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The effect of thymosin on glucocorticoid receptors in lymphoid cells
CELLULAR
IMMUNOLOGY
60,
376-385
(1981)
The Effect of Thymosin on Glucocorticoid in Lymphoid Cells PHYLLIS
Receptors
L. OSHEROFF
Biopolymer Research Department, Central Research Division, Hoffmann-LaRoche Nutley, New Jersey 07110
Inc.,
Received June 26, 1980: accepted November 25, 1980 The present study investigates the effect of a partially purified thymus factor, thymosin Fraction 5, and an homogeneous polypeptide component of Fraction 5, thymosin a,, on glucocorticoid resistance and glucocorticoid receptors in mouse thymocytes. Treatment of thymocytes in vitro with thymosin Fraction 5 or (Y, results in an increase in the percentage of glucocorticoid-resistant cells. Studies on the specific whole-cell binding of [‘Hldexamethasone and steroid competition experiments demonstrate the existence of high-affinity (& = I.0 X IO-” IV) specific glucocorticoid receptors in mouse thymocytes. Preincubation with thymosin Fraction 5 or (Y, appears to cause a reduction in the specific [3H]dexamethasone binding to intact thymocytes.
INTRODUCTION Numerous studies have suggested a role for a variety of thymic humoral factors or hormones in the maturation of thymus-derived lymphocytes (T cells). These studies suggested that the thymic hormones control the proliferation of precursor T cells in the bone marrow (1) and the subsequent differentiation and maturation of T cells in the thymus and peripheral lymphoid tissues (for reviews, see (2)). The various populations of cells which develop during T-cell maturation can be characterized by their sensitivity to killing by glucocorticoids. The precursors of T cells which have the capacity to repopulate the thymus after irradiation are relatively, but not completely, resistant to glucocorticoids (3, 4). The immunologically immature T cells, which are present mainly in the thymus cortex, are killed by glucocorticoids. The more mature, immunocompetent T cells, located primarily in the thymus medulla, are mostly resistant to glucocorticoids in vivo (5, 6); although it has been shown that the residual thymocyte population, isolated 48 hr after in vivo hydrocortisone injection, is partly sensitive to steroid treatment in vitro, as demonstrated by uridine incorporation inhibition (7). Biochemical studies have strongly suggested that many physiological responses to the glucocorticoid hormones are mediated through specific, intracellular binding proteins or receptors. In a generally accepted molecular mechanism the steroid binds strongly to the receptor in the cytoplasm of the target cell and acts as an allosteric effector molecule to increase the affinity of the receptor for nuclear binding sites. The interaction of the steroid-receptor complex with the genome then 376 OOOS-8749/81/080376-lOSOZ.OO/O Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
EFFECT
OF
THYMOSIN
ON
GLUCOCORTICOID
RECEPTORS
377
triggers the biological responses (for reviews see (8-10)). Using a cultured line of glucocorticoid-sensitive mouse lymphoma cells and their steroid-resistant variant clones, Tomkins and his colleagues (1 I-14) demonstrated that a number of clones resistant to steroid-induced cell death showed diminished numbers of glucocorticoid receptors or impaired translocation of the glucocorticoid receptor complex into the nucleus. On the other hand, Smith et al. (15) reported that in cultured human peripheral lymphocytes, mitogen-activated blast transformation was associated with a large increase in glucocorticoid-binding sites per cell, yet lymphocytes were equally sensitive to glucocorticoid inhibition of certain metabolic parameters (glucose transport, nucleoside incorporation), regardless of their state of activation. These observations have recently been extended to in uiwo antigen-stimulated lymph node lymphocytes ( 16). Lippman and Barr ( 17) and Fauci et al. ( 18) both reported a lack of direct correlation between cytoplasmic glucocorticoid receptors and the differential in vivo effects of glucocorticoids on the recirculation of lymphocyte subpopulations. It has been shown that preincubation (1 hr) or coincubation (2-10 hr) of mouse thymocytes with a thymic humoral factor leads to an increased number of hydrocortisone-resistant cells ( 19). Similar results were reported in 2- to 6-hr thymocyte cultures using a human serum thymic factor (20), or thymic epithelial culture supernatants (21). Bach et al. (22) reported that a serum thymic factor caused a 60-70s inhibition of steroid binding when thymocyte cytosol was incubated directly with the serum factor. Since incubation with the serum factor was carried out under cell-free conditions the observed decrease in steroid binding bore no relationship to the cell maturation process. It is possible that the reduced steroid binding was merely the result of nonspecific interference by the serum factor. In the present report we have attempted to explore the possible role of a partially purified thymic factor, thymosin Fraction 5 (23), and a homogeneous polypeptide component of Fraction 5, (Y~(24) in T-cell maturation by studying their effects on glucocorticoid resistance and glucocorticoid receptors of intact mouse thymus cells. The results suggest that thymosin Fraction 5 and (Y~may alter glucocorticoid resistance as well as glucocorticoid receptor activity in these thymus cells. MATERIALS
AND
METHODS
Mice Six- to ten-week-old male C57BL/6 were obtained from Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts. Intact mice were used in all experiments. Preparation
of Cell Suspensions
Mouse thymuses were removed aseptically and washed twice in HBSS. Thymocyte suspensions were prepared by gentle hand homogenization in a loose-fitting Dounce tissue grinder in HBSS, centrifuged (SOOg, 8 min), washed twice in HBSS at 0-4°C and resuspended in cold medium I (RPM1 1640 (Grand Island Biological Co. Grand Island, N.Y.) supplemented with 50 units/ml penicillin, 50 pg/ml streptomycin, 2 mM glutamine, and 10 mM Hepes (Calbiochem, La Jolla, Calif.). Cells were filtered through a metal sieve and kept on ice before incubation with thymosin.
378
PHYLLIS
L. OSHEROFF
Preparation of Calf and Mouse Thymosin Fractions
Calf thymosin Fraction 5(F-5) (23) used in these experiments was prepared by Dr. W. C. McGregor and co-workers, Biopolymer Research Department, Hoffmann-La Roche Inc., Nutley, New Jersey. Fraction 5 is purified from Fraction 3 (F-3, obtained by heat treatment (8O’C) of thymic tissue homogenates followed by acetone precipitation of the heated supernate) by ammonium sulfate precipitation (to 50% saturation), ultrafiltration of the solubilized precipitate, and desalting of the ultrafiltrate on Sephadex G-25 followed by lyophilization. The same procedures were used for the preparation of calf splenic F-5. Thymosin (Y~polypeptide is an homogenous acidic component of F-5 and contains 28 amino acid residues, the sequence of which is known (24). The thymosin o1 used in the present experiments was synthesized and kindly provided by Dr. S. S. Wang (Dept. of BioOrganic Chemistry, Hoffman-La Roche Inc., present address: Penninsula Laboratories, Inc., San Carlos, Calif., 94070). Mouse splenic F-3 was prepared as for calf F-3 from spleens obtained aseptically. All preparations were sterilized by filtration (0.22-Hrn Swinnex-13, Millipore Corp., Bedford, Mass.) before use. Preincubation with Thymosin and Assay for Specific Steroid Binding by Whole Cells
Preincubation of thymus cells with various concentrations of thymosin was carried out at 37°C in a humidified atmosphere of 5% C02-95% air for various periods of time as indicated under Results. The samples were prepared in 60 X 15-mm petri dishes in a total volume of 4 ml at 5-7.5 X lo6 cells/ml in medium I. Samples were prepared in duplicate for measuring total [3H]dexamethasone (dex) binding and nonspecific [3H]dex binding by whole cells. The assay for specific steroid binding by whole cells was modified from that of Sibley and Tomkins (12) and was carried out as follows. After incubation with thymosin, duplicate cultures were harvested in Falcon 12 X 75-mm polyethylene tubes and aliquots counted for viable cells by trypan blue exclusion in a hemacytometer. The cells were centrifuged (all centrifugations were carried out for 8 min at 800g) and washed three times with 3 ml of HBSS at 04°C. The cells were resuspended in 1 ml of medium I and 5 X 10m9 M [3H]dex (36-41 Ci/mmol, New England Nuclear, Boston, Mass., or 26 Ci/mmol, Amersham, Arlington Heights, Ill.) was added to each of the duplicate cultures. One of these cultures also received a lOOO-fold excess (5 X 10e6 M) of unlabeled dex (Sigma Chemical Co., St. Louis, MO.) to determine specific [3H]dex binding. This was taken as the difference in bound [‘H]dex between cells incubated in the absence (total) and presence (nonspecific) of unlabeled dex. After incubation at 37’C in a humidified atmosphere of 5% CO*--95% air for 45 min (an extended incubation period of 90 min did not result in increased binding), the cells were centrifuged, washed twice with 3 ml of HBSS at @-4”C resuspended in 23°C HBSS, and left to stand at room temperature for 10 min before centrifugation to dissociate most of the nonspecifically bound dex (25). The samples were resuspended in 0.2 ml of Dulbecco’s phosphate-buffered saline (PBS) and transferred to scintillation vials. The polyethylene tubes were rinsed twice with 0.2 ml PBS and the rinsings were combined in the scintillation vials. Biofluor (New England Nuclear) was added and the samples were counted in a Beckman LS-250 or Packard Tricarb liquid scintillation counter.
EFFECT OF THYMOSIN
ON GLUCOCORTICOID
RECEPTORS
379
Preincubation with Thymosin and Assay for Glucocorticoid Resistance of Mouse Thymocytes
Mouse thymocytes were resuspended in medium I at 5 to 10 X lo6 cells/ml. Preincubation with thymosin was carried out as described above for the steroidbinding experiments. Samples were prepared in 35 X IO-mm petri dishes in a total volume of 1.5 ml in quadruplicate, duplicate each for the determination of cell survival in the presence and absence of glucocorticoids. Following preincubation with thymosin for approximately 16 hr, hydrocortisone (HC, Sigma Chemical Co.) or dex was added to half of the quadruplicate cultures to a final concentration of 10e5 M (final concentrations of 10e6-5 X lop5 M had similar effect) and incubation was continued for periods of 4-10 hr. At the end of the incubation period, cultures were harvested into 12 X 75-mm Falcon polyethylene tubes. An aliquot (75-100 ~1) of each sample was diluted in 2 ml of PBS containing bovine serum albumin (crystallized, Miles Laboratories, Elkart, Ind.) at 1 mg/ml and 0.05% trypan blue (Grand Island Biological Co.) and counted immediately in a Model 6300A Cytograf cell counter (Bio/Physics Systems, Inc., presently Ortho Instruments) which gives the total number of cells and the percentage of cells excluding trypan blue. Numbers of viable cells (those excluding trypan blue) was calculated and percentage relative cell survival was expressed as: (no. of viable cells per ml in presence of HC or dex/ no. viable cells per ml in absence of HC or dex) X 100. RESULTS Effect of Thymosin on Glucocotricoid Resistance of Mouse Thymus Cells in a Serum-Free System
In the experiment described in Table 1, mouse thymus cells preincubated for approximately 16 hr with thymosin F-5 or thymosin (Y~(see Materials and Methods for a description of the various fractions of thymosin) were subsequently exposed to glucocorticoids (hydrocortisone (HC) or dexamethasone (dex)) for periods of 4-10 hr. It was noted that cells preincubated with thymosin F-5, splenic F-5, or mouse splenic Fraction 3( F-3) showed a higher spontaneous viability over untreated cells. Cells preincubated with thymosin (Y~showed viability similar to that of untreated cells. Section A in Table 1 shows that after a standard 5-hr exposure to the glucocorticoids, a higher percentage of thymosin F-5- or cut-treated cells survived the killing effect of glucocorticoids as compared to untreated cells. Mouse splenic F-3 or calf splenic F-5 did not show any effect. Sections B, C, and D show the actual number of viable cells following similar treatments in three experiments in which the period of exposure to HC was varied from 4 to 10 hr. In each case treatment with thymosin F-5 or (Y~increased the percentage of cells surviving the HC exposure over untreated cells. Specific Binding of [3H]dex to Intact Mouse Thymocytes
The specific binding of [3H]dex to intact mouse thymocytes was determined at increasing concentrations of [3H]dex in the absence and presence of 5 X 10d6 M competing unlabeled dex (see Materials and Methods for the determination of specific [ 3H Jdex binding). The results of two experiments are shown in Fig. 1. The Scatchard analysis (26) of the data (see inset) were linear, suggesting a single class
380
PHYLLIS
L. OSHEROFF TABLE
1
Effect of Thymosin F-5 and a, on Glucocorticoid Resistance of Mouse Thymus Cells under Serum-Free Conditions Percentage
relative
cell survival
Concentration Treatment
-
NOIE Thymosin
Thymosin
F-5
68.1 f
25 50 loo I50 200
(I,
Calf splenic
A0
(dml)
0.1 0.5 1.0 F-5
Mouse splenic
F-3
B”
f * f zk
P
I.3 (6)
78.3 (2.92/3.73)
68.1 (2.63/3.86)
43.6 (1.26/2.89)
3.6 1.4 2.0 2.9
82.2 90.8 96.3 90.7 92.7
79.2 80.3 90.4 85.7 82.5
51.1 58.6 56.4 64.0 63.7
82.0 91.5 88.7 88.0
Cb
(4) (4) (5) (3)
83.7 fc 0.8 (3) 89.0 f 3.9 (4)
(3.2413.94) (3.45/3.80) (3.66/3.80) (4.08/4.50) (3.93t4.24)
87.7 (2.93/3.34) 90.0 (3.01/3.34) 99.4 (3.48/3.50)
100
70.7 + 3.2 (3)
-
100 200
60.0 2 5.1 (2) 69.4 f 0.7 (2)
-
(3.35/4.23) (3.31/4.12) (4.0514.48) (3.97/4.63) (3.96/4.80)
85.3 (3.30/3.87) 90.6 (3.29/3.63) 65.0 (3.12/4.80) 68.8 (3.28/4.77)
(1.65/3.23) (1.94/3.31) (I .95 13.46) (2.1913.42) (2.42/3.80)
66.9 (1.58/2.36) 55.6 (I .44/2.59) -
Note. Mouse thymus cells were preincubated at 37°C for approximately I6 hr in the absence and presence of thymosin followed by an additional 4-10 hr of incubation (37°C) with and without IO-’ M HC or dex. The number of cells excluding trypan blue was determined at the end of the total incubation period by Cytograf cell counter analysis. Percentage relative cell survival was expressed as (viable cells with HC or dex/viable cells without HC or dex ) X 100. Incubation period in the presence and absence of HC or dex: A and C. 5 hr; B. 4 hr; D. IO hr. ‘Values shown are the mean k SE of two lo six separate experiments. The numkr of experiments is given in parentheses. ‘Values given in parenthesis in B. C. and D are numkr of viable cells/ml with HC (X10-‘) per no. of viable cells/ml without HC (x10-6).
of glucocorticoid-binding sites of uniform, high affinity. Analysis of several such experiments showed that the dissociation constant (I&) for mouse thymocytes was 1.O + 0.1 X 10T8 M, and that there was an average of 4800 binding sites per mouse thymus cell. The binding parameters for mouse thymocytes were comparable to those reported for rat thymus cells (27). Competition by Various Steroids with [3H]dex for Binding to Glucocoritcoid Receptors in Mouse Thymocytes
To determine if the binding of [3H]dex to mouse thymocytes was specific for glucocorticoid receptors, various steroids were tested for their ability to compete with [ 3H]dex for binding. The most effective competing steroid was dexamethasone, followed by hydrocortisone (cortisol) and progesterone (data not shown). Progesterone has been shown to act as a competitive inhibitor of glucocorticoid binding to receptor. Although progesterone binds, the hormone-receptor complex does not enter the nucleus and does not elicit glucocorticoid-specific biological responses (28). Cortisone showed a low level of competition which might be due to limited metabolic conversion to cortisol (25). The sex steroids estradiol and dihydrotestosterone did not compete efficiently for glucocorticoid-receptor binding.
Effect of
Thymosin on Specific Dex Binding to Intact Mouse Thymus Cells in a Serum-Free System
Preincubation of mouse thymocytes for 16.5-18.5 hr in serum-free medium with thymosin F-5 at concentrations of 50-200 Hg/ml of medium resulted in an average
EFFECT OF THYMOSIN
ON GLUCOCORTICOID
[““I
OEX CONCENTRATION
RECEPTORS
381
(IO-%)
FIG. 1. Specific binding of (‘H]dex to intact mouse thymus cells. Specific [3H]dex binding to intact mouse thymus cells at various [‘H]dex concentrations (the competing unlabeled dex was always 5 x 10m6 M) was measured as described under Materials and Methods. The Scatchard analysis of the data is shown in the inset: r = -0.997. See Results for binding parameters.
of 32% (at 150 Fg/ml) decrease in specific [3H]dex binding to whole cells (Table 2, column A). Nonspecific binding was usually between 6-10% of the total binding under the experimental conditions. In Table 2, columns B and C show similar data obtained in two separate experiments. Preincubation with thymosin (Y~at 1 and 5 pg/ml caused a 2&27% decrease in dex binding (Table 2, column B). Meanwhile mouse splenic F-5 or bovine serum albumin (BSA) at SO-200 pg protein/ml did not show any significant effect (Table 2, column C). The slight increases in dex binding (3311%) were not dose responsive and might have been due to non-specific interactions. To investigate the possibility that the mere presence of thymosin during the preincubation step (although presumably washed out at the end of the incubation period) could have affected the subsequent steroid-binding reaction and brought about the observed effects, mouse thymocytes were preincubated in the presence and absence (as control) of thymosin F-5 for a short period of time (30 min, presumably insufficient time for cell maturation to have taken place), washed as usual, and tested for specific [‘H]dex binding. No effect on [3H] dex-binding activity was observed in two separate experiments (Table 3). The specific binding of increasing concentrations of [ 3H]dex to mouse thymocytes preincubated for 18 hr in the absence (control) and presence of Thymosin F-5 ( 100 pg/ml) is shown in Fig. 2. The Scatchard analysis (inset) yields similar dissociation constants of 9.4 and 9.5 nM for control and thymosin-treated cells, respectively. There appeared to be a slight decrease in glucocorticoid-binding sites in thymosintreated cells (3700 sites per cell) as compared to control cells (4400 sites per cell). Whether this decrease is significant or not awaits further analysis using, for example, a cloned precursor T-cell population (see Discussion). DISCUSSION The present study investigates the possible role of a thymus factor, thymosin, in T-cell maturation by studying its effects on glucocorticoid resistance and glucocorticoid receptors in intact mouse thymocytes. All of the present experiments
382
PHYLLIS
L. OSHEROFF TABLE
2
Effect of Thymosin on Specific [‘H]dex Binding to Intact Mouse Thymocytes under Serum-Free Conditions [‘H]dex specifically bound (% of control) Treatment None Thymosin F-5
Thymosin a,
Mouse splenic F-3 BSA
Concentration Wml)
A”
B
-
100
100 (2204)*
50 100 I50 200 0.1 I.0 5.0 50 100 200 50 100 200
82.0 76.8 67.9 71.6
k f 2 + -
I.1 2.0 2.0 1.3
(4) (3) (3) (3)
81.3 67.0 70.9 83.5 80.5 72.6
C 100 (2689)’ 83.1 65.1 104.2 103.2 107.4 108.0 110.3 III.0
Note. 2-3 X IO’ cells per assay were preincubated for 16.5-18.5 hr with thymosin and assayed for specific [-‘H]dex binding to whole cells (see Materials and Methods for details). Results in cpm [‘Hldex specifically bound per 2 X 10’ viable cells were expressed as % of control. ’ Values shown are the mean + SE of three to four separate experiments. The number of experiments is given in parentheses. b Control values of cpm [‘Hldex specifically bound per 2 X IO’ cells.
were carried out under serum-free conditions to avoid nonspecific interference by proteins or steroid hormones (cortisol or progesterone) present in the serum. Most thymic lymphocytes (approximately 90-95%) reside in the cortex and are characterized by high levels of surface Thy-l and TL+, low levels of H-2 antigens, immunoincompetence, and glucocorticoid sensitivity (29). The thymus medulla contains about 3-596 of all thymocytes characterized by low Thy-l, TL-, high H2, higher glucocorticoid resistance, and immunocompetence. It has been shown by Weissman (30) that at least part of these medullary cells are derived from glucocorticoid-sensitive cortical precursors. It is also widely held that these medullary TABLE
3
Specific [‘H]dex Binding to Mouse Thymocytes after Short Incubation (30 min) with Thymosin F-5 under Serum-Free Conditions None Thymosin F-5
100 150
100 + 1.3 (7) 100 r 0.8 (7) 104 + I.6 (5)
Note. 2 X 10’ cells per assay were incubated with thymosin F-5 for 30 min. washed, and assayed for whole cell binding of [‘H]dex (see Materials and Methods for details). Results in cpm [3H]dex specifically bound per 2 X IO’ cells were expressed as % of control. Values shown are the mean f SE of five to seven determinations in two separate experiments. The number of determinations is given in parentheses.
EFFECT OF THYMOSIN
ON GLUCOCORTICOID
[“Ii]
OEX CONCENTRATION
RECEPTORS
383
db,
FIG. 2. The effect of thymosin F-5 on the specific binding of [jH]dex to intact mouse thymocytes under serum-free conditions. Mouse thymocytes were preincubated for 18 hr in the absence (control) and presence of thymosin F-S at 100 sg/ml. See legend to Fig. 1 and Materials and Methods for the specific [‘Hldex binding to control (0) and thymosin-treated cells (0). The Scatchard analysis is shown in the inset: r = -0.976 (0) and -0.972 (0). See Results for binding parameters.
cells are the source of peripheral T lymphocytes (3 1), although this is still controversial (32). Our experiments showed that when mouse thymocytes were pretreated for approximately 16 hr with thymosin F-5 or (Y~and then exposed to glucocorticoids, the percentage of cells resistant to the killing effect of steroids was higher than that found with untreated cells. Cells pretreated with splenic F-5 or mouse splenic F-3, on the other hand, showed no changes in steroid sensitivity. This is in agreement with previous reports ( 19-2 1) that incubation of mouse thymocytes with certain thymic or serum factors (2-6 hr or 2-10 hr in in vitro cultures with steroid present during the incubation) resulted in an increased number of steroid-resistant cells. It was noted that preincubation with thymosin F-5, splenic F-5, or mouse splenic F-3 maintained a higher spontaneous viability of thymocytes over control cells in culture. Thymosin (rl did not show such an effect. Whether this is due to the presence of certain growth factor(s) in thymosin and splenic F-5 and F-3 but not in the pure CY,polypeptide is not known. The present study also showed that mouse thymocytes contained high-affinity binding sites for glucocorticoids. Steroid competition experiments showed that the high-affinity [ 3H]dex binding to intact thymus cells was specific for glucocorticoid receptors. Our data showed that pretreatment of mouse thymocytes with thymosin F-5 or (Y, for 16-19 hr resulted in a reduction in specific whole-cell binding of [ 3H]dex by these thymocytes. Pretreatment with either mouse splenic F-3 or bovine serum albumin had no significant effect. Control experiments showed that if preincubation of thymocytes with thymosin F-5 was carried out for a brief period (30 min, presumably insufficient for cell maturation to have taken place) prior to the steroid-binding reaction, no change in dex binding was observed. This eliminates the possibility of nonspecific interference by thymosin in the steroid-binding reaction. It ought to be pointed out that most binding studies were carried out (following a preincubation with or without thymosin) in a cellular population where
384
PHYLLIS
L. OSHEROFF
cell viability was 38-45s for control cells and 54-6390 for thymosin-treated cells. It could only be postulated that some fractions of the thymocytes were responding to thymosin. The validity of the data was nevertheless supported at least in part by a similar reduction in [3H]dex binding (30%, data not shown) by thymosintreated BW 5 147 cells (a cultured line of mouse thymoma cells) where cell viability was more than 90% following preincubation. The effects of thymosin demonstrated in the present experiments, though not dramatic in magnitude, were nevertheless highly reproducible. Its effects in causing (i) an increase in mouse thymocyte survival in the presence of glucocorticoids; and (ii) a reduction in glucocorticoid receptors under the present experimental conditions, could possibly be two separate events, although resistance to glucocorticoid killing was in some instances associated with a decreased level of glucocorticoid receptors (12, 13, 33, 34). It is possible that some fractions of these thymocytes may respond to thymosin by either proliferating or maturing. To further investigate these possibilities, exploration of the effect of thymosin on glucocorticoid sensitivity and receptor activity in precursor T cells should prove useful. Preliminary data (not shown) indicate that thymosin F-5 causes an increase in dex binding to whole cells (18%) and crude nuclei (39%) of mouse bone marrow cells. However, the percentage of precursor T cells in bone marrow is relatively small, ranging from 3.4% evaluated by induction of Thy-l (35) to 16% estimated by peanut agglutinin binding (J. John Cohen, personal communication). Therefore, the thymosin effects on glucocorticoid sensitivity and receptors in precursor T cells could only be demonstrated when purer, cloned precursor T-cell populations (J. John Cohen, personal communication) are available. ACKNOWLEDGMENTS I am grateful to Dr. David Webb for his helpful suggestions and valuable discussions during this investigation. I also thank Dr. Armin H. Ramel for his encouragement and support throughout the course of this study. A critical reading of the manuscript by Drs. Michael Sherman, Dennis Stacy, and J. John Cohen is appreciated. Thanks are also due to Ms. Lyn Nelson for her assistance in the preparation of the manuscript.
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ON GLUCOCORTICOID
RECEPTORS
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IMMUNOLOGY
60,
376-385
(1981)
The Effect of Thymosin on Glucocorticoid in Lymphoid Cells PHYLLIS
Receptors
L. OSHEROFF
Biopolymer Research Department, Central Research Division, Hoffmann-LaRoche Nutley, New Jersey 07110
Inc.,
Received June 26, 1980: accepted November 25, 1980 The present study investigates the effect of a partially purified thymus factor, thymosin Fraction 5, and an homogeneous polypeptide component of Fraction 5, thymosin a,, on glucocorticoid resistance and glucocorticoid receptors in mouse thymocytes. Treatment of thymocytes in vitro with thymosin Fraction 5 or (Y, results in an increase in the percentage of glucocorticoid-resistant cells. Studies on the specific whole-cell binding of [‘Hldexamethasone and steroid competition experiments demonstrate the existence of high-affinity (& = I.0 X IO-” IV) specific glucocorticoid receptors in mouse thymocytes. Preincubation with thymosin Fraction 5 or (Y, appears to cause a reduction in the specific [3H]dexamethasone binding to intact thymocytes.
INTRODUCTION Numerous studies have suggested a role for a variety of thymic humoral factors or hormones in the maturation of thymus-derived lymphocytes (T cells). These studies suggested that the thymic hormones control the proliferation of precursor T cells in the bone marrow (1) and the subsequent differentiation and maturation of T cells in the thymus and peripheral lymphoid tissues (for reviews, see (2)). The various populations of cells which develop during T-cell maturation can be characterized by their sensitivity to killing by glucocorticoids. The precursors of T cells which have the capacity to repopulate the thymus after irradiation are relatively, but not completely, resistant to glucocorticoids (3, 4). The immunologically immature T cells, which are present mainly in the thymus cortex, are killed by glucocorticoids. The more mature, immunocompetent T cells, located primarily in the thymus medulla, are mostly resistant to glucocorticoids in vivo (5, 6); although it has been shown that the residual thymocyte population, isolated 48 hr after in vivo hydrocortisone injection, is partly sensitive to steroid treatment in vitro, as demonstrated by uridine incorporation inhibition (7). Biochemical studies have strongly suggested that many physiological responses to the glucocorticoid hormones are mediated through specific, intracellular binding proteins or receptors. In a generally accepted molecular mechanism the steroid binds strongly to the receptor in the cytoplasm of the target cell and acts as an allosteric effector molecule to increase the affinity of the receptor for nuclear binding sites. The interaction of the steroid-receptor complex with the genome then 376 OOOS-8749/81/080376-lOSOZ.OO/O Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
EFFECT
OF
THYMOSIN
ON
GLUCOCORTICOID
RECEPTORS
377
triggers the biological responses (for reviews see (8-10)). Using a cultured line of glucocorticoid-sensitive mouse lymphoma cells and their steroid-resistant variant clones, Tomkins and his colleagues (1 I-14) demonstrated that a number of clones resistant to steroid-induced cell death showed diminished numbers of glucocorticoid receptors or impaired translocation of the glucocorticoid receptor complex into the nucleus. On the other hand, Smith et al. (15) reported that in cultured human peripheral lymphocytes, mitogen-activated blast transformation was associated with a large increase in glucocorticoid-binding sites per cell, yet lymphocytes were equally sensitive to glucocorticoid inhibition of certain metabolic parameters (glucose transport, nucleoside incorporation), regardless of their state of activation. These observations have recently been extended to in uiwo antigen-stimulated lymph node lymphocytes ( 16). Lippman and Barr ( 17) and Fauci et al. ( 18) both reported a lack of direct correlation between cytoplasmic glucocorticoid receptors and the differential in vivo effects of glucocorticoids on the recirculation of lymphocyte subpopulations. It has been shown that preincubation (1 hr) or coincubation (2-10 hr) of mouse thymocytes with a thymic humoral factor leads to an increased number of hydrocortisone-resistant cells ( 19). Similar results were reported in 2- to 6-hr thymocyte cultures using a human serum thymic factor (20), or thymic epithelial culture supernatants (21). Bach et al. (22) reported that a serum thymic factor caused a 60-70s inhibition of steroid binding when thymocyte cytosol was incubated directly with the serum factor. Since incubation with the serum factor was carried out under cell-free conditions the observed decrease in steroid binding bore no relationship to the cell maturation process. It is possible that the reduced steroid binding was merely the result of nonspecific interference by the serum factor. In the present report we have attempted to explore the possible role of a partially purified thymic factor, thymosin Fraction 5 (23), and a homogeneous polypeptide component of Fraction 5, (Y~(24) in T-cell maturation by studying their effects on glucocorticoid resistance and glucocorticoid receptors of intact mouse thymus cells. The results suggest that thymosin Fraction 5 and (Y~may alter glucocorticoid resistance as well as glucocorticoid receptor activity in these thymus cells. MATERIALS
AND
METHODS
Mice Six- to ten-week-old male C57BL/6 were obtained from Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts. Intact mice were used in all experiments. Preparation
of Cell Suspensions
Mouse thymuses were removed aseptically and washed twice in HBSS. Thymocyte suspensions were prepared by gentle hand homogenization in a loose-fitting Dounce tissue grinder in HBSS, centrifuged (SOOg, 8 min), washed twice in HBSS at 0-4°C and resuspended in cold medium I (RPM1 1640 (Grand Island Biological Co. Grand Island, N.Y.) supplemented with 50 units/ml penicillin, 50 pg/ml streptomycin, 2 mM glutamine, and 10 mM Hepes (Calbiochem, La Jolla, Calif.). Cells were filtered through a metal sieve and kept on ice before incubation with thymosin.
378
PHYLLIS
L. OSHEROFF
Preparation of Calf and Mouse Thymosin Fractions
Calf thymosin Fraction 5(F-5) (23) used in these experiments was prepared by Dr. W. C. McGregor and co-workers, Biopolymer Research Department, Hoffmann-La Roche Inc., Nutley, New Jersey. Fraction 5 is purified from Fraction 3 (F-3, obtained by heat treatment (8O’C) of thymic tissue homogenates followed by acetone precipitation of the heated supernate) by ammonium sulfate precipitation (to 50% saturation), ultrafiltration of the solubilized precipitate, and desalting of the ultrafiltrate on Sephadex G-25 followed by lyophilization. The same procedures were used for the preparation of calf splenic F-5. Thymosin (Y~polypeptide is an homogenous acidic component of F-5 and contains 28 amino acid residues, the sequence of which is known (24). The thymosin o1 used in the present experiments was synthesized and kindly provided by Dr. S. S. Wang (Dept. of BioOrganic Chemistry, Hoffman-La Roche Inc., present address: Penninsula Laboratories, Inc., San Carlos, Calif., 94070). Mouse splenic F-3 was prepared as for calf F-3 from spleens obtained aseptically. All preparations were sterilized by filtration (0.22-Hrn Swinnex-13, Millipore Corp., Bedford, Mass.) before use. Preincubation with Thymosin and Assay for Specific Steroid Binding by Whole Cells
Preincubation of thymus cells with various concentrations of thymosin was carried out at 37°C in a humidified atmosphere of 5% C02-95% air for various periods of time as indicated under Results. The samples were prepared in 60 X 15-mm petri dishes in a total volume of 4 ml at 5-7.5 X lo6 cells/ml in medium I. Samples were prepared in duplicate for measuring total [3H]dexamethasone (dex) binding and nonspecific [3H]dex binding by whole cells. The assay for specific steroid binding by whole cells was modified from that of Sibley and Tomkins (12) and was carried out as follows. After incubation with thymosin, duplicate cultures were harvested in Falcon 12 X 75-mm polyethylene tubes and aliquots counted for viable cells by trypan blue exclusion in a hemacytometer. The cells were centrifuged (all centrifugations were carried out for 8 min at 800g) and washed three times with 3 ml of HBSS at 04°C. The cells were resuspended in 1 ml of medium I and 5 X 10m9 M [3H]dex (36-41 Ci/mmol, New England Nuclear, Boston, Mass., or 26 Ci/mmol, Amersham, Arlington Heights, Ill.) was added to each of the duplicate cultures. One of these cultures also received a lOOO-fold excess (5 X 10e6 M) of unlabeled dex (Sigma Chemical Co., St. Louis, MO.) to determine specific [3H]dex binding. This was taken as the difference in bound [‘H]dex between cells incubated in the absence (total) and presence (nonspecific) of unlabeled dex. After incubation at 37’C in a humidified atmosphere of 5% CO*--95% air for 45 min (an extended incubation period of 90 min did not result in increased binding), the cells were centrifuged, washed twice with 3 ml of HBSS at @-4”C resuspended in 23°C HBSS, and left to stand at room temperature for 10 min before centrifugation to dissociate most of the nonspecifically bound dex (25). The samples were resuspended in 0.2 ml of Dulbecco’s phosphate-buffered saline (PBS) and transferred to scintillation vials. The polyethylene tubes were rinsed twice with 0.2 ml PBS and the rinsings were combined in the scintillation vials. Biofluor (New England Nuclear) was added and the samples were counted in a Beckman LS-250 or Packard Tricarb liquid scintillation counter.
EFFECT OF THYMOSIN
ON GLUCOCORTICOID
RECEPTORS
379
Preincubation with Thymosin and Assay for Glucocorticoid Resistance of Mouse Thymocytes
Mouse thymocytes were resuspended in medium I at 5 to 10 X lo6 cells/ml. Preincubation with thymosin was carried out as described above for the steroidbinding experiments. Samples were prepared in 35 X IO-mm petri dishes in a total volume of 1.5 ml in quadruplicate, duplicate each for the determination of cell survival in the presence and absence of glucocorticoids. Following preincubation with thymosin for approximately 16 hr, hydrocortisone (HC, Sigma Chemical Co.) or dex was added to half of the quadruplicate cultures to a final concentration of 10e5 M (final concentrations of 10e6-5 X lop5 M had similar effect) and incubation was continued for periods of 4-10 hr. At the end of the incubation period, cultures were harvested into 12 X 75-mm Falcon polyethylene tubes. An aliquot (75-100 ~1) of each sample was diluted in 2 ml of PBS containing bovine serum albumin (crystallized, Miles Laboratories, Elkart, Ind.) at 1 mg/ml and 0.05% trypan blue (Grand Island Biological Co.) and counted immediately in a Model 6300A Cytograf cell counter (Bio/Physics Systems, Inc., presently Ortho Instruments) which gives the total number of cells and the percentage of cells excluding trypan blue. Numbers of viable cells (those excluding trypan blue) was calculated and percentage relative cell survival was expressed as: (no. of viable cells per ml in presence of HC or dex/ no. viable cells per ml in absence of HC or dex) X 100. RESULTS Effect of Thymosin on Glucocotricoid Resistance of Mouse Thymus Cells in a Serum-Free System
In the experiment described in Table 1, mouse thymus cells preincubated for approximately 16 hr with thymosin F-5 or thymosin (Y~(see Materials and Methods for a description of the various fractions of thymosin) were subsequently exposed to glucocorticoids (hydrocortisone (HC) or dexamethasone (dex)) for periods of 4-10 hr. It was noted that cells preincubated with thymosin F-5, splenic F-5, or mouse splenic Fraction 3( F-3) showed a higher spontaneous viability over untreated cells. Cells preincubated with thymosin (Y~showed viability similar to that of untreated cells. Section A in Table 1 shows that after a standard 5-hr exposure to the glucocorticoids, a higher percentage of thymosin F-5- or cut-treated cells survived the killing effect of glucocorticoids as compared to untreated cells. Mouse splenic F-3 or calf splenic F-5 did not show any effect. Sections B, C, and D show the actual number of viable cells following similar treatments in three experiments in which the period of exposure to HC was varied from 4 to 10 hr. In each case treatment with thymosin F-5 or (Y~increased the percentage of cells surviving the HC exposure over untreated cells. Specific Binding of [3H]dex to Intact Mouse Thymocytes
The specific binding of [3H]dex to intact mouse thymocytes was determined at increasing concentrations of [3H]dex in the absence and presence of 5 X 10d6 M competing unlabeled dex (see Materials and Methods for the determination of specific [ 3H Jdex binding). The results of two experiments are shown in Fig. 1. The Scatchard analysis (26) of the data (see inset) were linear, suggesting a single class
380
PHYLLIS
L. OSHEROFF TABLE
1
Effect of Thymosin F-5 and a, on Glucocorticoid Resistance of Mouse Thymus Cells under Serum-Free Conditions Percentage
relative
cell survival
Concentration Treatment
-
NOIE Thymosin
Thymosin
F-5
68.1 f
25 50 loo I50 200
(I,
Calf splenic
A0
(dml)
0.1 0.5 1.0 F-5
Mouse splenic
F-3
B”
f * f zk
P
I.3 (6)
78.3 (2.92/3.73)
68.1 (2.63/3.86)
43.6 (1.26/2.89)
3.6 1.4 2.0 2.9
82.2 90.8 96.3 90.7 92.7
79.2 80.3 90.4 85.7 82.5
51.1 58.6 56.4 64.0 63.7
82.0 91.5 88.7 88.0
Cb
(4) (4) (5) (3)
83.7 fc 0.8 (3) 89.0 f 3.9 (4)
(3.2413.94) (3.45/3.80) (3.66/3.80) (4.08/4.50) (3.93t4.24)
87.7 (2.93/3.34) 90.0 (3.01/3.34) 99.4 (3.48/3.50)
100
70.7 + 3.2 (3)
-
100 200
60.0 2 5.1 (2) 69.4 f 0.7 (2)
-
(3.35/4.23) (3.31/4.12) (4.0514.48) (3.97/4.63) (3.96/4.80)
85.3 (3.30/3.87) 90.6 (3.29/3.63) 65.0 (3.12/4.80) 68.8 (3.28/4.77)
(1.65/3.23) (1.94/3.31) (I .95 13.46) (2.1913.42) (2.42/3.80)
66.9 (1.58/2.36) 55.6 (I .44/2.59) -
Note. Mouse thymus cells were preincubated at 37°C for approximately I6 hr in the absence and presence of thymosin followed by an additional 4-10 hr of incubation (37°C) with and without IO-’ M HC or dex. The number of cells excluding trypan blue was determined at the end of the total incubation period by Cytograf cell counter analysis. Percentage relative cell survival was expressed as (viable cells with HC or dex/viable cells without HC or dex ) X 100. Incubation period in the presence and absence of HC or dex: A and C. 5 hr; B. 4 hr; D. IO hr. ‘Values shown are the mean k SE of two lo six separate experiments. The numkr of experiments is given in parentheses. ‘Values given in parenthesis in B. C. and D are numkr of viable cells/ml with HC (X10-‘) per no. of viable cells/ml without HC (x10-6).
of glucocorticoid-binding sites of uniform, high affinity. Analysis of several such experiments showed that the dissociation constant (I&) for mouse thymocytes was 1.O + 0.1 X 10T8 M, and that there was an average of 4800 binding sites per mouse thymus cell. The binding parameters for mouse thymocytes were comparable to those reported for rat thymus cells (27). Competition by Various Steroids with [3H]dex for Binding to Glucocoritcoid Receptors in Mouse Thymocytes
To determine if the binding of [3H]dex to mouse thymocytes was specific for glucocorticoid receptors, various steroids were tested for their ability to compete with [ 3H]dex for binding. The most effective competing steroid was dexamethasone, followed by hydrocortisone (cortisol) and progesterone (data not shown). Progesterone has been shown to act as a competitive inhibitor of glucocorticoid binding to receptor. Although progesterone binds, the hormone-receptor complex does not enter the nucleus and does not elicit glucocorticoid-specific biological responses (28). Cortisone showed a low level of competition which might be due to limited metabolic conversion to cortisol (25). The sex steroids estradiol and dihydrotestosterone did not compete efficiently for glucocorticoid-receptor binding.
Effect of
Thymosin on Specific Dex Binding to Intact Mouse Thymus Cells in a Serum-Free System
Preincubation of mouse thymocytes for 16.5-18.5 hr in serum-free medium with thymosin F-5 at concentrations of 50-200 Hg/ml of medium resulted in an average
EFFECT OF THYMOSIN
ON GLUCOCORTICOID
[““I
OEX CONCENTRATION
RECEPTORS
381
(IO-%)
FIG. 1. Specific binding of (‘H]dex to intact mouse thymus cells. Specific [3H]dex binding to intact mouse thymus cells at various [‘H]dex concentrations (the competing unlabeled dex was always 5 x 10m6 M) was measured as described under Materials and Methods. The Scatchard analysis of the data is shown in the inset: r = -0.997. See Results for binding parameters.
of 32% (at 150 Fg/ml) decrease in specific [3H]dex binding to whole cells (Table 2, column A). Nonspecific binding was usually between 6-10% of the total binding under the experimental conditions. In Table 2, columns B and C show similar data obtained in two separate experiments. Preincubation with thymosin (Y~at 1 and 5 pg/ml caused a 2&27% decrease in dex binding (Table 2, column B). Meanwhile mouse splenic F-5 or bovine serum albumin (BSA) at SO-200 pg protein/ml did not show any significant effect (Table 2, column C). The slight increases in dex binding (3311%) were not dose responsive and might have been due to non-specific interactions. To investigate the possibility that the mere presence of thymosin during the preincubation step (although presumably washed out at the end of the incubation period) could have affected the subsequent steroid-binding reaction and brought about the observed effects, mouse thymocytes were preincubated in the presence and absence (as control) of thymosin F-5 for a short period of time (30 min, presumably insufficient time for cell maturation to have taken place), washed as usual, and tested for specific [‘H]dex binding. No effect on [3H] dex-binding activity was observed in two separate experiments (Table 3). The specific binding of increasing concentrations of [ 3H]dex to mouse thymocytes preincubated for 18 hr in the absence (control) and presence of Thymosin F-5 ( 100 pg/ml) is shown in Fig. 2. The Scatchard analysis (inset) yields similar dissociation constants of 9.4 and 9.5 nM for control and thymosin-treated cells, respectively. There appeared to be a slight decrease in glucocorticoid-binding sites in thymosintreated cells (3700 sites per cell) as compared to control cells (4400 sites per cell). Whether this decrease is significant or not awaits further analysis using, for example, a cloned precursor T-cell population (see Discussion). DISCUSSION The present study investigates the possible role of a thymus factor, thymosin, in T-cell maturation by studying its effects on glucocorticoid resistance and glucocorticoid receptors in intact mouse thymocytes. All of the present experiments
382
PHYLLIS
L. OSHEROFF TABLE
2
Effect of Thymosin on Specific [‘H]dex Binding to Intact Mouse Thymocytes under Serum-Free Conditions [‘H]dex specifically bound (% of control) Treatment None Thymosin F-5
Thymosin a,
Mouse splenic F-3 BSA
Concentration Wml)
A”
B
-
100
100 (2204)*
50 100 I50 200 0.1 I.0 5.0 50 100 200 50 100 200
82.0 76.8 67.9 71.6
k f 2 + -
I.1 2.0 2.0 1.3
(4) (3) (3) (3)
81.3 67.0 70.9 83.5 80.5 72.6
C 100 (2689)’ 83.1 65.1 104.2 103.2 107.4 108.0 110.3 III.0
Note. 2-3 X IO’ cells per assay were preincubated for 16.5-18.5 hr with thymosin and assayed for specific [-‘H]dex binding to whole cells (see Materials and Methods for details). Results in cpm [‘Hldex specifically bound per 2 X 10’ viable cells were expressed as % of control. ’ Values shown are the mean + SE of three to four separate experiments. The number of experiments is given in parentheses. b Control values of cpm [‘Hldex specifically bound per 2 X IO’ cells.
were carried out under serum-free conditions to avoid nonspecific interference by proteins or steroid hormones (cortisol or progesterone) present in the serum. Most thymic lymphocytes (approximately 90-95%) reside in the cortex and are characterized by high levels of surface Thy-l and TL+, low levels of H-2 antigens, immunoincompetence, and glucocorticoid sensitivity (29). The thymus medulla contains about 3-596 of all thymocytes characterized by low Thy-l, TL-, high H2, higher glucocorticoid resistance, and immunocompetence. It has been shown by Weissman (30) that at least part of these medullary cells are derived from glucocorticoid-sensitive cortical precursors. It is also widely held that these medullary TABLE
3
Specific [‘H]dex Binding to Mouse Thymocytes after Short Incubation (30 min) with Thymosin F-5 under Serum-Free Conditions None Thymosin F-5
100 150
100 + 1.3 (7) 100 r 0.8 (7) 104 + I.6 (5)
Note. 2 X 10’ cells per assay were incubated with thymosin F-5 for 30 min. washed, and assayed for whole cell binding of [‘H]dex (see Materials and Methods for details). Results in cpm [3H]dex specifically bound per 2 X IO’ cells were expressed as % of control. Values shown are the mean f SE of five to seven determinations in two separate experiments. The number of determinations is given in parentheses.
EFFECT OF THYMOSIN
ON GLUCOCORTICOID
[“Ii]
OEX CONCENTRATION
RECEPTORS
383
db,
FIG. 2. The effect of thymosin F-5 on the specific binding of [jH]dex to intact mouse thymocytes under serum-free conditions. Mouse thymocytes were preincubated for 18 hr in the absence (control) and presence of thymosin F-S at 100 sg/ml. See legend to Fig. 1 and Materials and Methods for the specific [‘Hldex binding to control (0) and thymosin-treated cells (0). The Scatchard analysis is shown in the inset: r = -0.976 (0) and -0.972 (0). See Results for binding parameters.
cells are the source of peripheral T lymphocytes (3 1), although this is still controversial (32). Our experiments showed that when mouse thymocytes were pretreated for approximately 16 hr with thymosin F-5 or (Y~and then exposed to glucocorticoids, the percentage of cells resistant to the killing effect of steroids was higher than that found with untreated cells. Cells pretreated with splenic F-5 or mouse splenic F-3, on the other hand, showed no changes in steroid sensitivity. This is in agreement with previous reports ( 19-2 1) that incubation of mouse thymocytes with certain thymic or serum factors (2-6 hr or 2-10 hr in in vitro cultures with steroid present during the incubation) resulted in an increased number of steroid-resistant cells. It was noted that preincubation with thymosin F-5, splenic F-5, or mouse splenic F-3 maintained a higher spontaneous viability of thymocytes over control cells in culture. Thymosin (rl did not show such an effect. Whether this is due to the presence of certain growth factor(s) in thymosin and splenic F-5 and F-3 but not in the pure CY,polypeptide is not known. The present study also showed that mouse thymocytes contained high-affinity binding sites for glucocorticoids. Steroid competition experiments showed that the high-affinity [ 3H]dex binding to intact thymus cells was specific for glucocorticoid receptors. Our data showed that pretreatment of mouse thymocytes with thymosin F-5 or (Y, for 16-19 hr resulted in a reduction in specific whole-cell binding of [ 3H]dex by these thymocytes. Pretreatment with either mouse splenic F-3 or bovine serum albumin had no significant effect. Control experiments showed that if preincubation of thymocytes with thymosin F-5 was carried out for a brief period (30 min, presumably insufficient for cell maturation to have taken place) prior to the steroid-binding reaction, no change in dex binding was observed. This eliminates the possibility of nonspecific interference by thymosin in the steroid-binding reaction. It ought to be pointed out that most binding studies were carried out (following a preincubation with or without thymosin) in a cellular population where
384
PHYLLIS
L. OSHEROFF
cell viability was 38-45s for control cells and 54-6390 for thymosin-treated cells. It could only be postulated that some fractions of the thymocytes were responding to thymosin. The validity of the data was nevertheless supported at least in part by a similar reduction in [3H]dex binding (30%, data not shown) by thymosintreated BW 5 147 cells (a cultured line of mouse thymoma cells) where cell viability was more than 90% following preincubation. The effects of thymosin demonstrated in the present experiments, though not dramatic in magnitude, were nevertheless highly reproducible. Its effects in causing (i) an increase in mouse thymocyte survival in the presence of glucocorticoids; and (ii) a reduction in glucocorticoid receptors under the present experimental conditions, could possibly be two separate events, although resistance to glucocorticoid killing was in some instances associated with a decreased level of glucocorticoid receptors (12, 13, 33, 34). It is possible that some fractions of these thymocytes may respond to thymosin by either proliferating or maturing. To further investigate these possibilities, exploration of the effect of thymosin on glucocorticoid sensitivity and receptor activity in precursor T cells should prove useful. Preliminary data (not shown) indicate that thymosin F-5 causes an increase in dex binding to whole cells (18%) and crude nuclei (39%) of mouse bone marrow cells. However, the percentage of precursor T cells in bone marrow is relatively small, ranging from 3.4% evaluated by induction of Thy-l (35) to 16% estimated by peanut agglutinin binding (J. John Cohen, personal communication). Therefore, the thymosin effects on glucocorticoid sensitivity and receptors in precursor T cells could only be demonstrated when purer, cloned precursor T-cell populations (J. John Cohen, personal communication) are available. ACKNOWLEDGMENTS I am grateful to Dr. David Webb for his helpful suggestions and valuable discussions during this investigation. I also thank Dr. Armin H. Ramel for his encouragement and support throughout the course of this study. A critical reading of the manuscript by Drs. Michael Sherman, Dennis Stacy, and J. John Cohen is appreciated. Thanks are also due to Ms. Lyn Nelson for her assistance in the preparation of the manuscript.
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