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Effect of culturing on the human lymphocyte locomotion response to casein, C5a, and fMet-Leu-Phe
CELLULAR
IMMUNOLOGY
(1981)
a,4349
Effect of Culturing on the Human Lymphocyte Locomotion Response to Casein, C5a, and fMet-Leu-Phe’ ADEL K. EL-NAGGAR, DENNIS E. VAN EPPS,’ RALPH C. WILLIAMS, JR. Departments of Medicine and Microbiology, University o$ New Mexico, School of Medicine. Albuquerque, New Mexico 87131 Received
May
28, 1980. accepted September 19. 1980
Cultured and noncultured human peripheral blood monocyte-depleted lymphocytes were studied concurrently to determine the effect of culturing on lymphocyte locomotion in response to different chemokinetic (Net-Leu-Phe and casein) and chemotactic factors (C5a). This study demonstrates that culturing enhances lymphocyte locomotion as measured by the absolute distance migrated, but does not alter the ability of these cells to elicit a chemotactic or chemokinetic response. Furthermore, when lymphocyte locomotion is expressed as a migration index (difference between migration in response to a locomotor stimulus and that achieved in medium alone) the magnitude of the chemotactic or chemokinetic response observed was similar with both cultured and noncultured lymphocytes. We conclude from this study that culturing lymphocytes prior to the in vitro locomotion assay is not a prerequisite to measure chemotaxis, but only increases the distance that lymphocytes migrate.
INTRODUCTION Recently several investigators have demonstrated that lymphocytes migrate in in response to different chemokinetic and chemotactic factors (l-4). Some discrepancy exists as to whether it is essential to preculture lymphocytes in medium (4) or with mitogens (3) prior to measuring their responsiveness to different chemoattractants. Recently, the view that mitogen stimulation is essential to lymphocyte chemotaxis has been modified by Shields and Wilkinson (8) who have stated that lymphocyte culturing is essential to obtain an “optimal” locomotor response to different stimuli. In a previous study from this laboratory (5) we have shown that in the absence of culturing, human peripheral blood lymphocytes can respond to chemokinetic and chemotactic stimuli. In an effort to determine the influence of culturing on lymphocyte locomotion, we have conducted the current study. The data presented here demonstrate that in general culturing enhances lymphocyte locomotion but that the ability of lymphocytes to respond by chemokinesis or chemotaxis is not altered by the culturing procedure. vitro
’ This investigation was supported in part by Grant CA20819 awarded by the National Cancer Institute, DHEW and Grants AM13824-10 and AM13690-11 awarded by the Institute of Arthritis, Metabolism and Digestive Diseases, DHEW. * Recipient of Senior Investigatorship Award from the Arthritis Foundation. 43 0008-8749/81/070043-07$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
44
EL-NAGGAR,
VAN EPPS, AND WILLIAMS
MATERIALS
AND
METHODS
Cell preparation. Human peripheral blood mononuclear cells were separated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation. The mononuclear band was removed, washed twice in Hanks’ balanced salt solution (HBSS) and resuspended in 100% fetal calf serum (FCS). Monocytes were removed
C50 Concentration
‘“7
10% Cosrin
IO-'0 Molar
Concrntrotion
20%
40%
60%
Kre
10-7
Concenirotion
lO-g
of f-Met-Lw-ph0
FIG. 1. Migration of cultured and noncultured monocyte-depleted lymphocytes in response to varying concentrations of casein, CSa, andf-Met-Leu-Phe. The mean migration + 1 SD is indicated. Data for fMet-Leu-Phe are mean results from six different subjects and data for both casein and CSa are the mean results of seven subjects. Concentrations indicated for CSa and casein are dilutions of stock solution, (stock CSa preparations had an optical density of 1.3 at 280 nm and the stock casein solution contained approximately 2 mg/ml of casein).
HUMAN
LYMPHOCYTE
45
LOCOMOTION
by glass wool adherence as previously described (5). The nonadherent cells were washed twice and resuspended to 4 X lo6 cells/ml in HBSS containing 2% fetal calf serum. If these preparations contained greater than 1% monocytes as indicated by peroxidase-positive cells (5, 6), the glass wool adherence technique was repeated so that all lymphocyte preparations tested contained fewer than 1% peroxidasepositive cells. Such preparations are referred to as monocyte-depleted (MD) lymphocytes. Cultured cells. MD lymphocytes were resuspended in minimum essential medium (MEM) supplemented with 10% fetal calf serum, 2 mM L-gldtamine, 100 units/ ml penicillin, and 100 pg/ml of streptomycin. The suspensions were incubated for 24 hr without mitogens or antigens. After culture, cells were washed twice with HBSS and adjusted to 4 X lo6 lymphocytes/ml in HBSS containing 2% fetal calf serum. Viability as determined by trypan blue dye exclusion was greater than 98%. Chemotactic factors. Casein (Fisher, Pittsburgh, Pa.) preparations were made weekly in HBSS by incubating 2 mg/ml of casein for 2 hr at 37°C with frequent agitation. C5a was partially purified from fresh human serum after overnight incubation at 4°C with 1 M e-aminocaproic acid (Sigma Chemical Co., St. Louis, MO.). Activation of serum was accomplished by adding 20 mg/ml of boiled bakers yeast and incubating for 60 min at 37°C. Yeast was removed by centrifugation and the activated serum was then passed through a Sephadex G-50 gel filtration column.
1
I 10-O Molar
I IO-' Concentration
1 IO-*
1 to-'
of f-Met-bu-Phr
FIG. 2. Analysis of cultured and noncultured lymphocyte chemotactic or chemokinetic responses to fMet-Leu-Phe. Responses to each factor in the presence of a positive gradient (factor in lower compartment), a negative gradient (factor in upper compartment), and no gradient (factor in both compartments) are shown. Results in each case are the average of data obtained on five different subjects.
46
EL-NAGGAR,
VAN
EPPS,
AND
WILLIAMS
The low-molecular-weight eluent (<20,000 daltons) containing neutrophil chemotactic activity was concentrated to original volume by ultrafiltration using a micropore membrane which exluded molecules greater than 2000 daltons. The final preparation used for titration had an optical density of 1.35 at 280 nm. Locomotion assay. Lymphocyte locomotion assays were performed in blind-well chemotaxis chambers (Nucleopore, Pleasanton, Calif.) as previously described (5) using a modification of the leading front technique developed for neutrophils by Zigmond and Hirsch (7). This assay measures the distance in micrometers that cells migrate into a micropore membrane in a 3-hr incubation period at 37°C. The membrane used to separate the upper and lower compartments of the chamber had an 8-pm pore size (Sartorius, Hayward, Calif.). Assessment of lymphocyte locomotion was made after formaldehyde fixation, hematoxylin staining, and clearing of the micropore membranes with isopropyl alcohol and xylene (5). The distance that the leading three cells had migrated into the membrane was measured in five fields using the microscope micrometer and averaged. To determine if culturing altered the ability of lymphocytes to respond chemokinetically or chemotactically to different stimuli, lymphocyte locomotion in response to varying concentrations of stimuli in the upper cell compartment, the lower compartment, or both compartments was assessed. This technique has previously been shown to delineate directional from nondirectional locomotion (5) in a manner similar to the Zigmond and Hirsch checkerboard assay (7). In some cases the data are presented as a “migration index” which is the difference in micrometers between the distance that
Casein
FIG. 3. Analysis of cultured and casein. Responses to each factor in a negative gradient (factor in upper shown. Results in each case are the
Comxntration
noncultured lymphocyte chemotactic or chemokinetic responses to the presence of a positive gradient (factor in lower compartment), compartment), and no gradient (factor in both compartments) are average of data obtained on five different subjects.
HUMAN
LYMPHOCYTE
47
LOCOMOTION
lymphocytes migrate when stimulated with a chemokinetic and the distance they migrate in medium alone.
or chemotactic
stimulus,
RESULTS Monocyte-depleted lymphocytes were isolated, suspended to a concentration of 4 x lo6 lymphocytes/ml, and cultured overnight. The next day these cells and a preparation of freshly isolated monocyte-depleted lymphocytes from the same donor were tested in the locomotion assay. As shown in Fig. 1, the random unstimulated locomotion of lymphocytes cultured overnight was markedly enhanced (mean = 55 f 14.4 pm) as compared to lymphocyte preparations tested immediately after isolation (mean = 30 f 14 pm). This same enhanced locomotor activity was observed with cultured lymphocytes in the presence of casein, fMet-Leu-Phe or C5a present in the lower compartment as a migration stimulus (Fig. 1). The mean peak migration of cultured lymphocytes in response to these factors was 80 f 12, 66 ? 6, and 71 + 14, respectively, while the mean peak response of uncultured lymphocytes was 56 + 6.5, 48 f 6, and 44 f 11. These differences are statistically significant by paired t test analysis at p < 0.01. Since some investigators have claimed that culturing is essential to lymphocyte chemotaxis (2-4, 8) and since a previous report from this laboratory has shown that fMet-Leu-Phe and casein are chemokinetic factors and C5a is a chemotactic
-5 -10 I 10%
I 20%
I 50%
Concentration of C50 used FIG. 4. Analysis of cultured and noncultured lymphocyte chemotactic or chemokinetic responses to C5a. Responses to each factor in the presence of a positive gradient (factor in lower compartment), a negative gradient (factor in upper compartment), and no gradient (factor in both compartments) are shown. Results in each case are the average of data obtained on five different subjects.
48
EL-NAGGAR,
VAN
EPPS,
AND
WILLIAMS
factor for uncultured lymphocytes, experiments were conducted to determine if culturing alters the lymphocyte chemotactic or chemokinetic response. Both cultured and noncultured lymphocytes were tested in the locomotion assay by titrating the three migration-stimulating factors in the chemotaxis chamber. Directional and nondirectional enhanced random locomotion were evaluated by titrating each stimulus beyond its optimal dose in the upper cell compartment, the lower compartment, or both compartments of the chemotaxis chamber. If the factor stimulated a lymphocyte chemotactic response, the existence of a positive gradient (factor in lower compartment) would result in enhanced migration into the membrane as compared to the response in the presence of a negative gradient (factor in the upper compartment) or no gradient (factor in both compartments). If the factor stimulated a chemokinetic response, locomotion in the absence of a gradient (equal concentrations above and below the membrane) should be equal to or greater than the response in the presence of a positive gradient. As shown, the response of both cultured and noncultured lymphocytes to fMet-Leu-Phe (Fig. 2) and casein (Fig. 3) was a chemokinetic response whereas the response to C5a (Fig. 4) in each case was chemotactic. It is of interest to note that when the data are expressed as a migration index rather than the absolute migration, the magnitude of migration of cultured cells and noncultured cells is very similar indicating that culturing simply enhances locomotion in general and does not disproportionately magnify the effects of the chemotactic or chemokinetic stimulus. DISCUSSION It is well established that lymphocytes and their subsets show chemotactic and chemokinetic reactions to a variety of attractants (1, 2, 4, 5, 10). However, it is the contention of several investigators (4,8, 11) that lymphocytes must be cultured or be in a blast stage (3) in order to demonstrate in vitro chemotactic activity. In recent studies, (1, 5, 9) evidence has been presented indicating that noncultured cells are in fact capable of locomotion. Shields and Wilkinson (8) have stated that uncultured lymphocytes do not show optimal locomotor activity, but little data have been presented to compare the magnitude of cultured and noncultured lymphocyte responses. Recent studies by McCarty and Goetzl (9) have shown that culturing is not essential to T-lymphocyte chemokinesis in response to arachidonic acid. The current study presents data indicating that culturing lymphocytes does indeed enhance their absolute locomotor activity, although it does not appear to affect the chemokinetic or chemotactic nature of the response. Furthermore, as shown in Fig. 1, although cultured lymphocytes appear to move further into the membrane than noncultured lymphocytes, when data are expressed as a migration index to account for enhanced random locomotion in the absence of a stimulus, the actual effects of the chemotactic or chemokinetic agents on lymphocyte locomotion are very similar with cultured and noncultured lymphocytes. We conclude from these studies that: (i) culturing of lymphocytes prior to study is not essential to demonstrate a chemotactic response; (ii) culturing of lymphocytes does increase their absolute migration either in the presence or absence of a migration stimulus, but does not alter the response to a stimulus when data are expressed as a migration index; and (iii) culturing of lymphocytes does not change the nature of the migratory response to fMet-Leu-Phe, casein, or CSa. That is,
HUMAN
LYMPHOCYTE
LOCOMOTION
both cultured and noncultured lymphocytes show a chemokinetic and fMet-Leu-Phe, and a chemotactic response to C5a.
49 response to casein
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Ward, P. A., Unanue, G. R., Gonolnick, S. J., and Schreiner, G. F., J. Immunol. 119, 416, 1977. Wilkinson, P. C., Immunology 33, 407, 1977. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V., Nature (London) 256, 646, 1975. Parrott, M. V., Good, R. A., O’Neill, G. J., and Gupta, S., Proc. Natl. Acad. Sci. USA 75, 2392, 1978. El-Naggar, A. K., Van Epps, D., and Williams, R. C., Jr., Cell. Immunof., in press. Kaplow, L., Blood 26, 2, 1965. Zigmond, S. H., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. Shields, J. M., and Wilkinson, P. C., Clin. Exp. Immunol. 38, 598, 1979. McCarty, J., and Goetzl, E. J., Immunology 43, 103, 1979. O’Neill, G. J., and Parrott, M. V., Cell. Immunof.33, 257, 1977. Wilkinson, P. C., Roberts, J. A., Russell, R. J., and McLaughlin, M., Clin. Exp. Immunol. 25, 280, 1976.
IMMUNOLOGY
(1981)
a,4349
Effect of Culturing on the Human Lymphocyte Locomotion Response to Casein, C5a, and fMet-Leu-Phe’ ADEL K. EL-NAGGAR, DENNIS E. VAN EPPS,’ RALPH C. WILLIAMS, JR. Departments of Medicine and Microbiology, University o$ New Mexico, School of Medicine. Albuquerque, New Mexico 87131 Received
May
28, 1980. accepted September 19. 1980
Cultured and noncultured human peripheral blood monocyte-depleted lymphocytes were studied concurrently to determine the effect of culturing on lymphocyte locomotion in response to different chemokinetic (Net-Leu-Phe and casein) and chemotactic factors (C5a). This study demonstrates that culturing enhances lymphocyte locomotion as measured by the absolute distance migrated, but does not alter the ability of these cells to elicit a chemotactic or chemokinetic response. Furthermore, when lymphocyte locomotion is expressed as a migration index (difference between migration in response to a locomotor stimulus and that achieved in medium alone) the magnitude of the chemotactic or chemokinetic response observed was similar with both cultured and noncultured lymphocytes. We conclude from this study that culturing lymphocytes prior to the in vitro locomotion assay is not a prerequisite to measure chemotaxis, but only increases the distance that lymphocytes migrate.
INTRODUCTION Recently several investigators have demonstrated that lymphocytes migrate in in response to different chemokinetic and chemotactic factors (l-4). Some discrepancy exists as to whether it is essential to preculture lymphocytes in medium (4) or with mitogens (3) prior to measuring their responsiveness to different chemoattractants. Recently, the view that mitogen stimulation is essential to lymphocyte chemotaxis has been modified by Shields and Wilkinson (8) who have stated that lymphocyte culturing is essential to obtain an “optimal” locomotor response to different stimuli. In a previous study from this laboratory (5) we have shown that in the absence of culturing, human peripheral blood lymphocytes can respond to chemokinetic and chemotactic stimuli. In an effort to determine the influence of culturing on lymphocyte locomotion, we have conducted the current study. The data presented here demonstrate that in general culturing enhances lymphocyte locomotion but that the ability of lymphocytes to respond by chemokinesis or chemotaxis is not altered by the culturing procedure. vitro
’ This investigation was supported in part by Grant CA20819 awarded by the National Cancer Institute, DHEW and Grants AM13824-10 and AM13690-11 awarded by the Institute of Arthritis, Metabolism and Digestive Diseases, DHEW. * Recipient of Senior Investigatorship Award from the Arthritis Foundation. 43 0008-8749/81/070043-07$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
44
EL-NAGGAR,
VAN EPPS, AND WILLIAMS
MATERIALS
AND
METHODS
Cell preparation. Human peripheral blood mononuclear cells were separated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation. The mononuclear band was removed, washed twice in Hanks’ balanced salt solution (HBSS) and resuspended in 100% fetal calf serum (FCS). Monocytes were removed
C50 Concentration
‘“7
10% Cosrin
IO-'0 Molar
Concrntrotion
20%
40%
60%
Kre
10-7
Concenirotion
lO-g
of f-Met-Lw-ph0
FIG. 1. Migration of cultured and noncultured monocyte-depleted lymphocytes in response to varying concentrations of casein, CSa, andf-Met-Leu-Phe. The mean migration + 1 SD is indicated. Data for fMet-Leu-Phe are mean results from six different subjects and data for both casein and CSa are the mean results of seven subjects. Concentrations indicated for CSa and casein are dilutions of stock solution, (stock CSa preparations had an optical density of 1.3 at 280 nm and the stock casein solution contained approximately 2 mg/ml of casein).
HUMAN
LYMPHOCYTE
45
LOCOMOTION
by glass wool adherence as previously described (5). The nonadherent cells were washed twice and resuspended to 4 X lo6 cells/ml in HBSS containing 2% fetal calf serum. If these preparations contained greater than 1% monocytes as indicated by peroxidase-positive cells (5, 6), the glass wool adherence technique was repeated so that all lymphocyte preparations tested contained fewer than 1% peroxidasepositive cells. Such preparations are referred to as monocyte-depleted (MD) lymphocytes. Cultured cells. MD lymphocytes were resuspended in minimum essential medium (MEM) supplemented with 10% fetal calf serum, 2 mM L-gldtamine, 100 units/ ml penicillin, and 100 pg/ml of streptomycin. The suspensions were incubated for 24 hr without mitogens or antigens. After culture, cells were washed twice with HBSS and adjusted to 4 X lo6 lymphocytes/ml in HBSS containing 2% fetal calf serum. Viability as determined by trypan blue dye exclusion was greater than 98%. Chemotactic factors. Casein (Fisher, Pittsburgh, Pa.) preparations were made weekly in HBSS by incubating 2 mg/ml of casein for 2 hr at 37°C with frequent agitation. C5a was partially purified from fresh human serum after overnight incubation at 4°C with 1 M e-aminocaproic acid (Sigma Chemical Co., St. Louis, MO.). Activation of serum was accomplished by adding 20 mg/ml of boiled bakers yeast and incubating for 60 min at 37°C. Yeast was removed by centrifugation and the activated serum was then passed through a Sephadex G-50 gel filtration column.
1
I 10-O Molar
I IO-' Concentration
1 IO-*
1 to-'
of f-Met-bu-Phr
FIG. 2. Analysis of cultured and noncultured lymphocyte chemotactic or chemokinetic responses to fMet-Leu-Phe. Responses to each factor in the presence of a positive gradient (factor in lower compartment), a negative gradient (factor in upper compartment), and no gradient (factor in both compartments) are shown. Results in each case are the average of data obtained on five different subjects.
46
EL-NAGGAR,
VAN
EPPS,
AND
WILLIAMS
The low-molecular-weight eluent (<20,000 daltons) containing neutrophil chemotactic activity was concentrated to original volume by ultrafiltration using a micropore membrane which exluded molecules greater than 2000 daltons. The final preparation used for titration had an optical density of 1.35 at 280 nm. Locomotion assay. Lymphocyte locomotion assays were performed in blind-well chemotaxis chambers (Nucleopore, Pleasanton, Calif.) as previously described (5) using a modification of the leading front technique developed for neutrophils by Zigmond and Hirsch (7). This assay measures the distance in micrometers that cells migrate into a micropore membrane in a 3-hr incubation period at 37°C. The membrane used to separate the upper and lower compartments of the chamber had an 8-pm pore size (Sartorius, Hayward, Calif.). Assessment of lymphocyte locomotion was made after formaldehyde fixation, hematoxylin staining, and clearing of the micropore membranes with isopropyl alcohol and xylene (5). The distance that the leading three cells had migrated into the membrane was measured in five fields using the microscope micrometer and averaged. To determine if culturing altered the ability of lymphocytes to respond chemokinetically or chemotactically to different stimuli, lymphocyte locomotion in response to varying concentrations of stimuli in the upper cell compartment, the lower compartment, or both compartments was assessed. This technique has previously been shown to delineate directional from nondirectional locomotion (5) in a manner similar to the Zigmond and Hirsch checkerboard assay (7). In some cases the data are presented as a “migration index” which is the difference in micrometers between the distance that
Casein
FIG. 3. Analysis of cultured and casein. Responses to each factor in a negative gradient (factor in upper shown. Results in each case are the
Comxntration
noncultured lymphocyte chemotactic or chemokinetic responses to the presence of a positive gradient (factor in lower compartment), compartment), and no gradient (factor in both compartments) are average of data obtained on five different subjects.
HUMAN
LYMPHOCYTE
47
LOCOMOTION
lymphocytes migrate when stimulated with a chemokinetic and the distance they migrate in medium alone.
or chemotactic
stimulus,
RESULTS Monocyte-depleted lymphocytes were isolated, suspended to a concentration of 4 x lo6 lymphocytes/ml, and cultured overnight. The next day these cells and a preparation of freshly isolated monocyte-depleted lymphocytes from the same donor were tested in the locomotion assay. As shown in Fig. 1, the random unstimulated locomotion of lymphocytes cultured overnight was markedly enhanced (mean = 55 f 14.4 pm) as compared to lymphocyte preparations tested immediately after isolation (mean = 30 f 14 pm). This same enhanced locomotor activity was observed with cultured lymphocytes in the presence of casein, fMet-Leu-Phe or C5a present in the lower compartment as a migration stimulus (Fig. 1). The mean peak migration of cultured lymphocytes in response to these factors was 80 f 12, 66 ? 6, and 71 + 14, respectively, while the mean peak response of uncultured lymphocytes was 56 + 6.5, 48 f 6, and 44 f 11. These differences are statistically significant by paired t test analysis at p < 0.01. Since some investigators have claimed that culturing is essential to lymphocyte chemotaxis (2-4, 8) and since a previous report from this laboratory has shown that fMet-Leu-Phe and casein are chemokinetic factors and C5a is a chemotactic
-5 -10 I 10%
I 20%
I 50%
Concentration of C50 used FIG. 4. Analysis of cultured and noncultured lymphocyte chemotactic or chemokinetic responses to C5a. Responses to each factor in the presence of a positive gradient (factor in lower compartment), a negative gradient (factor in upper compartment), and no gradient (factor in both compartments) are shown. Results in each case are the average of data obtained on five different subjects.
48
EL-NAGGAR,
VAN
EPPS,
AND
WILLIAMS
factor for uncultured lymphocytes, experiments were conducted to determine if culturing alters the lymphocyte chemotactic or chemokinetic response. Both cultured and noncultured lymphocytes were tested in the locomotion assay by titrating the three migration-stimulating factors in the chemotaxis chamber. Directional and nondirectional enhanced random locomotion were evaluated by titrating each stimulus beyond its optimal dose in the upper cell compartment, the lower compartment, or both compartments of the chemotaxis chamber. If the factor stimulated a lymphocyte chemotactic response, the existence of a positive gradient (factor in lower compartment) would result in enhanced migration into the membrane as compared to the response in the presence of a negative gradient (factor in the upper compartment) or no gradient (factor in both compartments). If the factor stimulated a chemokinetic response, locomotion in the absence of a gradient (equal concentrations above and below the membrane) should be equal to or greater than the response in the presence of a positive gradient. As shown, the response of both cultured and noncultured lymphocytes to fMet-Leu-Phe (Fig. 2) and casein (Fig. 3) was a chemokinetic response whereas the response to C5a (Fig. 4) in each case was chemotactic. It is of interest to note that when the data are expressed as a migration index rather than the absolute migration, the magnitude of migration of cultured cells and noncultured cells is very similar indicating that culturing simply enhances locomotion in general and does not disproportionately magnify the effects of the chemotactic or chemokinetic stimulus. DISCUSSION It is well established that lymphocytes and their subsets show chemotactic and chemokinetic reactions to a variety of attractants (1, 2, 4, 5, 10). However, it is the contention of several investigators (4,8, 11) that lymphocytes must be cultured or be in a blast stage (3) in order to demonstrate in vitro chemotactic activity. In recent studies, (1, 5, 9) evidence has been presented indicating that noncultured cells are in fact capable of locomotion. Shields and Wilkinson (8) have stated that uncultured lymphocytes do not show optimal locomotor activity, but little data have been presented to compare the magnitude of cultured and noncultured lymphocyte responses. Recent studies by McCarty and Goetzl (9) have shown that culturing is not essential to T-lymphocyte chemokinesis in response to arachidonic acid. The current study presents data indicating that culturing lymphocytes does indeed enhance their absolute locomotor activity, although it does not appear to affect the chemokinetic or chemotactic nature of the response. Furthermore, as shown in Fig. 1, although cultured lymphocytes appear to move further into the membrane than noncultured lymphocytes, when data are expressed as a migration index to account for enhanced random locomotion in the absence of a stimulus, the actual effects of the chemotactic or chemokinetic agents on lymphocyte locomotion are very similar with cultured and noncultured lymphocytes. We conclude from these studies that: (i) culturing of lymphocytes prior to study is not essential to demonstrate a chemotactic response; (ii) culturing of lymphocytes does increase their absolute migration either in the presence or absence of a migration stimulus, but does not alter the response to a stimulus when data are expressed as a migration index; and (iii) culturing of lymphocytes does not change the nature of the migratory response to fMet-Leu-Phe, casein, or CSa. That is,
HUMAN
LYMPHOCYTE
LOCOMOTION
both cultured and noncultured lymphocytes show a chemokinetic and fMet-Leu-Phe, and a chemotactic response to C5a.
49 response to casein
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Ward, P. A., Unanue, G. R., Gonolnick, S. J., and Schreiner, G. F., J. Immunol. 119, 416, 1977. Wilkinson, P. C., Immunology 33, 407, 1977. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V., Nature (London) 256, 646, 1975. Parrott, M. V., Good, R. A., O’Neill, G. J., and Gupta, S., Proc. Natl. Acad. Sci. USA 75, 2392, 1978. El-Naggar, A. K., Van Epps, D., and Williams, R. C., Jr., Cell. Immunof., in press. Kaplow, L., Blood 26, 2, 1965. Zigmond, S. H., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. Shields, J. M., and Wilkinson, P. C., Clin. Exp. Immunol. 38, 598, 1979. McCarty, J., and Goetzl, E. J., Immunology 43, 103, 1979. O’Neill, G. J., and Parrott, M. V., Cell. Immunof.33, 257, 1977. Wilkinson, P. C., Roberts, J. A., Russell, R. J., and McLaughlin, M., Clin. Exp. Immunol. 25, 280, 1976.