a 5% CO2 moist atmosphere. The filters were fixed, stained, dehydrated, and mounted as described for eosinophil and polymorphonuclear leukocytes (16). Cell movement was quantitated by counting the total number of cells migrating beyond a distance of 50 pm, using 10 pm intervals, in five high power fields (hpf) in duplicate micropore filters. In any single experiment the duplicates differed by less than 20% of their mean and the results are expressed as the cumulative counts from 50 to 130 pm in 10 hpf. The total number of cells that had migrated in buffer alone represent the background (10076) and the effect of each variable is expressed relative to the background response. The effect on lymphocyte migration of each variable was examined in three to five separate experiments and the results are presented as a mean -C 1 SD. In order to categorize agents as chemotactic, chemokinetic, or both, a checkerboard assay was used (17, 1s). In this assay incremental doses of a reagent were placed on the cell side (upper chamber), chemotactic factor side (lower chamber), or both sides of the filters to establish varying concentration gradients in both directions. Migration was then analyzed as outlined above and also by the ZigmondHirsch leading front method (19) where the distance from the top of the filter to the point where two cells have migrated is determined in 5 hpf in duplicate filters. The theoretical distance that cells would have migrated if the effect were entirely chemokinetic is calculated from the experimental data (19) and compared to the actual distance traveled. In this way agents can be assessed for their chemokinetic effect (increased migration independent of a concentration gradient). chemotactic action (increased migration only in the presence of a positive gradient), and a combination of these qualities. Lumicolchicine was prepared by irradiating lo-? M colchicine in 95% ethanol in
328
CEiYTER,
WASSERMAN,
AKD
AUSTEN
a quartz crucible with ultraviolet light for 48 hr at 25°C. Conversion to lumicolchitine derivatives was assessed by dilution of this mixture to 2 x 10m5 M in 95% ethanol and measurement of absorbance at 200, 250, and 300 nanometers on a Beckman spectrophotometer. Conversion to lumicolchicine was considered nearly complete since absorbance of irradiated colchicine at 300 nm was 9% of a similar concentration of colchicine in 95% ethanol (20)) whereas absorbance at 200 and 250 nm was unchanged.
Che?+&inetic efject of colchicine. The chemokinetic effect of colchicine on rat splenic lymphocytes was assessed by placing equal concentrations of colchicine in a range of IO-* to 10m3 M in both the cell and stimulus compartments of modified Boyden chambers. Eight x lo6 cells/ml in M199-BSA were mixed with an equal volume of buffer containing 2 x lo-* to 2 x 10m3M colchicine, and 1 ml of each cell-colchicine mixture (4 x lo6 cells) was immediately introduced into the cell compartment of the chemotactic chamber at 25°C. Concentrations of colchicine identical to those on the cell side had been previously placed on the stimulus side in the same buffer. A dose-dependent chemokinetic response occurred with colchicine concentrations between 1 x lo-? and 4 X 10e7 M, with the latter giving the maximal stimulation (Fig. 1) . Higher concentrations were progressively less effective and there was no effect in the range of 10m4to 1O-3 izI. Preincubation of cells with the maximal stimulatory concentration of colchicine for 1 hr before placement into the chemotactic chambers containing that concentration of colchicine on the
FIG. 1. The cluzmokinctic response of rat splcuic lymphocytes to colchicine. The 100% line depicts the migration in buffer alone which ranged from 75 to 238 cells/l0 hpf, had a mean of 157 cells/l0 hpf and a standard deviation of 84 cells/l0 hpf. The effect of placing identical concentration of colchicine in the cell and stimulus compartments is presented as the mean percent of control response f 1 SD in four experiments.
DEPEiXDENCE
OF
LYMPIIOCYTE
MOTILITY
Oi’i
CELL COMPARTMENT COLCHICINE CM]
CELL COMPARTMENT COLCHICINE CM] 0
I xlo-7
2x10-’
4x10-7
329
MICROTLXULES
0
Eixlo-7
MO-‘,
l-l 67
8xlO-‘,
167
I41
2x10-’
8x10-’
4x10.’
98
~ 205
162
78
140
I 132
-4
1 125 1 144 p
I
FIG. 2. Checkerboard assay of the migrating response of rat splenic lymphocytes to colchicine. (A) Numbers indicate the mean distance to which the leading two cells migrated in 5 hpf into duplicate micropore filters. Numbers in parentheses are theoretical distances lymphocytes would have traveled if the effect of colchicine was solely chemokinetic. (B) Numbers indicate the total numbers of cells that migrated more than 50 pm into duplicate micropore filters in 10 hpf.
stimulus and cell sides did not decrease their movement as compared to cells that had not been preincubated. In order to determine whether colchicine could also have a chemotactic effect, a checkerboard assay was performed (Fig. 2A). The distances depicted in the vertical left hand column indicate the responses to gradients established from the stimulus side, the distances in the horizontal column across the top reflect the responses to gradients initiated on the cell side? and the distances in the diagonal from the upper left to the lower right represent the responses to increasing concentrations of colchicine in the absence of a gradient. Lymphocyte movement at each colchicine concentration was greatest when the agent was in equimolar concentrations on both sides of the filter, reflecting a chemokinetic cell response which was maximal at 4 X 10e7 M. The distances depicted in parentheses are theoretical and are based on the observed data depicted in the diagonal with the assumption that the effect of colchicine is entirely chemokinetic (19). In no instance where a positive gradient towards the stimulus compartment existed did the observed distance of the leading front exceed the theoretical, and thus there is no evidence that colchicine has a chemotactic action on rat splenic lymphocytes. The experiment was also analyzed by enumerating the total cells in 10 hpf which had moved more than 50 pm into duplicate filters (Fig. ZB). The maximum response for each concentration of colchicine was again observed when colchicine was placed in equimolar concentrations on both sides of the filter, so as to eliminate a gradient. Role of vvzzicrotubades in the chevzokinetic ejjfecCof colchicine. A second anti-mitotic agent, vinblastine, known to disassemblemicrotubules through a binding site different from that of colchicine, was examined for its dose-responseeffects on the migration of rat splenic lymphocytes. Preliminary experiments indicated that vinblastine, like colchicine, was chemokinetic and thus it was placed on both sides of the filter in the five experiments depicted in Fig. 3. A chemokinetic dose response was obtained in a concentration range of 1 X 10esto 2 X lo-? M. There was a broad plateau
330
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AND
AUSTEN
FIG. 3. The chemokinetic response of rat splenic lymphocytes to vinblastine. The 100% line depicts the migration in buffer alone which ranged from 84 to 180 cells/IO hpf, had a mean of 121.5 cells/l0 hpf and a standard deviation of 37 cells/l0 hpf. The effect of placing identical concentrations of vinblastine in the cell and stimulus compartments is presented as the mean percent of control response * 1 SD in five experiments.
j 0
I
I
I
10
20
30
HEAVY
WATER
I
I
40
50 f%l
FIG. 4. The dose response effect of heavy water 01, the random migration of rat splenic lymphocytes (e-0 ) and on their chemokinetic response to 4 X lo-’ M colchicine (0-O). The 100% line depicts the migration in buffer alone which ranged from 91 to 153 cells/l0 hpf, with a mean of 121 cells/l0 hpf and a standard deviation of 30 cells/l0 hpf. Results are expressed as the mean percent of control response * 1 SD in three experiments.
T)EPENDI’h’CE
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FIG. 5. Chemokinetic response of rat splenic lymphocytes to lumicolchicine (O-O), cdchicine ( O-O), and EtOH solvent (A-A). The 100% line depicts the migration in buffer alone and results are expressed as percent of baseline migration. in the chemokinetic response at concentrations from 2 X 10eT to 1 X 10-s M, with a Ioss of effect as the concentration was increased to 2 X 10m5 M. Inhibition was observed at 4 x lo-” M and was complete at lo-* M. Since the chemokinetic effect of vinblastine and colchicine suggested a role for microtubular disassembly in this response, heavy water, an agent that enhances
1
CYTOCHAL ASIN B (M] FIG. 6. The dose-response effect of cytochalasin B on the random migration of rat splenic lymphocytes. The 100% line depicts the migration in buffer alone which ranged from 84 cells/l0 hpf to 202 cells/l0 hpf, had a mean of 120 cells/l0 hpf and a standard deviation of 47 cells/l0 hpf. The effect of placing identical concentrations of cytochalasin B in the cell and stimulus compartments is presented as the mean percent of control response f 1 SD in five experiments.
332
CYTOCHAL
ASIN t? /M/
FIG. 7. The effect of cytochalasin B on the chemokinetic response of rat splenic lymphocytes to 4 X lo-’ M colchicine. The 100% line depicts the migration in buffer alone in four experiments which ranged from 84 cells/l0 hpf to 103 cells/l0 hpf, had a mean of 95.4 cells/l0 hpf and a standard deviation of 9.3 cells/l0 hpf. The effect of placing identical concentrations of cytochalasin B in the cell and stimulus compartments along with 4 X lo-’ M colchicine are presented as the mean percent of control response t 1 SD.
assembly of microtubules, was studied as a possible inhibitor of lymphocyte migration. D20 placed on both sides of the filter inhibited lymphocyte migration in final concentrations above 30% (Fig. 4), and this inhibition of 35% was not increased with concentrations of 40 and 50% DZO. In order to examine the effect of D20 on colchicine stimulation, rat splenic lymphocytes in a concentration of 8 x IO6 cells/ml were incubated with 8 x 1O-7 M colchicine for 30 min at 25°C. One-half ml of colchicine-stimulated cells were mixed with an equal volume of R/1199-BSA containing from zero to 100% DaO, and 1 ml of the cell suspension was then placed in a Boyden chamber. Concentrations of D,O and colchicine identical to those on the cell side had been placed on the stimulus side in the same buffer (Fig. 4). Colchicine alone in a final concentration of 4 x 10e7 121 gave a 340% increase in lymphocyte migration and this chemokinetic effect was reversed in a dose-response fashion by increasing concentrations of heavy water. A concentration of 50% D20 completely reversed the chemokinetic response to colchicine. Da0 similarly inhibited the chemokinetic effect of vinblastine (not shown). Lumicolchicine differs from colchicine in that it does not bind to monomeric tubulin and therefore cannot inhibit tubulin from assembling into microtubules. Colchicine and lumicolchicine at lo-* M in 95% EtOH were each diluted in M199-BSA to the concentrations of colchicine found to be chemokinetic. The effect of 95% EtOH diluted in parallel fashion was also evaluated. The colchicine gave a doseresponse stimulation from 2 to 4 X lo-? M, with the peak effect being 390% of baseline migration (Fig. 5). Lumicolchicine did not increase migration and the
inhil)ition observed at the highest concentration \vas cc~ml~arable to the effect of alcol1ul ;11011c. Efect of cytochrltrsin U on flzc chmokiwtic c#tjcr:c.t uf c.cllchicinc. The iiiotlulation of rat lymphocyte migration by cytochalasin B, an agent that inhibits microfilament function by preventing the interaction of actin-binding protein with subplasmalenma1 actin (21) , was assessedby placing cytochalasin B on both sides of the filter in a protocol similar to that used with heavy water. Cytochalasin B gave a dosedependent inhibition of lymphocyte migration in concentrations of 2 X lo-? to 2 x 10m9M (Fig. 6). Doses of 10-” and 10~4M resulted in complete inhibition of all lymphocyte migration and in these filters no cells were observed to have entered the filter to any depth beyond the stacking layer. In order to esamine the effect of cytochalasin B on colchicine stimulation, rat splenic lymphocytes were incubated in a concentration of 8 x 10G cells/ml with S x 10m7M colchicine in M199-BSA for 30 min at 25°C. One-half ml of colchicine-stimulated cells were mixed with an equal volume of M199-BSA containing from 2 X lOmato 2 X 10e4ill’ cytochalasin B and 1 ml of the cell suspensionwas then placed in a Boyden chamber. Concentrations of cytochalasin B and colchicine identical to those on the cell side had been placed on the stimulus side in the same buffer. Colchicine alone in a final concentration of 4 x 10e7M gave a 340% rise in lymphocyte migration and this chemokinetic effect was reversed by concentrations of cytochalasin B above IO-’ All (Fig. 7). Concentrations of cytochalasin U between 5 X 10m7and 4 X 10mF,%I reduced the chemokinetic responseto baseline, and concentrations of lo-” X, as in the studies on unstimulated migration, abolished all cell movement.
Rat splenic lymphocytes are stimulated to increase their chemokinetic migration by agents such as colchicine and vinblastine which disassembletheir microtubules. Both agents produce a dose-dependent increase in chemokinesis with the initial effects being observed at concentrations in the 10e8to 1O-7M range, and the peak effects occurring at 4 X 10m71Vl for colchicine (Fig. 1) and more broadly from 2 x 10m7to 2 x lo-” M for vinblastine (Fig. 3). The mean peak effect with 4 x lo-? M colchicine is 3.6 times baseline random migration of cells, and with 4 x 10m7M vinblastine the multiple is the same.As concentrations of colchicine and vinblastine are increased, there is a progressive decrease in chemokinesis such that migration returns to a baseline with 10m3Al colchicine and 2 X 10e5&I vinblastine. The nature of the augmented migration of rat splenic lymphocytes in the presence of colchicine was established as being chemokinetic and not chemotactic, as defined by the criteria developed for the study of polymorphonuclear leukocyte migration (18, 19). Since the maximum response for any concentration of colchicine by checkerboard analysis was observed when equimolar amounts of the agent were placed on both the cell and stimulus sides of the filters, a gradient was not necessary (Fig. 2A). Indeed, the checkerboard analysis showed that the movement observed in a series of gradients towards the stimulus side was no greater than the theoretical distance predicted by the chemokinetic effect alone and thus there was no evidence of a superimposed chemotactic effect (19). The interpretation is the samewhen the results are expressed as the total number of cells moving more than 50 pm into the filter. At any concentration of either colchicine (Fig. 2B) or vin-
334
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AND
ALl’STEN
blastine, the total cell movement was maximal in the absence of a gradient, indicating the chemokinetic stimulation by these microtubular depolymerizing agents. Two further lines of evidence indicate that the chemokinetic response of rat splenic lymphocytes is related to a shift in the dynamic state of microtubules toward disassembly. Lumicolchicine, which fails to ‘bind to monomeric tubulin (20)) had no effect on the movement of rat splenic lymphocytes over a dose range of lo-@ to 10m4M (Fig. 5). Heavy water, which is known to promote assembly of microtubules, partially suppressed random migration of rat splenic lymphocytes and in a dose-dependent fashion reversed the chemokinetic effect of 4 x 10e7 Al colchicine (Fig. 4). The shift of the microtubule state to favor disassembly would lessen any contribution to a rigid cytoskeleton of the resting lymphocyte and facilitate changes favoring membrane fluidity and motility. This relationship would offer an explanation for the observation that the resting non-motile lymphocyte is spherical, whereas the motile cell manifests a uropod extension and cytoplasmic rippling (4). An alternative interpretation, namely that the colchicine effect is not due to increased influx into but rather decreased efflux from the filters was not supported. Preincubation of cells for 1 hr with an optimal colchicine stimulus did not decrease their subsequent movement in chambers containing the same colchicine concentration as compared to cells not pretreated. Furthermore, the movement of both untreated and colchicine pretreated cells increased comparably at each hourly time point during the 3 hr period of migration. Studies with mouse splenic B lymphocytes (5) showed that cytochalasin B and agents which elevated intracellular levels of cyclic AMP decreased the development of motile forms in response to anti-Ig. In the present studies cytochalasin B inhibited the spontaneous migration of rat splenic lymphocytes in concentrations above 10m7M and completely inhibited all cell movement at a dose of 10-j M (Fig. 6). Cytochalasin B reversed the positive chemokinetic effect of rat splenic lymphocytes to 4 X 10m7M colchicine in concentrations of 1Omsto 10m6M and again completely inhibited cell movement at 10e5 NI (Fig. 7). The concentrations of cytochalasin B required to reverse colchicine-induced chemokinesis or inhibit random motility are generally less than those required to interfere with glucose (22) or nucleoside transport (23). Since ultrastructural studies were not carried out, the mechanism of the effects of the various agents on lymphocyte motility is speculative and based upon the observed effect of these agents on subcellular structures in other cell systems (20, 24, 25). The results are similar to those obtained with human and rabbit neutrophils where colchicine, vinblastine and vincristine increased random motility (26)) whereas cytochalasin B inhibited chemotaxis and spontaneous motility (27). REFERENCES 1. McCluskey, R. T., Benacerraf, B., and McCluskey, J. W., J. Immunol. 98, 466, 1963. 2. Wilkinson, P. C., Parrot, D. M. V., Russel, R. J., and Sless, F., J. Exp. Med. 145, 1158, 1977. 3. Ward, P. A., Unanue, E. R., Goralnick, S., and Schreiner, G. F., J. Zmmunol. 119, 416, 1977. 4. Unanue, E. R., Ault, K. A., and Karnovsky, M. J., J. Exp. Med. 139, 295, 1974. 5. Schreiner, G. F., and Unanue, E. R., J. Zmmunol. 114, 802, 1975. 6. Schreiner, G. F., and Unanue, E. R., J. Immulzol. 114, 809, 1975. 7. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V., Nature 256, 646, 1975. 8. Wilkinson, P. C., Roberts, J. A., Russell, R. J., and McLaughlin, M., Cl&. Exp. Immunol. 25, 280, 1976.
DEPENDENCE
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27.
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Johnson, G. S., Friedman, R. M., and Pastan, I., Proc. Satl. Acad. Sci. 68, 425, 1971. Kirkland, W. L., and Burton, P. R., Sature Stew Biol. 240, 205, 1972. Jarvis, S. C., Snyderman, R., and Cohen, H. J., Blood 48, 717, 1976. Fram, R. J., Sidman, C. L., and Unanue, E. R., J. Irrzmuuol. 117, 1456, 1976. Boyden, S., J. Exp. Med. 115, 453, 1962. Boyum, A., Scmd. J. Cl&. Lab. Invest. 21 (suppl 97), 31, 1968. Schlossman, S. F., and Hudson, L. J., J. I+n~zu,~ol. 110, 313, 1973. Kay, A. B., Clin. Exp. Imnzunol. 7, 723, 1970. Keller, H. U., Wilkinson, P. C., Abercrombie, M., Becker, E. L., Hirsch, J. G., Miller, M. E., Ramsey, W. S., and Zigmond, S. H., J. I?rtnzultol. 118, 1912, 1977. Wilkinson, P. C., and Allan, R. B., 11% “Leukocyte Chemotaxis: Methods, Physiology, and Clinical Implications” (J. A. Gallin and P. G. Quie, Eds.), pp. l-23. Raven Press, New York, 1978. Zigmond, S. A., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. Wilson, L., Bamburg, J. R., Mizel, S. B., Grisham, L. hf., and Creswell, K. M., Frd. Proc. 33, 158, 1974. Hartwig, J. H., and Stossel, T. P., J. Cell Biol. 71, 295, 1976. Estensen, R. D., and Plagemann, P. G. W., Proc. Natl. Acad. Sci. 69, 1430, 1972. Plagemann, P. G. W., and Estensen, R. D., J. Cell. Biol. 55, 179, 1972. Malawista, S. E., and Bensch, K. G., Science 156, 521, 1967. Padawer, J., J. Natl. Cancer Inst. 25, 731, 1960. Becker, E. L., and Showell, H. J., J. Imnzunol. 112, 2055, 1974. Becker, E. L., Davis, A. T., Estensen, R. D., and Quie, P. G., J. Iwwtunol. 108, 396, 1972.
IJIiWJKOLOGY
Alteration
DAVID Departments
M.
39, 325-335
(1978)
of Rat Splenic Lymphocyte by the State of Microtubule CENTER,~
of Medicine, of the AfFliuted
I.
STFTIIEN
Haward Hospitals
AND
WASSERMAN,~
Medical Cestrr, Rccei7d
Migration Integrity1 K.
in Vitro
FRANK
AUSTEN
School and the Xobcrt B. Brigham Inc., Boston, d~as.nacl~~~sctts 02120 April
Division
6, 19i8
Rat splenic lymphocytes exhibit a positive chemokinetic response to colchicine and vinblastine. Both agents elicit a dose-dependent increase in chemokinesis with their peak effect at 2 to 4 X IO-’ ill being 3.5 times baseline random migration. The distance traveled by the leading front and the total movement of rat splenic lymphocytes is maximal in the absence of a gradient at all effective concentrations of colchicine or vinblastine. Checkerboard analysis established this response as entirely chemokinetic without any chemotactic component. That this chemokinetic response was due to a shift in the dynamic state of microtubules toward disassembly was supported by the inactivity of lumicolchicine and the capacity of heavy water to reverse the effect in a dose-response fashion. Cytochalasin B suppressed baseline random migration and reversed the chemokinetic response of the rat splenic lymphocytes to 4 X lo-’ M colchicine. The chemokinetic motility of rat splenic lymphocytes may depend not only on microtubule disassembly hat also on the contractile activity of microfilaments.
INTRODUCTION The delayed type hypersensitivity skin reaction is characterized by a lymphocytic infiltrate composed predominantly of cells which are not specifically sensitized for the antigen eliciting the reaction ( 1) . Recent studies with rodent lymphocytes have revealed a capacity of T cells to migrate in response to specific antigen (2) and to lymphocyte products (3), and of B cells to be attracted to anti-Ig 4 (3). In the initial studies, mouse splenic B cells incubated with anti-Ig exhibited increased numbers of motile forms as assessed by direct microscopic observation (4, 5). This increase in numbers of motile forms required a metabolically active cell and was prevented by cytochalasin B even though surface capping of anti-IgG-IgG complexes was not diminished (4). Subsequent studies demonstrated that the motile responses to anti-Ig could he assessed by the Royden chamber technique and was 1 Supported by Grants AI-07722, AI-10356 and RR-OS669 from the National Institutes of Health. 2 Dr. Center is a Fellow of the American Lung Association. 3 Dr. Wasserman is a recipient of an Allergic Diseases Academic Award (AI-00254) from the National Institutes of Health. 4 Abbreviations used in this paper: BS.4, bovine serum albumin; cyclic AMP, cyclic 3’,5’adenosine monophosphate; hpf, high power field; Ig, immunoglobulin; IgG, immunoglobulin G; M, molar; M199, medium 199; and M199-BS.4, Ml99 containing 0.4%, l,ovillc s~-um albumin. 325 000%8749/78/0392-0325$02.00/O All
Copyright 0 1978 rights of reproduction
by
Academic Press, Inc. in any form reserved.
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chemokinetic and not chemotactic because a gradient of anti-Ig was not necessary (6). However, the response of rat splenic cells to low concentrations of anti-Ig in Boyden chambers was shown to be chemotactic while that to high concentrations was again chemokinetic (3). Further, human B lymphoblasts from cell lines maintained in continuous culture showed a chemotactic response to endotoxin-activated human plasma (7). Human peripheral blood T lymphocytes activated with phytohemaglutinin also responded chemotactically to endotoxin-activated human plasma (8). Mouse T lymphocytes taken from lymph nodes draining sites of challenge with specific antigenic proteins responded chemotactically to a gradient of the same antigen in a modified Boyden chamber assay (2). I n contrast, mouse lymphocytes taken from draining lymph nodes after contact sensitization with oxalazone responded chemokinetically to endotoxin-activated plasma (7). Finally, rat splenic T lymphocytes, but not B lymphocytes, manifested a chemotactic response to T cell lymphokines derived from mixed lymphocyte cultures (3). Thus, chemokinetic and chemotactic motile responses have been documented by in vitro studies with both B and T cells from several species. Colchicine, a microtubule depolymerizing agent, failed to inhibit the induction of motile forms in mouse splenic B cells by anti-Ig (5). However, pretreatment with colchicine did prevent the inhibition of the motile reaction observed with agents that elevated intracellular levels of cyclic 3’,5’-adenosine monophosphate (cyclic AMP) (5), suggesting that cyclic AMP-mediated microtubule assembly (9, 10) prevented lymphocyte motility. Colchicine treatment of cultured human B lymphoblasts prevented their chemotactic response to endotoxin-activated human plasma ; this effect was attributed to interference with the orientation required for directed locomotion, but not with the locomotor mechanism since chemokinesis was enhanced (7). It was subsequently shown that colchicine, vinblastine, and vincristine enhanced the motility .of normal and leukemic human lymphocytes, whereas cytochalasin B inhibited their migration ( 11). Colchicine concentrations in a dose range of 1O-4 M to 10m6M were also shown to increase the number of motile forms of mouse splenic T and B cells in CBA but not ,4 strain mice (12). The present studies demonstrate that colchicine alone induces a chemokinetic response in rat splenic lymphocytes. The optimal concentrations for eliciting the chemokinetic response are in the range of 2 x lo-7 M to 1 x 10m6ill, while concentrations in the range of 10m4 M to 1O-3 M do not affect migration. The action of colchicine is attributable to disassembly of microtubules in that it is not observed with the inactive form, lumicolchicine and is reversed in a dose-response fashion by the introduction of the microtubule stabilizing agent, heavy water. MATERIALS
AND
METHODS
Colchicine, vinblastine (Eli Lilly & Co., Indianapolis, Indiana), cytochalasin B, medium 199 (M199) heavy water (Sigma Chemical Co., St. Louis, Missouri), (Microbiological Associates, Bethesda, Maryland), bovine serum albumin (Miles Laboratories, Inc., Elkhart, Indiana), Sartorius nitrocellulose micropore filters (Beckman Instruments, Mountainside, New Jersey), polystyrene disposable chemotactic chambers (Adaps Corporation, Bedford, Massachusetts), Ficoll-paque@ (Pharmacia Fine Chemicals, Piscataway, New Jersey), and Sprague-Dawley rats (Charles River Breeders, Boston, Massachusetts) were obtained from the manufacturers and suppliers.
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Lymphocyte migration. The migration of rat splenic lymphocytes was assessed by a further modification of a Boyden chamber technique (3, 13). Sprague-Dawley rats weighing 250 to 300 g were sacrificed by COz intoxication and their spleens were removed. One to two spleens were placed in 100 ml of Ml99 and teased apart with surgical forceps. The resulting cell suspension was freed of cellular clumps and tissue debris by sieving through bolting cloth. The cell suspension was distributed in 10 ml volumes onto ten 10 ml Hypaque-Ficoll cushions (14) and centrifuged for 45 min at 4009 at 25°C. The bands of mononuclear cells at the interface were aspirated, washed twice in Ml99 and incubated in 20 ml of Ml99 containing 0.4% bovine serum albumin (M199-USA) and with carbonyl iron (15) in a shaking water bath for 30 min at 37°C. The cells were then layered on two additional 10 ml Hypaque-Ficoll cushions and were centrifuged for 45 min at 4009 at 25°C. The iron laden monocytes, contaminating polymorphonuclear leukocytes, and free iron particles were pelleted through the cushion. The cells at the Hypaque-Ficoll-Ml99 interface were routinely 95% lymphocytes (SD * 4% in 27 experiments as assessed by differential counts of 200 cells on smears stained with Giemsa or Wright’s stains). Four X lo6 lymphocytes in 1 ml of M199-BSA were placed in polystyrene disposable chemotactic chambers which utilized 8 pm nitrocellulose micropore filters to separate the cells from 1 ml of M199-BSA in a non-disposable lower compartment. For assessment of the chemokinetic response of lymphocytes, the stimulus was introduced into both compartments, whereas for chemotaxis it was placed in the lower compartment only. Migration experiments were carried out for 3 hr at 37°C in a 5% CO2 moist atmosphere. The filters were fixed, stained, dehydrated, and mounted as described for eosinophil and polymorphonuclear leukocytes (16). Cell movement was quantitated by counting the total number of cells migrating beyond a distance of 50 pm, using 10 pm intervals, in five high power fields (hpf) in duplicate micropore filters. In any single experiment the duplicates differed by less than 20% of their mean and the results are expressed as the cumulative counts from 50 to 130 pm in 10 hpf. The total number of cells that had migrated in buffer alone represent the background (10076) and the effect of each variable is expressed relative to the background response. The effect on lymphocyte migration of each variable was examined in three to five separate experiments and the results are presented as a mean -C 1 SD. In order to categorize agents as chemotactic, chemokinetic, or both, a checkerboard assay was used (17, 1s). In this assay incremental doses of a reagent were placed on the cell side (upper chamber), chemotactic factor side (lower chamber), or both sides of the filters to establish varying concentration gradients in both directions. Migration was then analyzed as outlined above and also by the ZigmondHirsch leading front method (19) where the distance from the top of the filter to the point where two cells have migrated is determined in 5 hpf in duplicate filters. The theoretical distance that cells would have migrated if the effect were entirely chemokinetic is calculated from the experimental data (19) and compared to the actual distance traveled. In this way agents can be assessed for their chemokinetic effect (increased migration independent of a concentration gradient). chemotactic action (increased migration only in the presence of a positive gradient), and a combination of these qualities. Lumicolchicine was prepared by irradiating lo-? M colchicine in 95% ethanol in
328
CEiYTER,
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a quartz crucible with ultraviolet light for 48 hr at 25°C. Conversion to lumicolchitine derivatives was assessed by dilution of this mixture to 2 x 10m5 M in 95% ethanol and measurement of absorbance at 200, 250, and 300 nanometers on a Beckman spectrophotometer. Conversion to lumicolchicine was considered nearly complete since absorbance of irradiated colchicine at 300 nm was 9% of a similar concentration of colchicine in 95% ethanol (20)) whereas absorbance at 200 and 250 nm was unchanged.
Che?+&inetic efject of colchicine. The chemokinetic effect of colchicine on rat splenic lymphocytes was assessed by placing equal concentrations of colchicine in a range of IO-* to 10m3 M in both the cell and stimulus compartments of modified Boyden chambers. Eight x lo6 cells/ml in M199-BSA were mixed with an equal volume of buffer containing 2 x lo-* to 2 x 10m3M colchicine, and 1 ml of each cell-colchicine mixture (4 x lo6 cells) was immediately introduced into the cell compartment of the chemotactic chamber at 25°C. Concentrations of colchicine identical to those on the cell side had been previously placed on the stimulus side in the same buffer. A dose-dependent chemokinetic response occurred with colchicine concentrations between 1 x lo-? and 4 X 10e7 M, with the latter giving the maximal stimulation (Fig. 1) . Higher concentrations were progressively less effective and there was no effect in the range of 10m4to 1O-3 izI. Preincubation of cells with the maximal stimulatory concentration of colchicine for 1 hr before placement into the chemotactic chambers containing that concentration of colchicine on the
FIG. 1. The cluzmokinctic response of rat splcuic lymphocytes to colchicine. The 100% line depicts the migration in buffer alone which ranged from 75 to 238 cells/l0 hpf, had a mean of 157 cells/l0 hpf and a standard deviation of 84 cells/l0 hpf. The effect of placing identical concentration of colchicine in the cell and stimulus compartments is presented as the mean percent of control response f 1 SD in four experiments.
DEPEiXDENCE
OF
LYMPIIOCYTE
MOTILITY
Oi’i
CELL COMPARTMENT COLCHICINE CM]
CELL COMPARTMENT COLCHICINE CM] 0
I xlo-7
2x10-’
4x10-7
329
MICROTLXULES
0
Eixlo-7
MO-‘,
l-l 67
8xlO-‘,
167
I41
2x10-’
8x10-’
4x10.’
98
~ 205
162
78
140
I 132
-4
1 125 1 144 p
I
FIG. 2. Checkerboard assay of the migrating response of rat splenic lymphocytes to colchicine. (A) Numbers indicate the mean distance to which the leading two cells migrated in 5 hpf into duplicate micropore filters. Numbers in parentheses are theoretical distances lymphocytes would have traveled if the effect of colchicine was solely chemokinetic. (B) Numbers indicate the total numbers of cells that migrated more than 50 pm into duplicate micropore filters in 10 hpf.
stimulus and cell sides did not decrease their movement as compared to cells that had not been preincubated. In order to determine whether colchicine could also have a chemotactic effect, a checkerboard assay was performed (Fig. 2A). The distances depicted in the vertical left hand column indicate the responses to gradients established from the stimulus side, the distances in the horizontal column across the top reflect the responses to gradients initiated on the cell side? and the distances in the diagonal from the upper left to the lower right represent the responses to increasing concentrations of colchicine in the absence of a gradient. Lymphocyte movement at each colchicine concentration was greatest when the agent was in equimolar concentrations on both sides of the filter, reflecting a chemokinetic cell response which was maximal at 4 X 10e7 M. The distances depicted in parentheses are theoretical and are based on the observed data depicted in the diagonal with the assumption that the effect of colchicine is entirely chemokinetic (19). In no instance where a positive gradient towards the stimulus compartment existed did the observed distance of the leading front exceed the theoretical, and thus there is no evidence that colchicine has a chemotactic action on rat splenic lymphocytes. The experiment was also analyzed by enumerating the total cells in 10 hpf which had moved more than 50 pm into duplicate filters (Fig. ZB). The maximum response for each concentration of colchicine was again observed when colchicine was placed in equimolar concentrations on both sides of the filter, so as to eliminate a gradient. Role of vvzzicrotubades in the chevzokinetic ejjfecCof colchicine. A second anti-mitotic agent, vinblastine, known to disassemblemicrotubules through a binding site different from that of colchicine, was examined for its dose-responseeffects on the migration of rat splenic lymphocytes. Preliminary experiments indicated that vinblastine, like colchicine, was chemokinetic and thus it was placed on both sides of the filter in the five experiments depicted in Fig. 3. A chemokinetic dose response was obtained in a concentration range of 1 X 10esto 2 X lo-? M. There was a broad plateau
330
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FIG. 3. The chemokinetic response of rat splenic lymphocytes to vinblastine. The 100% line depicts the migration in buffer alone which ranged from 84 to 180 cells/IO hpf, had a mean of 121.5 cells/l0 hpf and a standard deviation of 37 cells/l0 hpf. The effect of placing identical concentrations of vinblastine in the cell and stimulus compartments is presented as the mean percent of control response * 1 SD in five experiments.
j 0
I
I
I
10
20
30
HEAVY
WATER
I
I
40
50 f%l
FIG. 4. The dose response effect of heavy water 01, the random migration of rat splenic lymphocytes (e-0 ) and on their chemokinetic response to 4 X lo-’ M colchicine (0-O). The 100% line depicts the migration in buffer alone which ranged from 91 to 153 cells/l0 hpf, with a mean of 121 cells/l0 hpf and a standard deviation of 30 cells/l0 hpf. Results are expressed as the mean percent of control response * 1 SD in three experiments.
T)EPENDI’h’CE
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FIG. 5. Chemokinetic response of rat splenic lymphocytes to lumicolchicine (O-O), cdchicine ( O-O), and EtOH solvent (A-A). The 100% line depicts the migration in buffer alone and results are expressed as percent of baseline migration. in the chemokinetic response at concentrations from 2 X 10eT to 1 X 10-s M, with a Ioss of effect as the concentration was increased to 2 X 10m5 M. Inhibition was observed at 4 x lo-” M and was complete at lo-* M. Since the chemokinetic effect of vinblastine and colchicine suggested a role for microtubular disassembly in this response, heavy water, an agent that enhances
1
CYTOCHAL ASIN B (M] FIG. 6. The dose-response effect of cytochalasin B on the random migration of rat splenic lymphocytes. The 100% line depicts the migration in buffer alone which ranged from 84 cells/l0 hpf to 202 cells/l0 hpf, had a mean of 120 cells/l0 hpf and a standard deviation of 47 cells/l0 hpf. The effect of placing identical concentrations of cytochalasin B in the cell and stimulus compartments is presented as the mean percent of control response f 1 SD in five experiments.
332
CYTOCHAL
ASIN t? /M/
FIG. 7. The effect of cytochalasin B on the chemokinetic response of rat splenic lymphocytes to 4 X lo-’ M colchicine. The 100% line depicts the migration in buffer alone in four experiments which ranged from 84 cells/l0 hpf to 103 cells/l0 hpf, had a mean of 95.4 cells/l0 hpf and a standard deviation of 9.3 cells/l0 hpf. The effect of placing identical concentrations of cytochalasin B in the cell and stimulus compartments along with 4 X lo-’ M colchicine are presented as the mean percent of control response t 1 SD.
assembly of microtubules, was studied as a possible inhibitor of lymphocyte migration. D20 placed on both sides of the filter inhibited lymphocyte migration in final concentrations above 30% (Fig. 4), and this inhibition of 35% was not increased with concentrations of 40 and 50% DZO. In order to examine the effect of D20 on colchicine stimulation, rat splenic lymphocytes in a concentration of 8 x IO6 cells/ml were incubated with 8 x 1O-7 M colchicine for 30 min at 25°C. One-half ml of colchicine-stimulated cells were mixed with an equal volume of R/1199-BSA containing from zero to 100% DaO, and 1 ml of the cell suspension was then placed in a Boyden chamber. Concentrations of D,O and colchicine identical to those on the cell side had been placed on the stimulus side in the same buffer (Fig. 4). Colchicine alone in a final concentration of 4 x 10e7 121 gave a 340% increase in lymphocyte migration and this chemokinetic effect was reversed in a dose-response fashion by increasing concentrations of heavy water. A concentration of 50% D20 completely reversed the chemokinetic response to colchicine. Da0 similarly inhibited the chemokinetic effect of vinblastine (not shown). Lumicolchicine differs from colchicine in that it does not bind to monomeric tubulin and therefore cannot inhibit tubulin from assembling into microtubules. Colchicine and lumicolchicine at lo-* M in 95% EtOH were each diluted in M199-BSA to the concentrations of colchicine found to be chemokinetic. The effect of 95% EtOH diluted in parallel fashion was also evaluated. The colchicine gave a doseresponse stimulation from 2 to 4 X lo-? M, with the peak effect being 390% of baseline migration (Fig. 5). Lumicolchicine did not increase migration and the
inhil)ition observed at the highest concentration \vas cc~ml~arable to the effect of alcol1ul ;11011c. Efect of cytochrltrsin U on flzc chmokiwtic c#tjcr:c.t uf c.cllchicinc. The iiiotlulation of rat lymphocyte migration by cytochalasin B, an agent that inhibits microfilament function by preventing the interaction of actin-binding protein with subplasmalenma1 actin (21) , was assessedby placing cytochalasin B on both sides of the filter in a protocol similar to that used with heavy water. Cytochalasin B gave a dosedependent inhibition of lymphocyte migration in concentrations of 2 X lo-? to 2 x 10m9M (Fig. 6). Doses of 10-” and 10~4M resulted in complete inhibition of all lymphocyte migration and in these filters no cells were observed to have entered the filter to any depth beyond the stacking layer. In order to esamine the effect of cytochalasin B on colchicine stimulation, rat splenic lymphocytes were incubated in a concentration of 8 x 10G cells/ml with S x 10m7M colchicine in M199-BSA for 30 min at 25°C. One-half ml of colchicine-stimulated cells were mixed with an equal volume of M199-BSA containing from 2 X lOmato 2 X 10e4ill’ cytochalasin B and 1 ml of the cell suspensionwas then placed in a Boyden chamber. Concentrations of cytochalasin B and colchicine identical to those on the cell side had been placed on the stimulus side in the same buffer. Colchicine alone in a final concentration of 4 x 10e7M gave a 340% rise in lymphocyte migration and this chemokinetic effect was reversed by concentrations of cytochalasin B above IO-’ All (Fig. 7). Concentrations of cytochalasin U between 5 X 10m7and 4 X 10mF,%I reduced the chemokinetic responseto baseline, and concentrations of lo-” X, as in the studies on unstimulated migration, abolished all cell movement.
Rat splenic lymphocytes are stimulated to increase their chemokinetic migration by agents such as colchicine and vinblastine which disassembletheir microtubules. Both agents produce a dose-dependent increase in chemokinesis with the initial effects being observed at concentrations in the 10e8to 1O-7M range, and the peak effects occurring at 4 X 10m71Vl for colchicine (Fig. 1) and more broadly from 2 x 10m7to 2 x lo-” M for vinblastine (Fig. 3). The mean peak effect with 4 x lo-? M colchicine is 3.6 times baseline random migration of cells, and with 4 x 10m7M vinblastine the multiple is the same.As concentrations of colchicine and vinblastine are increased, there is a progressive decrease in chemokinesis such that migration returns to a baseline with 10m3Al colchicine and 2 X 10e5&I vinblastine. The nature of the augmented migration of rat splenic lymphocytes in the presence of colchicine was established as being chemokinetic and not chemotactic, as defined by the criteria developed for the study of polymorphonuclear leukocyte migration (18, 19). Since the maximum response for any concentration of colchicine by checkerboard analysis was observed when equimolar amounts of the agent were placed on both the cell and stimulus sides of the filters, a gradient was not necessary (Fig. 2A). Indeed, the checkerboard analysis showed that the movement observed in a series of gradients towards the stimulus side was no greater than the theoretical distance predicted by the chemokinetic effect alone and thus there was no evidence of a superimposed chemotactic effect (19). The interpretation is the samewhen the results are expressed as the total number of cells moving more than 50 pm into the filter. At any concentration of either colchicine (Fig. 2B) or vin-
334
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blastine, the total cell movement was maximal in the absence of a gradient, indicating the chemokinetic stimulation by these microtubular depolymerizing agents. Two further lines of evidence indicate that the chemokinetic response of rat splenic lymphocytes is related to a shift in the dynamic state of microtubules toward disassembly. Lumicolchicine, which fails to ‘bind to monomeric tubulin (20)) had no effect on the movement of rat splenic lymphocytes over a dose range of lo-@ to 10m4M (Fig. 5). Heavy water, which is known to promote assembly of microtubules, partially suppressed random migration of rat splenic lymphocytes and in a dose-dependent fashion reversed the chemokinetic effect of 4 x 10e7 Al colchicine (Fig. 4). The shift of the microtubule state to favor disassembly would lessen any contribution to a rigid cytoskeleton of the resting lymphocyte and facilitate changes favoring membrane fluidity and motility. This relationship would offer an explanation for the observation that the resting non-motile lymphocyte is spherical, whereas the motile cell manifests a uropod extension and cytoplasmic rippling (4). An alternative interpretation, namely that the colchicine effect is not due to increased influx into but rather decreased efflux from the filters was not supported. Preincubation of cells for 1 hr with an optimal colchicine stimulus did not decrease their subsequent movement in chambers containing the same colchicine concentration as compared to cells not pretreated. Furthermore, the movement of both untreated and colchicine pretreated cells increased comparably at each hourly time point during the 3 hr period of migration. Studies with mouse splenic B lymphocytes (5) showed that cytochalasin B and agents which elevated intracellular levels of cyclic AMP decreased the development of motile forms in response to anti-Ig. In the present studies cytochalasin B inhibited the spontaneous migration of rat splenic lymphocytes in concentrations above 10m7M and completely inhibited all cell movement at a dose of 10-j M (Fig. 6). Cytochalasin B reversed the positive chemokinetic effect of rat splenic lymphocytes to 4 X 10m7M colchicine in concentrations of 1Omsto 10m6M and again completely inhibited cell movement at 10e5 NI (Fig. 7). The concentrations of cytochalasin B required to reverse colchicine-induced chemokinesis or inhibit random motility are generally less than those required to interfere with glucose (22) or nucleoside transport (23). Since ultrastructural studies were not carried out, the mechanism of the effects of the various agents on lymphocyte motility is speculative and based upon the observed effect of these agents on subcellular structures in other cell systems (20, 24, 25). The results are similar to those obtained with human and rabbit neutrophils where colchicine, vinblastine and vincristine increased random motility (26)) whereas cytochalasin B inhibited chemotaxis and spontaneous motility (27). REFERENCES 1. McCluskey, R. T., Benacerraf, B., and McCluskey, J. W., J. Immunol. 98, 466, 1963. 2. Wilkinson, P. C., Parrot, D. M. V., Russel, R. J., and Sless, F., J. Exp. Med. 145, 1158, 1977. 3. Ward, P. A., Unanue, E. R., Goralnick, S., and Schreiner, G. F., J. Zmmunol. 119, 416, 1977. 4. Unanue, E. R., Ault, K. A., and Karnovsky, M. J., J. Exp. Med. 139, 295, 1974. 5. Schreiner, G. F., and Unanue, E. R., J. Zmmunol. 114, 802, 1975. 6. Schreiner, G. F., and Unanue, E. R., J. Immulzol. 114, 809, 1975. 7. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V., Nature 256, 646, 1975. 8. Wilkinson, P. C., Roberts, J. A., Russell, R. J., and McLaughlin, M., Cl&. Exp. Immunol. 25, 280, 1976.
DEPENDENCE
9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27.
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MICROTUXULES
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Johnson, G. S., Friedman, R. M., and Pastan, I., Proc. Satl. Acad. Sci. 68, 425, 1971. Kirkland, W. L., and Burton, P. R., Sature Stew Biol. 240, 205, 1972. Jarvis, S. C., Snyderman, R., and Cohen, H. J., Blood 48, 717, 1976. Fram, R. J., Sidman, C. L., and Unanue, E. R., J. Irrzmuuol. 117, 1456, 1976. Boyden, S., J. Exp. Med. 115, 453, 1962. Boyum, A., Scmd. J. Cl&. Lab. Invest. 21 (suppl 97), 31, 1968. Schlossman, S. F., and Hudson, L. J., J. I+n~zu,~ol. 110, 313, 1973. Kay, A. B., Clin. Exp. Imnzunol. 7, 723, 1970. Keller, H. U., Wilkinson, P. C., Abercrombie, M., Becker, E. L., Hirsch, J. G., Miller, M. E., Ramsey, W. S., and Zigmond, S. H., J. I?rtnzultol. 118, 1912, 1977. Wilkinson, P. C., and Allan, R. B., 11% “Leukocyte Chemotaxis: Methods, Physiology, and Clinical Implications” (J. A. Gallin and P. G. Quie, Eds.), pp. l-23. Raven Press, New York, 1978. Zigmond, S. A., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. Wilson, L., Bamburg, J. R., Mizel, S. B., Grisham, L. hf., and Creswell, K. M., Frd. Proc. 33, 158, 1974. Hartwig, J. H., and Stossel, T. P., J. Cell Biol. 71, 295, 1976. Estensen, R. D., and Plagemann, P. G. W., Proc. Natl. Acad. Sci. 69, 1430, 1972. Plagemann, P. G. W., and Estensen, R. D., J. Cell. Biol. 55, 179, 1972. Malawista, S. E., and Bensch, K. G., Science 156, 521, 1967. Padawer, J., J. Natl. Cancer Inst. 25, 731, 1960. Becker, E. L., and Showell, H. J., J. Imnzunol. 112, 2055, 1974. Becker, E. L., Davis, A. T., Estensen, R. D., and Quie, P. G., J. Iwwtunol. 108, 396, 1972.