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The patterns and distribution of euchrysine-binding grains in nonstimulated human peripheral T and non-T lymphocytes
CELLULAR
IMMUNOLOGY
42, 170- 176 (1979)
SHORT COMMUNICATIONS The Patterns and Distribution of Euchrysine-Binding Grains in Nonstimulated Human Peripheral T and Non-T Lymphocytes1 BOGDAN MDZEWSKI,* *Department
of Laboratory
02497, and t Defiartment
KRYSTYNA SWIERKOWSKA,~ EUGENIA KLING,~ JAN STEFFEN t Diagnostics, Postgraduate Centre of Immunology, M. SklodowskaXurie Oncology, Warsaw 00-973, Poland Received
of Medical Memorial
AND
School, Warsazv Institute of
June 15,1978
The patterns of supravital staining with euchrysine, a fluorescent stain thought to bind selectively to lysosomal membranes, were evaluated in resting human lymphocytes separated on the basis of their ability to form spontaneous rosettes with sheep red blood cells in thymus-dependent (T) and thymus-independent (non-T) subpopulations. Two basic staining patterns were found in unseparated lymphocyte populations: type I, small fluorescent granules in a conglomerate form, usually located in a single spot close to the cell membrane; type II, discrete fluorescent granules dispersed over the entire cytoplasm. The overwhelming majority of non-T lymphocytes displayed the type II pattern. Within the T-cell subpopulations both type I and type II patterns were found in proportions which were subject to donor-to-donor variability.
INTRODUCTION Human peripheral lymphocytes stained supravitally with euchrysine, a stain thought to bind preferentially to the membranes of lysosomes (l), display one of the two basic fluorescence patterns: a single conglomerate of fluorescent granules situated close to the cell membrane (type I pattern) or many fine granules dispersed within the entire cytoplasm (type II pattern) (2). In our previous study on lymphocytes from patients with chronic lymphocytic leukemia (CLL) we found a predominance of cells displaying type II euchrysine staining patterns (3). Since it is known that CLL in most instances is a monoclonal proliferation of B lymphocytes, the question was posed whether the above fluorescence pattern is also typical for normal peripheral B lymphocytes. This prompted us to study the euchrysine staining patterns of human peripheral lymphocytes from normal donors, separated into thymus-dependent (T) and thymus-independent (non-T) subpopulations on the basis of the ability of the former to form spontaneous rosettes with sheep red blood cells. 1 Supported by Grants 0219 and 1302 from the Polish National Cancer Program PR-6. 170
FIG. 1. A preparation of unseparated human peripheral lymphocytes stained supravitally indicate three types of fluorescent staining patterns. X1000.
with euchrysine by the method of Blume et al. (8). Arrows 5
172
SHORT
COMMUNICATIONS
MATERIALS
AND
METHODS
Mononuclear leukocytes were separated from the heparinized blood of 13 healthy donors following centrifugation on Ficoll-Uropoline (a mixture of sodium amidotrizoate and N-methyl gluconium amidotrizoate) density 1.078, in principle as described by B#yum (4). Phagocytic cells were removed from the suspension of leukocytes in Hanks’ solution following incubation with carbonyl-iron and application of a strong magnet (5). Separation of T from non-T lymphocytes, based on the ability of the former to bind spontaneously sheep red blood cells (SRBC), was carried out in principle as described by Wybran et al. (6), usually in a two-step procedure. In the first step 1 X lo7 lymphocytes, washed three times in Hanks’ solution containing EDTA (0.04 M for the first wash and 0.0027 it4 for the second and third washes), suspended in 1 ml of calf serum, and mixed with 1 ml of a lo/O suspension of SRBC in Hanks’ solution, were centrifuged at 5Og, kept 20 min at room temperature, and, following centrifugation on Ficoll-Uropoline at 950 g, separated into bottdm and interface fractions. SRBC in the bottom fraction were lysed by a 0.83% solution of ammonium chloride. The second step was performed as described above, only with interface cells but the mixture of lymphocytes and SRBC was kept 1 hr at 0 to 4°C prior to centrifugation on Ficoll-Uropoline and only second interface cells were collected. The percentages of cells forming spontaneous rosettes with SRBC and the percentages of those binding FITC-conjugated polyvalent rabbit anti-human Ig after a 30-min preincubation at 37°C (7) were determined in the unseparated, bottom (T) , and second interface (non-T) lymphocyte populations. The staining of living cells was performed with euchrysine 3RX Gurr-London, following three washes in Hanks’ solution of unseparated, T, and non-T lymphocytes, according to the method of Blume et al. (8). Powdered aminoacridine euchrysine was dissolved in 0.01 M phosphate-buffered saline (pH 7.4) and diluted in phenol red-free Eagle’s Spinner minimal essential medium to a final concentration of 1 pg/ml. This solution, in a volume of 0.4 ml, was added to a l-ml suspension of 1 x lo6 lymphocytes. Following a 30-min incubation at 37°C in darkness, the cells were spread on glass slides using a cytocentrifuge and examined with a Leitz fluorescent microscope equipped with a halogen illuminator HBO 200, a dark-field condenser, and a BG 12/3 mm filter as well as a yellow barrier filter. RESULTS Three types of staining patterns were distinguished on the basis of the number, distribution, and morphology of the euchrysine-binding granules in preparations of unseparated lymphocytes (Fig. 1 and Table 1) . Type 1: Small, orange fluorescent granules in a single conglomerate form, usually placed close to the cell membrane (mean : 42.9% ; range : 31.5-40.0%). Type II: Numerous, discrete orange fluorescent granules, dispersed:all over the cytoplasm (mean : 53.7% ; range : 46.6-62.5 % ) . Type III: Several large orange fluorescent granules, usually in clusters, dispersed throughout the whole cytoplasm (mean : 3.4% ; range : 0.4-6.8% ) . Similarly, in the subpopulation of T cells all three types of staining patterns were
80.5 73.0 77.5 74.5 79.0 74.0 64.0 75.0 67.5 74.0 61.0 77.0 78.0
73.5
Mean
E-RFC (%I
1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment
13.3
12.4 15.0 18.0 14.0 14.0 16.0 14.8 14.0 13.0 ND 9.0 11.0 9.0
SIg+ cells !%)
of Unseparated Type
42.9
36.6 40.0 46.6 31.5 47.8 45.8 43.0 46.3 42.3 48.0 43.0 39.0 47.5
I
53.7
56.8 55.0 46.6 62.5 51.8 52.6 54.8 52.7 55.3 49.0 52.3 60.0 49.0
II
3.4
6.6 5.0 6.8 6.0 0.4 1.6 2.2 1.0 2.4 3.0 4.7 1.0 3.5
III
Cells (ye) exhibiting specific type of euchrysine binding
lymphocytes
Frequencies
Unseparated
The Relative
1
91.4
96.0 97.0 90.5 92.3 93.0 84.0 96.0 92.0 90.7 92.0 76.0 95.0 94.0
E-RFC (%I
1.0
0.5 1.5 2.0 2.8 5.0 1.0 0.0 1.0 1.0 ND 6.0 0.5 0.5
SIg+ cells (%I
51.6
21.0 53.0 60.3 53.0 58.8 53.8 53.8 53.5 57.2 60.0 49.6 39.5 55.0
I
45.1
77.5 37.0 35.2 43.0 39.4 43.2 45.0 44.5 38.0 40.0 45.6 57.5 40.5
II
3.4
1.5 10.0 4.5 4.0 1.8 1.0 1.2 2.0 4.8 0.0 4.8 3.0 5.0
III
Cells (70) exhibiting specific type of euchrysine binding
T lymphocytes
Human Peripheral Lymphocytes and Separated I, II, and III Euchrysine Binding Patterns
TABLE
8.0
5.0 6.5 6.5 4.0 7.0 4.0 0.5 4.0 8.0 1.5 6.0 7.0 13.5
E-RFC (%)
T and Non-T
3.0 2.0 1.5 13.1 5.5 15.0 11.0 5.0 5.0
50.5 50.0 53.0 41.5 34.0 ND 16.0 28.0 34.0
5.4
2.6 3.0 1.5
39.5 28.0 46.0
40.6
2.3
I
91.2
94.4 98.0 95.0 83.0 89.0 79.0 87.3 90.0 91.5
91.4 97.0 95.5
94.4
II
3.4
2.6 0.0 3.5 4.0 5.5 6.0 1.7 5.0 3.5
6.0 0.0 3.0
3.5
III
Cells (ye) exhibiting specific type of euchrysine binding
lymphocytes
66.0
SIg+ cells (%I
Non-T
Cells Displaying
‘
z ;: 3 ;; 1: cn
s
?I n \
FIG. 2. The patterns of fluorescence of human peripheral non-T lymphocytes following supravital staining with euchrysine. All cells display numerous discrete fluorescent granules scattered all over the cytoplasm. X900.
SHORT
COMMUNICATIONS
175
encountered, though in slightly different proportions (Table I) : type I, 51.6% (range: 2160.3%) ; type II, 45.1% (range: 35.2-77.5s) ; type III, 3.4% (range: O&10.0%). The considerable variability in the relative frequencies of type I and type II cells in the T-lymphocyte subpopulations of different donors could not be accounted for by different admixture of cells which did not form the E-rosettes. Within the subpopulation of non-T cells type II patterns were predominant (mean: 91.2% ; range: 79.0-9&O%), whereas only a low percentage of cells displayed either type I (mean: 5.4% ; range: 1.5-15.0%) or type III (mean: 3.4%; range : O&6.0%) staining patterns (Fig. 2 and Table 1). In the majority of experiments the relative frequency of type I patterns in the non-T-cell population was lower than the percentage of E rosette-forming cells which have not been removed by the separation procedure. DISCUSSION The results reported above demonstrate that, of the two basic patterns of euchrysine staining of human peripheral lymphocytes, type I is displayed by the overwhelming majority of non-T cells, consisting of true B lymphocytes which express easily detectable surface immunoglobulins following preincubation at 37°C and cells which in absence of this marker display the Fc fragment of immunoglobulins (9, IO) ; both types I and II are present in roughly equal, though donorto-donor variable, proportions in the population of T lymphocytes. The more dispersed and coarse type III staining pattern, which was found in a variable but always minor fraction of cells in both the T and non-T subpopulations, was always associated with cells displaying monocyte morphology in preparations from the whole blood. We assume therefore that resting cells displaying this pattern of euchrysine staining belong to the macrophage-monocyte lineage, since it has been demonstrated that part of these cells in peripheral blood does not display phagocytic properties (11). It was demonstrated that the distribution of euchrysine-binding grains corresponds to the sites of acid phosphatase activity and, as visualized by electron microscopy, the stain is associated with cytoplasmic membrane-bound bodies and therefore believed to bind preferentially to membranes of lysosomes (1) . However the distribution of different types of euchrysine binding patterns among cells of the purified peripheral T- and non-T-lymphocyte subpopulations does not correspond with the patterns of lysosomal enzyme activities so far investigated. In particular, nonspecific cy-naphtyl esterase activity, which has been reported to be a useful marker for mature T lymphocytes (ll-14), is present in at least 80 to 90% of these cells in peripheral blood and disappears during the first 2 days in mitogenstimulated cultures (13)) whereas type I and II euchrysine staining patterns are encountered in roughly equal proportions in nonactivated peripheral T lymphocytes. Following stimulation by PHA, Con A, and PWM most cells undergoing blast transformation display after 48 hr the more dispersed and coarse type III pattern of staining (unpublished observations). Of the two other lysosomal enzymes so far studied both p-glucuronidase and acid phosphatase activity are present in roughly equal numbers of T and B cells from human peripheral blood (14). The relationships between the types of euchrysine staining patterns and the known heterogeneity of peripheral T lymphocytes, in terms of their immunological function and membrane markers (15) and/or in viva life span and potential to
176
SHORT
COMMUNICATIONS
respond to non-specific mitogens (16), are at present unknown. Evaluation of relationships between the euchrysine staining patterns and the in vitro proliferative potential of T lymphocytes might be of particular interest, since lysosomal changes are closely related to subsequent blast transformation induced in vitro by nonspecific mitogens (17, 18) and only a donor-to-donor variable fraction of human peripheral T lymphocytes is responsive to these stimuli (16). REFERENCES 1. Biberfeld, P., Acta Patlzol. Microbial. Scand. A 223 (Suppl.), 1, 1971. 2. Allison, A. C., and Young, M. R., Life Sci. 3, 1407, 1974. 3. Mdzewski, B., Diagn. Lab. 13, 6, 1977. 4. Bplyum, A., Stand. J. Clin. Lab. Invest. 21, (Suppl. 97), 1968. 5. Kuper, S. A. W., Bignall, J. R., and Luckcock, E. D., Lancet 1, 852, 1961. 6. Wybran, J., Chantler, S., and Fudenberg, H. H., Lancet 1, 126, 1973. 7. Lobo, P., Westervelt, F. B., and Horwitz, D. A., /. Immunol. 114, 116, 1975. 8. Blume, R. S., Glade, P. R., and Chessin, L. N., Blood 33, 1, 1969. 9. Horwitz, D. A., and Lobo, P., J. Clin. Invest. 56, 1464, 1975. 10. Wisl#ff, F., Frfiland, S. S., and Michaelsen, T. E., Znt. Arch. Allergy 47, 139, 1974. 11. Horwitz, D. A., Allison, A. C., Ward, P., and Kight, N., Cl&. Exp. Immunol. 30, 289, 1977.
12. Mueller, J., Brun de1 Re, G., Buerki, H., Keller, H. II., Hess, M. W., and Cottier, H., Eur. J. Immunol. 5, 270, 1975. 13. Kulenkampff, H. J., Janossy, G., and Greaves, M. F., B&t. J. Haematol. 36, 231, 1977. 14. Pangalis, G. A., Waldman, S. R., and Rappaport, H., Amer. J. Clin. Pathol. 69, 314, 1978. 15. Shiku, H., Kisielow, P., Boyse, E. A., and Oetgen, H. F., Transplant. Rev. 8, 381, 1976. 16. Steffen, J. A., Swierkowska, K., Michalowski, A., Kling, E., and Nowakowska, A., In “Mutagen-Induced Chromosome Damage in Man.” (H. J. Evans and D. C. Lloyd, Eds.) , pp. 89-107. Edinburgh Univ. Press, Edinburgh, 1978. 17. Hirschhorn, R., Brittinger, G., Hirschhorn, K., and Weissmann, G., J. Cell. Biol. 37, 412, 1968. 18. Nadler, H. L., Dowben, R. M., and Hsia, D. Y. Y., Blood 34, 52, 1969.
IMMUNOLOGY
42, 170- 176 (1979)
SHORT COMMUNICATIONS The Patterns and Distribution of Euchrysine-Binding Grains in Nonstimulated Human Peripheral T and Non-T Lymphocytes1 BOGDAN MDZEWSKI,* *Department
of Laboratory
02497, and t Defiartment
KRYSTYNA SWIERKOWSKA,~ EUGENIA KLING,~ JAN STEFFEN t Diagnostics, Postgraduate Centre of Immunology, M. SklodowskaXurie Oncology, Warsaw 00-973, Poland Received
of Medical Memorial
AND
School, Warsazv Institute of
June 15,1978
The patterns of supravital staining with euchrysine, a fluorescent stain thought to bind selectively to lysosomal membranes, were evaluated in resting human lymphocytes separated on the basis of their ability to form spontaneous rosettes with sheep red blood cells in thymus-dependent (T) and thymus-independent (non-T) subpopulations. Two basic staining patterns were found in unseparated lymphocyte populations: type I, small fluorescent granules in a conglomerate form, usually located in a single spot close to the cell membrane; type II, discrete fluorescent granules dispersed over the entire cytoplasm. The overwhelming majority of non-T lymphocytes displayed the type II pattern. Within the T-cell subpopulations both type I and type II patterns were found in proportions which were subject to donor-to-donor variability.
INTRODUCTION Human peripheral lymphocytes stained supravitally with euchrysine, a stain thought to bind preferentially to the membranes of lysosomes (l), display one of the two basic fluorescence patterns: a single conglomerate of fluorescent granules situated close to the cell membrane (type I pattern) or many fine granules dispersed within the entire cytoplasm (type II pattern) (2). In our previous study on lymphocytes from patients with chronic lymphocytic leukemia (CLL) we found a predominance of cells displaying type II euchrysine staining patterns (3). Since it is known that CLL in most instances is a monoclonal proliferation of B lymphocytes, the question was posed whether the above fluorescence pattern is also typical for normal peripheral B lymphocytes. This prompted us to study the euchrysine staining patterns of human peripheral lymphocytes from normal donors, separated into thymus-dependent (T) and thymus-independent (non-T) subpopulations on the basis of the ability of the former to form spontaneous rosettes with sheep red blood cells. 1 Supported by Grants 0219 and 1302 from the Polish National Cancer Program PR-6. 170
FIG. 1. A preparation of unseparated human peripheral lymphocytes stained supravitally indicate three types of fluorescent staining patterns. X1000.
with euchrysine by the method of Blume et al. (8). Arrows 5
172
SHORT
COMMUNICATIONS
MATERIALS
AND
METHODS
Mononuclear leukocytes were separated from the heparinized blood of 13 healthy donors following centrifugation on Ficoll-Uropoline (a mixture of sodium amidotrizoate and N-methyl gluconium amidotrizoate) density 1.078, in principle as described by B#yum (4). Phagocytic cells were removed from the suspension of leukocytes in Hanks’ solution following incubation with carbonyl-iron and application of a strong magnet (5). Separation of T from non-T lymphocytes, based on the ability of the former to bind spontaneously sheep red blood cells (SRBC), was carried out in principle as described by Wybran et al. (6), usually in a two-step procedure. In the first step 1 X lo7 lymphocytes, washed three times in Hanks’ solution containing EDTA (0.04 M for the first wash and 0.0027 it4 for the second and third washes), suspended in 1 ml of calf serum, and mixed with 1 ml of a lo/O suspension of SRBC in Hanks’ solution, were centrifuged at 5Og, kept 20 min at room temperature, and, following centrifugation on Ficoll-Uropoline at 950 g, separated into bottdm and interface fractions. SRBC in the bottom fraction were lysed by a 0.83% solution of ammonium chloride. The second step was performed as described above, only with interface cells but the mixture of lymphocytes and SRBC was kept 1 hr at 0 to 4°C prior to centrifugation on Ficoll-Uropoline and only second interface cells were collected. The percentages of cells forming spontaneous rosettes with SRBC and the percentages of those binding FITC-conjugated polyvalent rabbit anti-human Ig after a 30-min preincubation at 37°C (7) were determined in the unseparated, bottom (T) , and second interface (non-T) lymphocyte populations. The staining of living cells was performed with euchrysine 3RX Gurr-London, following three washes in Hanks’ solution of unseparated, T, and non-T lymphocytes, according to the method of Blume et al. (8). Powdered aminoacridine euchrysine was dissolved in 0.01 M phosphate-buffered saline (pH 7.4) and diluted in phenol red-free Eagle’s Spinner minimal essential medium to a final concentration of 1 pg/ml. This solution, in a volume of 0.4 ml, was added to a l-ml suspension of 1 x lo6 lymphocytes. Following a 30-min incubation at 37°C in darkness, the cells were spread on glass slides using a cytocentrifuge and examined with a Leitz fluorescent microscope equipped with a halogen illuminator HBO 200, a dark-field condenser, and a BG 12/3 mm filter as well as a yellow barrier filter. RESULTS Three types of staining patterns were distinguished on the basis of the number, distribution, and morphology of the euchrysine-binding granules in preparations of unseparated lymphocytes (Fig. 1 and Table 1) . Type 1: Small, orange fluorescent granules in a single conglomerate form, usually placed close to the cell membrane (mean : 42.9% ; range : 31.5-40.0%). Type II: Numerous, discrete orange fluorescent granules, dispersed:all over the cytoplasm (mean : 53.7% ; range : 46.6-62.5 % ) . Type III: Several large orange fluorescent granules, usually in clusters, dispersed throughout the whole cytoplasm (mean : 3.4% ; range : 0.4-6.8% ) . Similarly, in the subpopulation of T cells all three types of staining patterns were
80.5 73.0 77.5 74.5 79.0 74.0 64.0 75.0 67.5 74.0 61.0 77.0 78.0
73.5
Mean
E-RFC (%I
1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment
13.3
12.4 15.0 18.0 14.0 14.0 16.0 14.8 14.0 13.0 ND 9.0 11.0 9.0
SIg+ cells !%)
of Unseparated Type
42.9
36.6 40.0 46.6 31.5 47.8 45.8 43.0 46.3 42.3 48.0 43.0 39.0 47.5
I
53.7
56.8 55.0 46.6 62.5 51.8 52.6 54.8 52.7 55.3 49.0 52.3 60.0 49.0
II
3.4
6.6 5.0 6.8 6.0 0.4 1.6 2.2 1.0 2.4 3.0 4.7 1.0 3.5
III
Cells (ye) exhibiting specific type of euchrysine binding
lymphocytes
Frequencies
Unseparated
The Relative
1
91.4
96.0 97.0 90.5 92.3 93.0 84.0 96.0 92.0 90.7 92.0 76.0 95.0 94.0
E-RFC (%I
1.0
0.5 1.5 2.0 2.8 5.0 1.0 0.0 1.0 1.0 ND 6.0 0.5 0.5
SIg+ cells (%I
51.6
21.0 53.0 60.3 53.0 58.8 53.8 53.8 53.5 57.2 60.0 49.6 39.5 55.0
I
45.1
77.5 37.0 35.2 43.0 39.4 43.2 45.0 44.5 38.0 40.0 45.6 57.5 40.5
II
3.4
1.5 10.0 4.5 4.0 1.8 1.0 1.2 2.0 4.8 0.0 4.8 3.0 5.0
III
Cells (70) exhibiting specific type of euchrysine binding
T lymphocytes
Human Peripheral Lymphocytes and Separated I, II, and III Euchrysine Binding Patterns
TABLE
8.0
5.0 6.5 6.5 4.0 7.0 4.0 0.5 4.0 8.0 1.5 6.0 7.0 13.5
E-RFC (%)
T and Non-T
3.0 2.0 1.5 13.1 5.5 15.0 11.0 5.0 5.0
50.5 50.0 53.0 41.5 34.0 ND 16.0 28.0 34.0
5.4
2.6 3.0 1.5
39.5 28.0 46.0
40.6
2.3
I
91.2
94.4 98.0 95.0 83.0 89.0 79.0 87.3 90.0 91.5
91.4 97.0 95.5
94.4
II
3.4
2.6 0.0 3.5 4.0 5.5 6.0 1.7 5.0 3.5
6.0 0.0 3.0
3.5
III
Cells (ye) exhibiting specific type of euchrysine binding
lymphocytes
66.0
SIg+ cells (%I
Non-T
Cells Displaying
‘
z ;: 3 ;; 1: cn
s
?I n \
FIG. 2. The patterns of fluorescence of human peripheral non-T lymphocytes following supravital staining with euchrysine. All cells display numerous discrete fluorescent granules scattered all over the cytoplasm. X900.
SHORT
COMMUNICATIONS
175
encountered, though in slightly different proportions (Table I) : type I, 51.6% (range: 2160.3%) ; type II, 45.1% (range: 35.2-77.5s) ; type III, 3.4% (range: O&10.0%). The considerable variability in the relative frequencies of type I and type II cells in the T-lymphocyte subpopulations of different donors could not be accounted for by different admixture of cells which did not form the E-rosettes. Within the subpopulation of non-T cells type II patterns were predominant (mean: 91.2% ; range: 79.0-9&O%), whereas only a low percentage of cells displayed either type I (mean: 5.4% ; range: 1.5-15.0%) or type III (mean: 3.4%; range : O&6.0%) staining patterns (Fig. 2 and Table 1). In the majority of experiments the relative frequency of type I patterns in the non-T-cell population was lower than the percentage of E rosette-forming cells which have not been removed by the separation procedure. DISCUSSION The results reported above demonstrate that, of the two basic patterns of euchrysine staining of human peripheral lymphocytes, type I is displayed by the overwhelming majority of non-T cells, consisting of true B lymphocytes which express easily detectable surface immunoglobulins following preincubation at 37°C and cells which in absence of this marker display the Fc fragment of immunoglobulins (9, IO) ; both types I and II are present in roughly equal, though donorto-donor variable, proportions in the population of T lymphocytes. The more dispersed and coarse type III staining pattern, which was found in a variable but always minor fraction of cells in both the T and non-T subpopulations, was always associated with cells displaying monocyte morphology in preparations from the whole blood. We assume therefore that resting cells displaying this pattern of euchrysine staining belong to the macrophage-monocyte lineage, since it has been demonstrated that part of these cells in peripheral blood does not display phagocytic properties (11). It was demonstrated that the distribution of euchrysine-binding grains corresponds to the sites of acid phosphatase activity and, as visualized by electron microscopy, the stain is associated with cytoplasmic membrane-bound bodies and therefore believed to bind preferentially to membranes of lysosomes (1) . However the distribution of different types of euchrysine binding patterns among cells of the purified peripheral T- and non-T-lymphocyte subpopulations does not correspond with the patterns of lysosomal enzyme activities so far investigated. In particular, nonspecific cy-naphtyl esterase activity, which has been reported to be a useful marker for mature T lymphocytes (ll-14), is present in at least 80 to 90% of these cells in peripheral blood and disappears during the first 2 days in mitogenstimulated cultures (13)) whereas type I and II euchrysine staining patterns are encountered in roughly equal proportions in nonactivated peripheral T lymphocytes. Following stimulation by PHA, Con A, and PWM most cells undergoing blast transformation display after 48 hr the more dispersed and coarse type III pattern of staining (unpublished observations). Of the two other lysosomal enzymes so far studied both p-glucuronidase and acid phosphatase activity are present in roughly equal numbers of T and B cells from human peripheral blood (14). The relationships between the types of euchrysine staining patterns and the known heterogeneity of peripheral T lymphocytes, in terms of their immunological function and membrane markers (15) and/or in viva life span and potential to
176
SHORT
COMMUNICATIONS
respond to non-specific mitogens (16), are at present unknown. Evaluation of relationships between the euchrysine staining patterns and the in vitro proliferative potential of T lymphocytes might be of particular interest, since lysosomal changes are closely related to subsequent blast transformation induced in vitro by nonspecific mitogens (17, 18) and only a donor-to-donor variable fraction of human peripheral T lymphocytes is responsive to these stimuli (16). REFERENCES 1. Biberfeld, P., Acta Patlzol. Microbial. Scand. A 223 (Suppl.), 1, 1971. 2. Allison, A. C., and Young, M. R., Life Sci. 3, 1407, 1974. 3. Mdzewski, B., Diagn. Lab. 13, 6, 1977. 4. Bplyum, A., Stand. J. Clin. Lab. Invest. 21, (Suppl. 97), 1968. 5. Kuper, S. A. W., Bignall, J. R., and Luckcock, E. D., Lancet 1, 852, 1961. 6. Wybran, J., Chantler, S., and Fudenberg, H. H., Lancet 1, 126, 1973. 7. Lobo, P., Westervelt, F. B., and Horwitz, D. A., /. Immunol. 114, 116, 1975. 8. Blume, R. S., Glade, P. R., and Chessin, L. N., Blood 33, 1, 1969. 9. Horwitz, D. A., and Lobo, P., J. Clin. Invest. 56, 1464, 1975. 10. Wisl#ff, F., Frfiland, S. S., and Michaelsen, T. E., Znt. Arch. Allergy 47, 139, 1974. 11. Horwitz, D. A., Allison, A. C., Ward, P., and Kight, N., Cl&. Exp. Immunol. 30, 289, 1977.
12. Mueller, J., Brun de1 Re, G., Buerki, H., Keller, H. II., Hess, M. W., and Cottier, H., Eur. J. Immunol. 5, 270, 1975. 13. Kulenkampff, H. J., Janossy, G., and Greaves, M. F., B&t. J. Haematol. 36, 231, 1977. 14. Pangalis, G. A., Waldman, S. R., and Rappaport, H., Amer. J. Clin. Pathol. 69, 314, 1978. 15. Shiku, H., Kisielow, P., Boyse, E. A., and Oetgen, H. F., Transplant. Rev. 8, 381, 1976. 16. Steffen, J. A., Swierkowska, K., Michalowski, A., Kling, E., and Nowakowska, A., In “Mutagen-Induced Chromosome Damage in Man.” (H. J. Evans and D. C. Lloyd, Eds.) , pp. 89-107. Edinburgh Univ. Press, Edinburgh, 1978. 17. Hirschhorn, R., Brittinger, G., Hirschhorn, K., and Weissmann, G., J. Cell. Biol. 37, 412, 1968. 18. Nadler, H. L., Dowben, R. M., and Hsia, D. Y. Y., Blood 34, 52, 1969.