Life Sciences, Vol. Printed in the USA
51, pp.
i033-i039
Pergamon
Press
INHIBITION OF IMMUNOREACTIVE GROWTH HORMONE SECRETION
FROM LYMPHOID CELL LINES BY DEXAMETHASONE Ting-Lin Kao and Walter J. Meyer, III
Departments of Human Biological Chemistry & Genetics and Psychiatry & Behavioral Sciences, The University of Texas Medical Branch, Galveston, TX, USA
(Received
in final
form July 14,
1992)
Summar~ The regulation of irGH secretion by the immune system was examined using lymphoid cell lines, H9 and IM9. Using a highly sensitive immunoassay, irGH secretion by H9 was negatively regulated by dexamethasone, whereas many other regulators of hGH secretion, including hormones, monoamines, and second messenger, had no measurable effect on irGH secretion. Treatment of H9 cells with dexamethasone for 48 hours could cause as high as 70% reduction in irGH secretion without affecting either cell numbers or viability. Using IM9, neither growth hormone releasing hormone nor thyrotropin releasing hormone had significant effect on either irGH steady-state level transcripts or irGH secretion. These findings suggest that irGH secretion by lymphocytes was regulated in a different manner from that by pituitary cells. Production of neuroendocrine peptides by lymphoid cells is one proposed mechanism for the communication between the neuroendocrine and the immune systems. It has been demonstrated that lymphocytes produce pituitary hormones such as ACTH (1), TSH (2), endorphin (3), prolactin (4), and growth hormone (5,6). Recently, immunoreactive growth hormone (irGH) production by human peripheral blood lymphocytes has been shown independently by two groups (5,6). In addition, Hattori et a_l. have quantitated irGH secretion by human lymphocytes and have shown that such secretion is regulated in a different manner from that in the anterior pituitary (6). Our group has recently developed model systems for study of irGH secretion using human lymphoid cell lines and have successfully quantitated irGH secretion from H9, a T-cell line, using a highly sensitive double-antibody immunoassay (7). In the present study, we study the regulation of irGH secretion from H9 using the hGH immunoassay and from IM9, a B-cell line, using reverse hemolytic plaque assay and dot blot analysis.
Author to whom reprint requests should be addressed: Walter J. Meyer, III, M.D., Professor and Vice Chairman for Research, Department of Psychiatry & Behavioral Sciences, University of Texas Medical Branch, Galveston, TX 77555-0429 Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1034
Dexamethasone
Inhibits
irGH S e c r e t i o n
Vol.
51, No.
13, 1992
Materials and Methods The B-cell line, IM9, was supplied by Dr. E. Brad Thompson at the University of Texas Medical Branch (UTMB), Galveston, and the T-cell line, H9, was supplied by Dr. Miles Cloyd, UTMB. Cells were cultured in RPMI 1640 or DMEM supplemented with 5% fetal calf serum and antibiotics. Viability was determined by trypan blue exclusion. After incubation with or without various pharmacological agents for 24-48 hours, ceils were harvested by centrifugation (150-200 x g) for 10 minutes. Immunoassay. Secretion of irGH was measured by a highly sensitive double antibody immunoassay as previous described (7). Briefly, blank medium containing various concentrations of hGH or conditioned cultured medium was concentrated 50X by dialysis and lyophilization. Similarly concentrated blank medium with hGH was used to set up standard curves. Concentrated samples (100/xl) were assayed using a commercial hGH kit (Nichols Institute, Allegro hGH kit). The detection limit was lpg/ml. Reverse Hemolytic Plaque Assay. The assay was performed as previously described with several minor modifications (8). Sheep red blood cells (SRBC) were coupled to staphylococcal protein A (Sigma) as previous described (9). Briefly, the coupled SRBC was mixed with an equal volume of lymphocytes resuspended in RPMI-0.1% BSA-antibiotics in a concentration of 1 X 106 cells/ml, antihuman growth hormone antiserum (Chemicon 1:50), and guinea pig complement (Cordis Labs, 1:20). The resulting mixture was infused by capillary action into Cunningham slide chambers in duplicate. The plaques were counted at 10X magnification after overnight incubation. Statistical analysis. The data were expressed as means + SD. Student's t-test and paired t-test were used for statistical analysis. Dot blot analysis. Cytoplasmic RNA was extracted from the lymphocytes using a method described by Gough (10). Bacteria carrying plasmids which contain either the rat or human GH cDNA insert were provided by Dr. Weigent of the University of Alabama, who transformed the bacteria using plasmids donated by Dr. Denoto of UCSF. A 500-bp pvuII fragment containing either the rat or human GH coding region was isolated as probe. Hybridization was carried out according to a standard protocol (1 I). After total RNA was transferred onto 0.45/xm nitrocellulose filter, the blot was incubated with prehybridization buffer (50% formamide, 5X Denhardt's solution, 0.1% SDS, 100/xg/ml of salmon sperm DNA, 5X SSPE) at 42 degrees for 1-2 hours. For hybridization 1'32 nick-translated probe with specific activity of 1-5 x 108 cpm/#g was added to the hybridization buffer (prehybridization buffer with 10% dextran sulfate added) at 1-5 x 106 cpm/ml. After overnight incubation, the blot was washed extensively and autoradiographed for a week.
Vol.
51, No.
13,
1992
Dexamethasone
Inhibits
irGH S e c r e t i o n
1035
Results The secretion of irGH by H9 cells was measured by a highly sensitive double-antibody immunoassay. Typically, H9 cells secret irGH in the order of 10 pg/107 cells in 24-48 hours (7). Cells treated with dexamethasone (10-6M-10-8M) for 48 hours was inhibited in a dosedependent fashion (p < 0.05; Figure 1). Under the culture condition used in our experiment, dexamethasone was not cytotoxic to the cells, as neither the cell number nor the viability changed significantly with treatment of dexamethasone (Figure 2). The secretion of irGH by H9 cells treated with same doses of dexamethasone for only 24 hours was not affected (data not shown). Treatment of H9 cells for 24-48 hours by a variety of agents, including physiological stimuli or inhibitors of pituitary GH secretion (GH releasing hormone [GHRH], somatostatin, somatomedin), monoamines, mitogen, second messenger, and hormones which stimulate GH secretion in vitro, had no appreciable effect on irGH secretion by H9. Table 1 lists all the pharmacological agents tested which have no appreciable effects on H9 secretion of irGH.
200
r-i 0 14 ¢:1¢1 O0
ra~
100
q.,10 01.4 U I,J Q 0
QJ.I
0
0
IO~M
1 0 a M lO'SM lO~Vl
lOa°M
Dex Concentration
FIG 1 H9 irGH secretion in response to various concentrations of dexamethasone. Cells were incubated in 10-6M dexamethasone for 48 hours and the culture supernatants were assayed for irGH content. Average of 3 experiments.
1036
Dexamethasone
I
Inhibits
irGH S e c r e t i o n
Vol.
51, No.
Viability
Rel. Cell #
200~ t-I
0
0 U m
,,
1OO
" I
@
o,,-4
14,-i GI @
mo
O
0
lO'6M
lO'TM
104~M
Dox Concentration FIG 2
H9 cell number and viability after 48 hours of incubation with 106M dexamethasone. Average of 3 experiments. TABLE 1 Pharmacological Stimuli Which Do Not Affect irGH Secretion by H9 Cells. Stimuli
Dose Tested
TRH Somatostatin Somatomedin Epinephrine Norepinephrine Isoproterenol Clonidine Serotonin Insulin Triiodothyronine Lipopolysaccharide Testosterone Estrogen Dibuteryl AMP Thymosin fraction 5
3XlO'TM, 3X lO'9M 1/2g/ml, 10 ng/ml 100 ng/ml, 10 ng/ml, 1 ng/ml 106M, lO-8M 10-6M, 10-8M 10-6M, IO-SM IO-6M, IO-SM 10-6M, 104M, 10-1°M 10-SM, 1OTM, 10-9M 10-TM, 10-9M 50 gg/ml, 5/xg/ml 250 ng/ml, 25 ng/ml 10 ng/ml, 1 ng/ml, 0.1 ng/ml 10-SM, 10-6M 1 /zg/ml, 10 ng/ml, 0.1 ng/ml
13, 1992
Vol.
51, No.
13,
1992
Dexamethasone
Inhibits
irGH S e c r e t i o n
1037
As previously reported, IM9, a B-cell line, secreted irGH at an extremely low level such that its irGH secretion could not be accurately quantitated using the double-antibody immunoassay (7). We examined the effects of 10SM GHRH and .275 x 10-6M thyrotropin releasing hormone (TRH) on irGH production and secretion by IM9 cells using reverse hemolytic plaque assay and dot blot analysis. In a hemolytic plaque assay using 5X104 cells per slide, cells incubated in plain media produced 32 _+ 13 plaques, compared to cells incubated in media with GHRH providing 62 _+ 23 plaques. The differences were not statistically significant (p=. 13 by paired t-test, n=3). TRH stimulation had no effect on the number of plaque-producing lymphocytes. Consistent with this finding, dot blot analysis revealed no significant increase in the amount of message detected with either rat of human GH cDNA probe after GHRH or TRH treatment (Figure 3).
A
B
GH3
GH3
L
L
MOCK
MOCK
GHRH
GHRH
TRH
TRH FIG 3
Dot blot of RNA with either rGH or hGH cDNA probes using GH 3 cells, L cells, and IM9 cells. The IM9 cells were incubated with plain media (MOCK), plain media plus 1OSM GHRH, and plain media plus .275 x 10-6M TRH. A. Rat probe; GH 3 RNA, 2/~g, l#g; L cell RNA: 10/xg, 5/xg; IM9 RNA, 40~g, 20~g. B. Human probe; GH 3 RNA, 2/xg, ltxg, 0.5~g; L cell RNA, 10/xg, 5~g, 2.5#g; IM9 RNA: 40/~g, 20/~g, 10#g.
Discussion In this study, we have tested a wide spectrum of naturally-occurring and synthetic compounds which affected hGH secretion in vitro and/or in vivo and found that most of the agents
1038
Dexamethasone
Inhibits
irGH S e c r e t i o n
Vol.
51, No.
13, 1992
examined have no measurable effect on irGH secretion by H9 cells. This observation confirmed the earlier report by Hattori et. al. (6), whose group showed that neither GHRH nor a somatostatin analogue had effect on irGH secretion by peripheral blood lymphocytes. In addition to GHRH and somatostatin, we have also studied the effects of several other neuropeptides, monoamines, regulators of GH gene expression, and second messenger, among which only dexamethasone affected irGH secretion by H9 significantly. Apparently, irGH secretion by lymphocytes is regulated differently from hGH secretion by pituitary cells. One possible explanation is that even though lymphocytes do have GHRH receptors (12), they may lack some of the other receptors which are present in the pituitary. Another possibility is the existence of tissue-specific regulatory mechanisms. Analogous reports can be found in the studies of prolactin secretion by various types of tissues and cells. When human decidual tissues were cultured, known regulators of prolactin secretion in the pituitary such as dopamine or TRH did not affect prolactin secretion from the decidual cells (13). Neither was prolactin secretion by the cell line IM-9-P3, a variant subclone of IM9, affected by most of the hormones known to modulate prolactin secretion in the pituitary (14). It is possible that certain components in the fetal bovine serum may have influences on the results of our assay. For example, somatomedin binding proteins in serum may have bound the somatomedin and led to the negative results. We have performed preliminary experiments by adapting the H9 cells to serum-free H9 cells grown in serum-free medium (data not shown). Similar experiments in the future may help us to separate the effects of serum components from exogenously added stimuli. The concentrations used were in the physiologic range or in a range known to affect pituitary GH secretion. However, it is also possible that the compounds tested may be able to produce an effect on the cells at concentrations much lower than those used in the experiments. It is of interest to note that dexamethasone is the only known regulator of prolactin secretion by IM-9-P3 cells. Similar to our observation, dexamethasone caused a reduction in the pituitary neuropeptide secretion by lymphocytes. Since the cell number and viability did not change significantly after dexamethasone treatment under our culture condition, this reduction in irGH secretion by H9 cells could not be attributed to cytotoxic effects of glucocorticoids. The mechanism of glucocorticoid action on H9 lymphocytes is definitely different from that on pituitary cells, since dexamethasone could stimulate hGH mRNA production (15, 16) and hydrocortisone could stimulate hGH release in pituitary cells (17). In view of general immunosuppressive effects of glucocorticoids, even when dexamethasone was not directly causing lymphoid cell lysis, it may still influence production and secretion of certain proteins by the cells, which may in turn affect the metabolic state of the lymphocytes. There are numerous intricate communications between the neuroendocrine and the immune systems. Previous works in our laboratory and in others (1-7) have supported the idea that pituitary and hypothalamic neuropeptides produced by lymphocytes may be one mode of communication for the neuroendocrine-immune network. In fact, several of the lymphocytederived neuropeptides, such as ACTH (1) and TSH (18), have been found to be regulated by stimulatory or inhibitory factors which control their pituitary counterparts. However, this does not necessarily imply that lymphocyte production of neuropeptides are regulated in exactly the same way as their neuroendocrine counterparts are. For instance, both ACTH and TSH secretions by lymphocytes are also stimulated by T-cell or B-cell mitogens, suggesting that even though the neuroendocrine and the immune systems are related in the anatomical as well as developmental levels and closely interact with each other, the two systems still differ in regulatory mechanisms and functions in a complicated manner. Our
Vol.
51, No.
13, 1992
Dexamethasone
Inhibits
irGH S e c r e t i o n
1039
report that irGH secretion by lymphocyte is regulated differently from pituitary hGH serves as additional illustration of such difference. Acknowledgments This grant was supported partially by the George and Mary Josephine Hamman Foundation and the James W, McLaughlin Fellowship Fund. The authors wish to thank Nita Brannon for help in preparing the manuscript. References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16 17. 18.
E.M. SMITH, A.C. MORRILL, W.J. MEYER, and J.E. BLALOCK. (1986) Nature 322, 881-882. E.M. SMITH, M. PHAN, D. COOPENHAVER, T.E. KRUGER, and J.E. BLALOCK. (1983) Proc. Natl. Acad. Sci. USA 80, 6016-6023. D. HARBOUR-MCMENAMIN, E.M. SMITH, and J.E. BLALOCK. (1986) ICSU Short Reports. Advances in Gene Technology: Molecular Biology of the Endocrine System 4, 378-379. P.C. HIESTAND, P. MEKLER, R. NORDMANN, A. GRIEDER, and C. PERMMONGKOL. (1986) Proc. Natl. Acad. Sci. USA 83, 2599-2603. D.A. WEIGANT, J.B. BAXTER, W.E. WEAN, L.R. SMITH, K.L. BOST, and J.E. BLALOCK. (1988) FASEB J. 2, 2812-2818. N. HATTORI, A. SHIMATSU, M. SUGITA, S. KUMAGI, AND H. IMMURA. (1990) Biochem. Biophys. Res. Commun. 168, 396-401. T.-L. KAO, S.C. SUPOWIT, E.A. THOMPSON, and W.J. MEYER, III. (1992) Cell. Mol. Neurobiol. 12. 483-498. P.F. SMITH, L.S. FRAWLEY, and J.D. NEILL. (1984) Endocrinology 115, 24842486. B.B. MISHELL and S.M. SHIIGI, (eds.). (1980) Selected Methods in Cellular Immunology. W. H. Freeman & Co., San Franscisco. N.M. GOUGH. (1988) Anal. Biochem. 73, 93-95. G.M. WANL, E. ONG, J. MEINKOTH, R. FRANCO and M. BARINAGA. (1981) Method for the Transfer of DNA, RNA and Protein to Nitrocellulose and Diazotized Paper Solid Supports. Schleicher and Schuell, Keene, NH. V. GUARCELLO, D.A. WEIGENT and J.E. BLALOCK. Cell Immunol. 136:291302, 1991. A. GOLANDER, J. BARRETT, T. HURLEY, S. BARRY, and S. HANDWERGER. (1979) J. Clin. Endocrinol. Metab. 49, 787-789. B. GELLERSEN, G.E. DIMATTA, G. FRIESEN, and H.G. BOHNET. (1989) Endocrinology 125, 2853-2861. R.M. EVANS, N.C. BIRNBERG, and M.G. ROSENFELD. (1982) Proc. Natl. Acad. Sci. USA 79, 7659-63. J.R. DAVIS, E.M. WILSON, M.E. VIDAL, A.P. JOHNSON, S.S. LYNCH, and SHEPPARD, M.C. (1989) J. Clin. Endocrinol. Metab. 69, 704-708. E.F. ADAMS, I.E. BRAJKOVICH, and K. MASHITER. (1981) J. Clin. Endocrinol. Metab. 261, 13367-13372. D.V. HARBOUR, T.E. KRUGER, D. COPPENHAVER, E.M. SMITH, and W.J. MEYER. (1989) Moh Cell. Endocrinol. 64, 229-241.
51, pp.
i033-i039
Pergamon
Press
INHIBITION OF IMMUNOREACTIVE GROWTH HORMONE SECRETION
FROM LYMPHOID CELL LINES BY DEXAMETHASONE Ting-Lin Kao and Walter J. Meyer, III
Departments of Human Biological Chemistry & Genetics and Psychiatry & Behavioral Sciences, The University of Texas Medical Branch, Galveston, TX, USA
(Received
in final
form July 14,
1992)
Summar~ The regulation of irGH secretion by the immune system was examined using lymphoid cell lines, H9 and IM9. Using a highly sensitive immunoassay, irGH secretion by H9 was negatively regulated by dexamethasone, whereas many other regulators of hGH secretion, including hormones, monoamines, and second messenger, had no measurable effect on irGH secretion. Treatment of H9 cells with dexamethasone for 48 hours could cause as high as 70% reduction in irGH secretion without affecting either cell numbers or viability. Using IM9, neither growth hormone releasing hormone nor thyrotropin releasing hormone had significant effect on either irGH steady-state level transcripts or irGH secretion. These findings suggest that irGH secretion by lymphocytes was regulated in a different manner from that by pituitary cells. Production of neuroendocrine peptides by lymphoid cells is one proposed mechanism for the communication between the neuroendocrine and the immune systems. It has been demonstrated that lymphocytes produce pituitary hormones such as ACTH (1), TSH (2), endorphin (3), prolactin (4), and growth hormone (5,6). Recently, immunoreactive growth hormone (irGH) production by human peripheral blood lymphocytes has been shown independently by two groups (5,6). In addition, Hattori et a_l. have quantitated irGH secretion by human lymphocytes and have shown that such secretion is regulated in a different manner from that in the anterior pituitary (6). Our group has recently developed model systems for study of irGH secretion using human lymphoid cell lines and have successfully quantitated irGH secretion from H9, a T-cell line, using a highly sensitive double-antibody immunoassay (7). In the present study, we study the regulation of irGH secretion from H9 using the hGH immunoassay and from IM9, a B-cell line, using reverse hemolytic plaque assay and dot blot analysis.
Author to whom reprint requests should be addressed: Walter J. Meyer, III, M.D., Professor and Vice Chairman for Research, Department of Psychiatry & Behavioral Sciences, University of Texas Medical Branch, Galveston, TX 77555-0429 Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1034
Dexamethasone
Inhibits
irGH S e c r e t i o n
Vol.
51, No.
13, 1992
Materials and Methods The B-cell line, IM9, was supplied by Dr. E. Brad Thompson at the University of Texas Medical Branch (UTMB), Galveston, and the T-cell line, H9, was supplied by Dr. Miles Cloyd, UTMB. Cells were cultured in RPMI 1640 or DMEM supplemented with 5% fetal calf serum and antibiotics. Viability was determined by trypan blue exclusion. After incubation with or without various pharmacological agents for 24-48 hours, ceils were harvested by centrifugation (150-200 x g) for 10 minutes. Immunoassay. Secretion of irGH was measured by a highly sensitive double antibody immunoassay as previous described (7). Briefly, blank medium containing various concentrations of hGH or conditioned cultured medium was concentrated 50X by dialysis and lyophilization. Similarly concentrated blank medium with hGH was used to set up standard curves. Concentrated samples (100/xl) were assayed using a commercial hGH kit (Nichols Institute, Allegro hGH kit). The detection limit was lpg/ml. Reverse Hemolytic Plaque Assay. The assay was performed as previously described with several minor modifications (8). Sheep red blood cells (SRBC) were coupled to staphylococcal protein A (Sigma) as previous described (9). Briefly, the coupled SRBC was mixed with an equal volume of lymphocytes resuspended in RPMI-0.1% BSA-antibiotics in a concentration of 1 X 106 cells/ml, antihuman growth hormone antiserum (Chemicon 1:50), and guinea pig complement (Cordis Labs, 1:20). The resulting mixture was infused by capillary action into Cunningham slide chambers in duplicate. The plaques were counted at 10X magnification after overnight incubation. Statistical analysis. The data were expressed as means + SD. Student's t-test and paired t-test were used for statistical analysis. Dot blot analysis. Cytoplasmic RNA was extracted from the lymphocytes using a method described by Gough (10). Bacteria carrying plasmids which contain either the rat or human GH cDNA insert were provided by Dr. Weigent of the University of Alabama, who transformed the bacteria using plasmids donated by Dr. Denoto of UCSF. A 500-bp pvuII fragment containing either the rat or human GH coding region was isolated as probe. Hybridization was carried out according to a standard protocol (1 I). After total RNA was transferred onto 0.45/xm nitrocellulose filter, the blot was incubated with prehybridization buffer (50% formamide, 5X Denhardt's solution, 0.1% SDS, 100/xg/ml of salmon sperm DNA, 5X SSPE) at 42 degrees for 1-2 hours. For hybridization 1'32 nick-translated probe with specific activity of 1-5 x 108 cpm/#g was added to the hybridization buffer (prehybridization buffer with 10% dextran sulfate added) at 1-5 x 106 cpm/ml. After overnight incubation, the blot was washed extensively and autoradiographed for a week.
Vol.
51, No.
13,
1992
Dexamethasone
Inhibits
irGH S e c r e t i o n
1035
Results The secretion of irGH by H9 cells was measured by a highly sensitive double-antibody immunoassay. Typically, H9 cells secret irGH in the order of 10 pg/107 cells in 24-48 hours (7). Cells treated with dexamethasone (10-6M-10-8M) for 48 hours was inhibited in a dosedependent fashion (p < 0.05; Figure 1). Under the culture condition used in our experiment, dexamethasone was not cytotoxic to the cells, as neither the cell number nor the viability changed significantly with treatment of dexamethasone (Figure 2). The secretion of irGH by H9 cells treated with same doses of dexamethasone for only 24 hours was not affected (data not shown). Treatment of H9 cells for 24-48 hours by a variety of agents, including physiological stimuli or inhibitors of pituitary GH secretion (GH releasing hormone [GHRH], somatostatin, somatomedin), monoamines, mitogen, second messenger, and hormones which stimulate GH secretion in vitro, had no appreciable effect on irGH secretion by H9. Table 1 lists all the pharmacological agents tested which have no appreciable effects on H9 secretion of irGH.
200
r-i 0 14 ¢:1¢1 O0
ra~
100
q.,10 01.4 U I,J Q 0
QJ.I
0
0
IO~M
1 0 a M lO'SM lO~Vl
lOa°M
Dex Concentration
FIG 1 H9 irGH secretion in response to various concentrations of dexamethasone. Cells were incubated in 10-6M dexamethasone for 48 hours and the culture supernatants were assayed for irGH content. Average of 3 experiments.
1036
Dexamethasone
I
Inhibits
irGH S e c r e t i o n
Vol.
51, No.
Viability
Rel. Cell #
200~ t-I
0
0 U m
,,
1OO
" I
@
o,,-4
14,-i GI @
mo
O
0
lO'6M
lO'TM
104~M
Dox Concentration FIG 2
H9 cell number and viability after 48 hours of incubation with 106M dexamethasone. Average of 3 experiments. TABLE 1 Pharmacological Stimuli Which Do Not Affect irGH Secretion by H9 Cells. Stimuli
Dose Tested
TRH Somatostatin Somatomedin Epinephrine Norepinephrine Isoproterenol Clonidine Serotonin Insulin Triiodothyronine Lipopolysaccharide Testosterone Estrogen Dibuteryl AMP Thymosin fraction 5
3XlO'TM, 3X lO'9M 1/2g/ml, 10 ng/ml 100 ng/ml, 10 ng/ml, 1 ng/ml 106M, lO-8M 10-6M, 10-8M 10-6M, IO-SM IO-6M, IO-SM 10-6M, 104M, 10-1°M 10-SM, 1OTM, 10-9M 10-TM, 10-9M 50 gg/ml, 5/xg/ml 250 ng/ml, 25 ng/ml 10 ng/ml, 1 ng/ml, 0.1 ng/ml 10-SM, 10-6M 1 /zg/ml, 10 ng/ml, 0.1 ng/ml
13, 1992
Vol.
51, No.
13,
1992
Dexamethasone
Inhibits
irGH S e c r e t i o n
1037
As previously reported, IM9, a B-cell line, secreted irGH at an extremely low level such that its irGH secretion could not be accurately quantitated using the double-antibody immunoassay (7). We examined the effects of 10SM GHRH and .275 x 10-6M thyrotropin releasing hormone (TRH) on irGH production and secretion by IM9 cells using reverse hemolytic plaque assay and dot blot analysis. In a hemolytic plaque assay using 5X104 cells per slide, cells incubated in plain media produced 32 _+ 13 plaques, compared to cells incubated in media with GHRH providing 62 _+ 23 plaques. The differences were not statistically significant (p=. 13 by paired t-test, n=3). TRH stimulation had no effect on the number of plaque-producing lymphocytes. Consistent with this finding, dot blot analysis revealed no significant increase in the amount of message detected with either rat of human GH cDNA probe after GHRH or TRH treatment (Figure 3).
A
B
GH3
GH3
L
L
MOCK
MOCK
GHRH
GHRH
TRH
TRH FIG 3
Dot blot of RNA with either rGH or hGH cDNA probes using GH 3 cells, L cells, and IM9 cells. The IM9 cells were incubated with plain media (MOCK), plain media plus 1OSM GHRH, and plain media plus .275 x 10-6M TRH. A. Rat probe; GH 3 RNA, 2/~g, l#g; L cell RNA: 10/xg, 5/xg; IM9 RNA, 40~g, 20~g. B. Human probe; GH 3 RNA, 2/xg, ltxg, 0.5~g; L cell RNA, 10/xg, 5~g, 2.5#g; IM9 RNA: 40/~g, 20/~g, 10#g.
Discussion In this study, we have tested a wide spectrum of naturally-occurring and synthetic compounds which affected hGH secretion in vitro and/or in vivo and found that most of the agents
1038
Dexamethasone
Inhibits
irGH S e c r e t i o n
Vol.
51, No.
13, 1992
examined have no measurable effect on irGH secretion by H9 cells. This observation confirmed the earlier report by Hattori et. al. (6), whose group showed that neither GHRH nor a somatostatin analogue had effect on irGH secretion by peripheral blood lymphocytes. In addition to GHRH and somatostatin, we have also studied the effects of several other neuropeptides, monoamines, regulators of GH gene expression, and second messenger, among which only dexamethasone affected irGH secretion by H9 significantly. Apparently, irGH secretion by lymphocytes is regulated differently from hGH secretion by pituitary cells. One possible explanation is that even though lymphocytes do have GHRH receptors (12), they may lack some of the other receptors which are present in the pituitary. Another possibility is the existence of tissue-specific regulatory mechanisms. Analogous reports can be found in the studies of prolactin secretion by various types of tissues and cells. When human decidual tissues were cultured, known regulators of prolactin secretion in the pituitary such as dopamine or TRH did not affect prolactin secretion from the decidual cells (13). Neither was prolactin secretion by the cell line IM-9-P3, a variant subclone of IM9, affected by most of the hormones known to modulate prolactin secretion in the pituitary (14). It is possible that certain components in the fetal bovine serum may have influences on the results of our assay. For example, somatomedin binding proteins in serum may have bound the somatomedin and led to the negative results. We have performed preliminary experiments by adapting the H9 cells to serum-free H9 cells grown in serum-free medium (data not shown). Similar experiments in the future may help us to separate the effects of serum components from exogenously added stimuli. The concentrations used were in the physiologic range or in a range known to affect pituitary GH secretion. However, it is also possible that the compounds tested may be able to produce an effect on the cells at concentrations much lower than those used in the experiments. It is of interest to note that dexamethasone is the only known regulator of prolactin secretion by IM-9-P3 cells. Similar to our observation, dexamethasone caused a reduction in the pituitary neuropeptide secretion by lymphocytes. Since the cell number and viability did not change significantly after dexamethasone treatment under our culture condition, this reduction in irGH secretion by H9 cells could not be attributed to cytotoxic effects of glucocorticoids. The mechanism of glucocorticoid action on H9 lymphocytes is definitely different from that on pituitary cells, since dexamethasone could stimulate hGH mRNA production (15, 16) and hydrocortisone could stimulate hGH release in pituitary cells (17). In view of general immunosuppressive effects of glucocorticoids, even when dexamethasone was not directly causing lymphoid cell lysis, it may still influence production and secretion of certain proteins by the cells, which may in turn affect the metabolic state of the lymphocytes. There are numerous intricate communications between the neuroendocrine and the immune systems. Previous works in our laboratory and in others (1-7) have supported the idea that pituitary and hypothalamic neuropeptides produced by lymphocytes may be one mode of communication for the neuroendocrine-immune network. In fact, several of the lymphocytederived neuropeptides, such as ACTH (1) and TSH (18), have been found to be regulated by stimulatory or inhibitory factors which control their pituitary counterparts. However, this does not necessarily imply that lymphocyte production of neuropeptides are regulated in exactly the same way as their neuroendocrine counterparts are. For instance, both ACTH and TSH secretions by lymphocytes are also stimulated by T-cell or B-cell mitogens, suggesting that even though the neuroendocrine and the immune systems are related in the anatomical as well as developmental levels and closely interact with each other, the two systems still differ in regulatory mechanisms and functions in a complicated manner. Our
Vol.
51, No.
13, 1992
Dexamethasone
Inhibits
irGH S e c r e t i o n
1039
report that irGH secretion by lymphocyte is regulated differently from pituitary hGH serves as additional illustration of such difference. Acknowledgments This grant was supported partially by the George and Mary Josephine Hamman Foundation and the James W, McLaughlin Fellowship Fund. The authors wish to thank Nita Brannon for help in preparing the manuscript. References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16 17. 18.
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