- Email: [email protected]
The effect of growth hormone on the proliferation of human Th cell clones
Life Sciawes, Vol. 66, No. 20, pp. 1929-1935,ZooO Copyright 0 2000 Elsevier Science Inc. Rinted in the USA. All ri@s mewed 0024-3205/006-see timt matter
PII 50024-3205(00)00518-X
THE EFFECT OF GROWTH HORMONE ON THE PROLIFERATION CELL CLONES
OF HUMAN Th
Naomi Yamashita’, Yoshihide Hashimoto ‘, Masaru Honjo3 and Naohide Yamashita4 ‘Department of Medicine, Teikyo University School of Medicine, Tokyo 173-8045 Japan; * Institute of Biological Science, Mitsui Pharmaceuticals, Mobara, Chiba, 297-00 17 Japan; 3Life Science Laboratory, Mitui Chemicals, Mobara, Chiba 297-0017, Japan; 4Department of Advanced Medical Science, Institute of Medical Science University of Tokyo, Tokyo 108-8639 Japan (Received in final form December 10,1999)
Summary The effects of human growth hormone (hGH) on human Th cell clones were examined. Both 20K and 22K hGH stimulated the proliferation of Th2 and ThO cells in the presence of mite antigen, whereas they did not stimulate the proliferation of Thl cells. Because the effect of 20K hGH was almost the same as that of 22KhGH, it was suggested that the action of hGH was not mediated through The application of growth prolactin receptor but through hGH receptors. In Th2 hormone binding protein (GHBP) inhibited the cell growth of Thl clones. However, and ThO cells GHBP inihibited the hGH-stimulated cell proliferation. GHBP alone did not affect the proliferation of Th2 and ThO cells. hGH was detected in the supematant of Thl clones in the presence of mite antigen but it was hGH was detected in one out of 4 batches of ThO not detected in Th2 clones. clones. These data indicated that hGH was secreted from Thl clones, and that ThO clones possessed characteristics of both Th2 and ThO clones. Key WCW-ds:Thl, Th2, ThO, growth hormone, human growth hormone
It has been demonstrated that receptors for growth hormone (GH) exist on the membrane of lymphocytes (l-3), and that GH participates in the differentiation of T cells in the thymus (1,4,5), where GH is synthesized at the epithelial cells (4). The main source of the circulating GH is anterior pituitary. However, much attention has recently been paid to the relationship between neuroendocrine and immune systems (1,6), because lymphocytes synthesize and secrete GH (7,8), and because GH is important for the proliferation of lymphocytes (9,lO). CD4+ T lymphocyte populations can be characterized by the marked variation in their cytokine production patterns, and they are divided into three different subsets: Thl, Th2 and ThO cells (1 l13). Thl cells, which secrete IL-2 and INF-y, are involved in the cellular immunity, and Th2 cells, which secrete IL-4, IL-5 and IL-13, are responsible for humoral immunity and IgE-mediated allergic responses (1 l-1 3). ThO cells produce both Thl and Th2 cytokines (13). Although Address correspondence: Naohide Yamashita, M.D., Ph.D. Department of Advanced Medical Science, Institute of Medical Science University 1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan TEL:xx-81-3-5449-5698, FAXxx-81-3-5449-5456, Email:[email protected]
of Tokyo, 4-3-
1930
Effect of GH on Human ‘I%Cell Clones
Vol. 66. No. 20,200O
Hattori et al. have reported that GH affects cytokine production in T lymphocytes (7), the effect of GH on T cell subpopulations has not been investigated. Because T cell subpopulations possess their own functions, it is necessary to examine the effect of GH in the respective populations. In the present study, therefore, we explored the effect of human GH (hGH) on Thl , ll-12 and ThO cells using human antigen-specific T cell clones. There are two forms of hGH. The majority is 22K hGH, whereas 20K hGH, a variant form of hGH (14), is about 10 % of circulating hGH. In the present study, we investigated the effects of both forms of hGH on T cell clones. Methods
Reagents 22K hGH (Genotropin) was purchased from Sumitomo Pharmaceuticals (Osaka, Japan). 20K hGH was produced as described previously (14) and hGH-binding protein (GHBP) was prepared as described by Wada et al. (15). Donors Th2 and ThO clones were established from three Dermatophagoides farinae (Der f)-sensitive atopic asthmatic patients. Thl clones were obtained from three purified protein derivative (PPD)-sensitive normal volunteers. Human studies committee approval and individual informed consent from each donor was obtained before we conducted these studies. T cell cloning Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrihrgation. Cells were incubated with 10 @ml Der f (kindly gifted by Torii Pharmaceutical Co, Ltd., Tokyo, Japan) or 5 &ml PPD (Kyowa Pharmaceutical Co, Tokyo, Japan) in flatbottomed 24-well plates in RPM11640 medium supplemented with 10% human AB serum (C-six Diagnostics Inc., Mequon, WI). After 7-day culture, blast cells were harvested, and cloned by limiting dilution method or micropipetting method in round-bottomed 96-well plates, as previously described (16). Cloning was carried out in the same medium supplemented with 10% human AB serum, antigen (Ag) (Df or PPD), 5 ng/mi recombinant IL-2 (Takeda Chemical Industries Ltd., Osaka, Japan), and autologous irradiated (4000 rad) PBMCs as a source of antigen-presenting cells (APCs). Cells were fed weekly with 2 ng/ml IL-2. On day 21, cultures were expanded in 24well plates with irradiated autologous APCs, Ag, and IL-2 at 2 @ml. Clones were further incubated for 7 days, and tested for antigen-specificity by 72-hour .proliferation assay of the incorporation of [3H]-TdR (Amersham Corp., Arlington Heights, IL) for the last 16 hours of the The clones, which exhibited a stimulation index of more than five and were culture. unresponsive to irrelevant Ags, were regarded as antigen-specific T cell clones (stimulation index = the mean cpm of triplicate cultures with stimuli / the mean cpm of triplicate cultures without any stimuli). Three types of T cell clones, ThO (which produce both Thl and Th2 cytokines), Thl, and Th2 cells were determined by their ability to produce IL-4, IL-5, IFN-y and TNF-g. T cell clones, which produced IL-4 and IL-5 with no production of IFN-)Iand TNF-P, were included in Th2. The clones, which secreted IFN-y and TNF-P with no IL-4 and IL-5, were categorized into Thl cells. The clones, which produced both Thl and Th2 cytokines, were categorized into ThO cells as reported previously (16). To investigate the effects of hGH on cell proliferation, the clones with irradiated APCs in RPM11640 medium containing 2% fetal calf serum were rested for 12 hours, followed by the stimulation of relevant antigen in the presence or absence of 22K or 20K hGH for 72 hours. The proliferation was assessed by the incorporation of [‘HI-TdR for the last 16 hours of the culture. Measurement of hGH by ELI&A The content of hGH in the supematant of the culture was measured using ELISA as described previously (17), with minor modifications. In brief, the microtiter plates were coated with 10
Effect of GH on Human Th Cell Clones
Vol. 66, No. 20,200O
1931
&ml monoclonal anti-hGH antibody A36020047P (BiosPacific Inc., Emeryville, CA). Remaining protein-binding sites were blocked with Block Ace (Dainippon Pharmaceuticals, Osaka, Japan). After incubation with the sample and washing, monoclonal anti-hGH antibody D14 (0.5 &ml, Mitsui Pharmaceuticals, Tokyo, Japan) was applied, followed by the addition of 3,3’,5,5’The reaction was terminated tetramethyl-benzidine (TMB) substrate (Mitsui Pharmaceuticals). with 1N sufliuic acid and the absorbance was determined at 450 run against a reference of 620 nm. The detection limit of hGH was 10 pg/ml.
Statistical analysis For comparisons of multiple parameters, we used analysis of variance (ANOVA). ~0.05 were considered to be statistically significant.
Values of p
Results
Egects of hGH on the mite antigen-stimulated proliferation of the Th cell clones Mite antigen stimulated the Th2 cell Fig. 1A shows the effect of hGH on Th2 cell proliferation. hGH at concentrations of I and 10 rig/ml significantly growth about by 130 times of the control. enhanced the mite antigen-stimulated Th2 cell proliferation, and the effects of 20K and 22K hGH were not different. At lower or higher concentration of hGH, the stimulatory effect was not significant. hGH did not affect the proliferation of Th2 cells in the absence of mite antigen (data not shown). Because 5 r&I GHBP significantly inhibited the hGH-stimulated proliferation of Ba/F3 cells (15), we used 5 nM GHBP in this study. The addition of GHBP (5 nM) diminished the effect of both 20K and 22K hGH (Fig. IB), indicating that the effect of hGH on Th2 cells was The presence of GHBP in itself did not change the mediated through specific receptors. proliferation of Th2 cells. A
Th2
cm
1'
clone *pc00t
/
B wm
*p
Effects of hGH and GHBP on the proliferation of Th2 clone. A, effects of 22K and 20K hGH on the proliferation of Th2 cells. Mean f SE (n=3) is shown. B, effects of GHBP on hGH-stimulated Th2 cell proliferation. “mite Ag” indicates the addition of mite antigen. Mean f SE (n=4) is shown. *, statistically significant, pcO.01 or ~~0.05 as indicated in the figure.
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Effect of GH on Human Tb Cell Clones
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The effect of hGH on Thl cell proliferation is depicted in Fig. 2. Both 20K and 22K hGH did not stimulate the mite antigen-stimulated cell ‘growth of Thl clone. However, the addition of GHBP significantly inhibited the mite antigen-stimulated cell proliferation. hGH did not affect the proliferation of Thl cells in the absence of mite antigen (data not shown). cpm
2
Thl
* 0c0.01
clone
oe+4
1.5e+4
1 .os+4
5.0e+3
o.oe+o c 0 n tro
I
m GHBP
22KG 100
ite
A g
H
10
20KG 1
1
100
10
H 1
1
np,m
GHBP
GHBP
Fig.2 Effects of hGH and GHBP on the proliferation of Thl clone. “mite Ag” indicates the addition of mite antigen. Mean f SE (n=4) is shown *, statistically significant, pco.0 1.
ThO
clone
Ill
GHBP
100
22KGH 10
Ita Al 1
1 GH5P
too
20KGH 10
1
1 nplml GHBP
Fig.3 Effects of hGH and GHBP on the proliferation of ThO clone. “mite Ag” indicates the addition of mite antigen. Mean f SE (n=4) is shown *, statistically significant, pco.01.
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Effect of GH on Human Th Cell Clones
Vol. 66, No. 20,200O
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The effect of hGH on ThO cells was similar to that on Th2 cells. As shown in Fig. 3, hGH at a concentration of 1 @ml significantly stimulated the proliferation of ThO cells, and the addition of GHBP inhibited this stimulation. Again, hGH did not affect the cell growth in the absence of mite antigen (data not shown).
Secretion of hGHfrom Th clones Because GHBP inhibited the cell proliferation of Thl cells (Fig. 2), it was suggested that hGH was secreted from Thl cells. In order to clarify this notion we measured hGH in the supematant of culture media of Th clones. Thl cells did not secrete detectable hGH in the absence of mite antigen, whereas hGH release was stimulated in the presence of mite antigen (Table 1). Mean value of hGH secreted from mite antigen-stimulated Thl cells was 39.0 f 6.87 pg/m1/24hr/106 cells (mean f SE)(n=8). hGH was not detected in the supematant of mite antigen-stimulated Th2 cells. In ThO cells hGH was detectable only in 1. batch, but was undetectable in other 3 batches. TABLE 1 ThO
Thl
Th2
38 49 74 42 14 13 41 41
t
GH secreted from Th clones (pg/m1/24hr/106 cells)
Discussion The results of the present study revealed that both 20K and 22K hGH stimulated the proliferation of Th2 and ThO cells in the presence of mite antigen. Although the proliferation of Thl cells was not stimulated by hGH, the experiments of GHBP and hormone assay revealed that hGH was secreted from Thl cells. It has been reported hGH is produced by human peripheral blood (7). However, this is the first report that hGH is mainly produced by Thl cells. Thl cells participate in the cellular immunity, and Th2 cells, in humoral immunity. ThO cells are intermediate between Thl and Th2 cells (13). ThO cells change their characteristics to those of Thl or Th2 cells by various cytokines (18). In the present study ThO cells showed similar characteristics to Th2 cells in the case of cell proliferation. However, hGH secretion pattern of ThO cells was different from that of Th2 cells and in one batch hGH was dectected similar to the case of Thl cells. Thus ThO cells possessed intermediate characteristics of both Thl and Th2 cells in the present results. hGH significantly stimulated the proliferation of Th2 cells at the concentrations of 1 and 10 ng/ml. In the case of ThO cells statistical significance was obtained only at 1 ng/ml hGH probably because the stimulatory effect of hGH on Th2 cells was greater than that of ThO cells. The finding that the addition of GHBP abolished the effect of hGH indicates the specific action of hGH, which was mediated through its specific receptor. The majority of circulating hGH is 22K hGH. The 20K hGH is formed by alternative splicing of mRNA from pituitary-expressed hGH-N gene and consequently lacks residue 32-46 of 22K hGH (19,20). This deleted region is involved in the interface with both the GH receptor and the
1934
Effect of GH on Human Th Cell Clones
Vol. 66,No.20,2000
pro&tin receptor in 22K hGH (21). Thus, 20K hGH poorly binds to prolactin receptors, Three mechanisms have although 22K hGH binds to both hGH and prolactin receptors (22). The first is been postulated in concerning with the effects of hGH on immune system (1). through the effects of prolactin receptors, the second is through insulin-like growth factor-l (IGFAlthough prolactin also augments T cell 1) and the third is the direct effects of hGH. proliferation (l), the effects on Th cell clone proliferation were not different between 20K and 22K The effects via IGF-1 and hGH. Therefore, the effects through prolactin receptors are unlikely. the direct effects of hGH are probable. Although many effects of GH on immune systems have been reported through the effects of IGF-1 (1,10,23), there exist direct effects of GH (24,25). Administration of hGH at the time of The precise mechanism should be fUrther investigated. human immuno-deficiency virus envelope protein (HIV-1-gp120) provokes a change in the In mice a cytokine production pattern with a preferential switch from Th2 to Thl cells (26). conversion of Th cell populations from Thl to Th2 occurs after burn and administration of GH can reverse this change (27). Thus GH appears to control Th cell responses in other systems.
1. 2. 3.
4. 5. 6. 7. 8. 9. 10. Il. 12. 13. ’ 14.
15. 16. 17.
18. 19. 20.
References B. VELKENIERS, Z. DOGOUSAN, F. NAESSENS, R. HOOGHE and E.L. HOOGHEPETERS, Cell. Mol. Life Sci. 54 1102-l 108 (1998). R. BADOLATO, H.E. BOND, G. VALERIO, A. PETRELLA, G. MARRONE, S. VENUTA, M.J. WALTERS and A. TERANORE, J. Clin. Endocrinol. Metab. 79 984-990 (1994). R. RAPAPORT, I.N. SILLS, L. GREEN, P. BARRETT, J. LABUS, K.A. SKUSA, A. CHARTOFF, L. GOODE, M. STENE and B. PETERSEN, J. Clin. Endocrinol. Metab. 80 2612-2629 (1995). K.W. KELLEY, Biochem. Pharmacol. 38 705 713 (1989). N. MAGGIANO, M. PIANTELLI, R. RICCI, L.M. LAROCCA, A. CAPELLI and F.O. RANELLETTI, J. Histochem. Cytochem. 42 1349-1354 (1994). R. CLARK, Endocrine Rev. 18 157-179 (1997). N. HATTORI, K. IKEKUBO, T. ISHIHARA, K. MORIDERA, M. HINO and H. KURAHACHI, J. Clin. Endocrinol. Metab. 79 1678-1680 (1994). S. VARMA, P. SABHARWAL, J.F. SHERIDAN and W.B. MALARKEY, J. Clin. Endocrinol. Metab. 76 49-53 (1993). K.E. MERCOLA, M.J. CLINE and D.W. GOLDE, Blood 117 596-599 (1990). M.E. GEFFNER, N. BERSCH, B.M. LIPPE, R.G. ROSENFELD, R.L. HINTZ and D.W. GOLDE. J. Clin. Endocrinol. Metab. 71 464-469 (1990) T.R. MOSMANN, M.W. CHERWINSKI, M.A. BOND, M.A. GIEDLIN and R.L. COFFMAN, J. Immunol. 136 2348-2357 (1986). T.R. MOSMANN and R.L. COFFMAN, Ann. Rev. Immunol. 7 145-173 (1989). S. ROMAGNANI, Ann. Rev. Immunol. 12 227-257 (1994). H. UCHIDA, N. NAITO, N. ASADA, M. WADA, M. IKEDA, H. KOBAYASHI, M. ASANAGI, K. MORI, Y. FUJITA, K. KONDA, N. KUSUHARA, T. KAMIOKA, N. NAKASHIMA and M. HONJO. J. Biotechnol. 55 10 1- 112 (1997). M. WADA, H. UCHIDA, M. IKEDA, B. TSUNEKAWA, N. NAITO, S. BABA, E. TANAKA, Y. HASHIMOTO and M. HONJO, Mol. Endocrinol. 12 146-156 (1997) N. YAMASHITA, M. TAKENO, S. KANEKO, and T. SAKANE, Clin. Exp. Immunol. 107 332-345 (1997). T. TSUSHIMA, Y. KATO, Y. MIYACHI, K. CHIHARA, A. TERAMOTO, M. IRIE, Y. HASHIMOTO and STUDY GROUP OF 20K hGH, J. Clin Endocrinol. Metab. 84 317-322 (1999). G. TRINCHIERI, Ann. Rev. Immunol. 13 25 l-76 (1995). D.D. DENOTE, D.D. MOORE and H.M. GOODMAN, Nucleic Acid Res. 9 3719-3730 (1981). U.J. LEWIS, L.F. BONEWALD and L.J LEWIS, Biochem. Biophys. Res. Commun. 92 511-
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516 (1980). 21. B.C. CUNNINGHAM, S. BASS, G. FUH and J.A. WELLS, Science 250 1709-1712 (1990). 22. H. RUI, J.Y. DJEU, G.A. EVANS, P.A. KELLY and W.L. FARRAR, J. Biol. Chem. 267 24076-2408 1 (1992) 23. W.D. SALMON and V.J. BURKHALTER, J. Lab. Clin. Med. 129 430-438 (1997). 24. W.J. MURPHY, S.K. DURUM and D.L. LONGO, J. Exp. Med. 178 231-236 (1993). 25. R.A. GJERSET, J. YEARGIN, S.K VOLKMAN, V. VILA, J. ARYA and M. HAAS, J. Irnmunol. 145 3497-3501 (1990). 26. M. MELLADO, M. LLORENTE, J.M. RODRIGUES-FRADE, P. LUCAS, C. MARTINEZ and G. DEL REAL, Vaccine 16 1 1 1 l- 1115 (1998). 27. K. TAKAGI, F. SUZUKI, R. E. BARROW, S. E. WOLF and D.N. HERNDON, Ann. Surg. 228 106-111 (1998).
PII 50024-3205(00)00518-X
THE EFFECT OF GROWTH HORMONE ON THE PROLIFERATION CELL CLONES
OF HUMAN Th
Naomi Yamashita’, Yoshihide Hashimoto ‘, Masaru Honjo3 and Naohide Yamashita4 ‘Department of Medicine, Teikyo University School of Medicine, Tokyo 173-8045 Japan; * Institute of Biological Science, Mitsui Pharmaceuticals, Mobara, Chiba, 297-00 17 Japan; 3Life Science Laboratory, Mitui Chemicals, Mobara, Chiba 297-0017, Japan; 4Department of Advanced Medical Science, Institute of Medical Science University of Tokyo, Tokyo 108-8639 Japan (Received in final form December 10,1999)
Summary The effects of human growth hormone (hGH) on human Th cell clones were examined. Both 20K and 22K hGH stimulated the proliferation of Th2 and ThO cells in the presence of mite antigen, whereas they did not stimulate the proliferation of Thl cells. Because the effect of 20K hGH was almost the same as that of 22KhGH, it was suggested that the action of hGH was not mediated through The application of growth prolactin receptor but through hGH receptors. In Th2 hormone binding protein (GHBP) inhibited the cell growth of Thl clones. However, and ThO cells GHBP inihibited the hGH-stimulated cell proliferation. GHBP alone did not affect the proliferation of Th2 and ThO cells. hGH was detected in the supematant of Thl clones in the presence of mite antigen but it was hGH was detected in one out of 4 batches of ThO not detected in Th2 clones. clones. These data indicated that hGH was secreted from Thl clones, and that ThO clones possessed characteristics of both Th2 and ThO clones. Key WCW-ds:Thl, Th2, ThO, growth hormone, human growth hormone
It has been demonstrated that receptors for growth hormone (GH) exist on the membrane of lymphocytes (l-3), and that GH participates in the differentiation of T cells in the thymus (1,4,5), where GH is synthesized at the epithelial cells (4). The main source of the circulating GH is anterior pituitary. However, much attention has recently been paid to the relationship between neuroendocrine and immune systems (1,6), because lymphocytes synthesize and secrete GH (7,8), and because GH is important for the proliferation of lymphocytes (9,lO). CD4+ T lymphocyte populations can be characterized by the marked variation in their cytokine production patterns, and they are divided into three different subsets: Thl, Th2 and ThO cells (1 l13). Thl cells, which secrete IL-2 and INF-y, are involved in the cellular immunity, and Th2 cells, which secrete IL-4, IL-5 and IL-13, are responsible for humoral immunity and IgE-mediated allergic responses (1 l-1 3). ThO cells produce both Thl and Th2 cytokines (13). Although Address correspondence: Naohide Yamashita, M.D., Ph.D. Department of Advanced Medical Science, Institute of Medical Science University 1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan TEL:xx-81-3-5449-5698, FAXxx-81-3-5449-5456, Email:[email protected]
of Tokyo, 4-3-
1930
Effect of GH on Human ‘I%Cell Clones
Vol. 66. No. 20,200O
Hattori et al. have reported that GH affects cytokine production in T lymphocytes (7), the effect of GH on T cell subpopulations has not been investigated. Because T cell subpopulations possess their own functions, it is necessary to examine the effect of GH in the respective populations. In the present study, therefore, we explored the effect of human GH (hGH) on Thl , ll-12 and ThO cells using human antigen-specific T cell clones. There are two forms of hGH. The majority is 22K hGH, whereas 20K hGH, a variant form of hGH (14), is about 10 % of circulating hGH. In the present study, we investigated the effects of both forms of hGH on T cell clones. Methods
Reagents 22K hGH (Genotropin) was purchased from Sumitomo Pharmaceuticals (Osaka, Japan). 20K hGH was produced as described previously (14) and hGH-binding protein (GHBP) was prepared as described by Wada et al. (15). Donors Th2 and ThO clones were established from three Dermatophagoides farinae (Der f)-sensitive atopic asthmatic patients. Thl clones were obtained from three purified protein derivative (PPD)-sensitive normal volunteers. Human studies committee approval and individual informed consent from each donor was obtained before we conducted these studies. T cell cloning Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrihrgation. Cells were incubated with 10 @ml Der f (kindly gifted by Torii Pharmaceutical Co, Ltd., Tokyo, Japan) or 5 &ml PPD (Kyowa Pharmaceutical Co, Tokyo, Japan) in flatbottomed 24-well plates in RPM11640 medium supplemented with 10% human AB serum (C-six Diagnostics Inc., Mequon, WI). After 7-day culture, blast cells were harvested, and cloned by limiting dilution method or micropipetting method in round-bottomed 96-well plates, as previously described (16). Cloning was carried out in the same medium supplemented with 10% human AB serum, antigen (Ag) (Df or PPD), 5 ng/mi recombinant IL-2 (Takeda Chemical Industries Ltd., Osaka, Japan), and autologous irradiated (4000 rad) PBMCs as a source of antigen-presenting cells (APCs). Cells were fed weekly with 2 ng/ml IL-2. On day 21, cultures were expanded in 24well plates with irradiated autologous APCs, Ag, and IL-2 at 2 @ml. Clones were further incubated for 7 days, and tested for antigen-specificity by 72-hour .proliferation assay of the incorporation of [3H]-TdR (Amersham Corp., Arlington Heights, IL) for the last 16 hours of the The clones, which exhibited a stimulation index of more than five and were culture. unresponsive to irrelevant Ags, were regarded as antigen-specific T cell clones (stimulation index = the mean cpm of triplicate cultures with stimuli / the mean cpm of triplicate cultures without any stimuli). Three types of T cell clones, ThO (which produce both Thl and Th2 cytokines), Thl, and Th2 cells were determined by their ability to produce IL-4, IL-5, IFN-y and TNF-g. T cell clones, which produced IL-4 and IL-5 with no production of IFN-)Iand TNF-P, were included in Th2. The clones, which secreted IFN-y and TNF-P with no IL-4 and IL-5, were categorized into Thl cells. The clones, which produced both Thl and Th2 cytokines, were categorized into ThO cells as reported previously (16). To investigate the effects of hGH on cell proliferation, the clones with irradiated APCs in RPM11640 medium containing 2% fetal calf serum were rested for 12 hours, followed by the stimulation of relevant antigen in the presence or absence of 22K or 20K hGH for 72 hours. The proliferation was assessed by the incorporation of [‘HI-TdR for the last 16 hours of the culture. Measurement of hGH by ELI&A The content of hGH in the supematant of the culture was measured using ELISA as described previously (17), with minor modifications. In brief, the microtiter plates were coated with 10
Effect of GH on Human Th Cell Clones
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1931
&ml monoclonal anti-hGH antibody A36020047P (BiosPacific Inc., Emeryville, CA). Remaining protein-binding sites were blocked with Block Ace (Dainippon Pharmaceuticals, Osaka, Japan). After incubation with the sample and washing, monoclonal anti-hGH antibody D14 (0.5 &ml, Mitsui Pharmaceuticals, Tokyo, Japan) was applied, followed by the addition of 3,3’,5,5’The reaction was terminated tetramethyl-benzidine (TMB) substrate (Mitsui Pharmaceuticals). with 1N sufliuic acid and the absorbance was determined at 450 run against a reference of 620 nm. The detection limit of hGH was 10 pg/ml.
Statistical analysis For comparisons of multiple parameters, we used analysis of variance (ANOVA). ~0.05 were considered to be statistically significant.
Values of p
Results
Egects of hGH on the mite antigen-stimulated proliferation of the Th cell clones Mite antigen stimulated the Th2 cell Fig. 1A shows the effect of hGH on Th2 cell proliferation. hGH at concentrations of I and 10 rig/ml significantly growth about by 130 times of the control. enhanced the mite antigen-stimulated Th2 cell proliferation, and the effects of 20K and 22K hGH were not different. At lower or higher concentration of hGH, the stimulatory effect was not significant. hGH did not affect the proliferation of Th2 cells in the absence of mite antigen (data not shown). Because 5 r&I GHBP significantly inhibited the hGH-stimulated proliferation of Ba/F3 cells (15), we used 5 nM GHBP in this study. The addition of GHBP (5 nM) diminished the effect of both 20K and 22K hGH (Fig. IB), indicating that the effect of hGH on Th2 cells was The presence of GHBP in itself did not change the mediated through specific receptors. proliferation of Th2 cells. A
Th2
cm
1'
clone *pc00t
/
B wm
*p
Effects of hGH and GHBP on the proliferation of Th2 clone. A, effects of 22K and 20K hGH on the proliferation of Th2 cells. Mean f SE (n=3) is shown. B, effects of GHBP on hGH-stimulated Th2 cell proliferation. “mite Ag” indicates the addition of mite antigen. Mean f SE (n=4) is shown. *, statistically significant, pcO.01 or ~~0.05 as indicated in the figure.
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The effect of hGH on Thl cell proliferation is depicted in Fig. 2. Both 20K and 22K hGH did not stimulate the mite antigen-stimulated cell ‘growth of Thl clone. However, the addition of GHBP significantly inhibited the mite antigen-stimulated cell proliferation. hGH did not affect the proliferation of Thl cells in the absence of mite antigen (data not shown). cpm
2
Thl
* 0c0.01
clone
oe+4
1.5e+4
1 .os+4
5.0e+3
o.oe+o c 0 n tro
I
m GHBP
22KG 100
ite
A g
H
10
20KG 1
1
100
10
H 1
1
np,m
GHBP
GHBP
Fig.2 Effects of hGH and GHBP on the proliferation of Thl clone. “mite Ag” indicates the addition of mite antigen. Mean f SE (n=4) is shown *, statistically significant, pco.0 1.
ThO
clone
Ill
GHBP
100
22KGH 10
Ita Al 1
1 GH5P
too
20KGH 10
1
1 nplml GHBP
Fig.3 Effects of hGH and GHBP on the proliferation of ThO clone. “mite Ag” indicates the addition of mite antigen. Mean f SE (n=4) is shown *, statistically significant, pco.01.
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The effect of hGH on ThO cells was similar to that on Th2 cells. As shown in Fig. 3, hGH at a concentration of 1 @ml significantly stimulated the proliferation of ThO cells, and the addition of GHBP inhibited this stimulation. Again, hGH did not affect the cell growth in the absence of mite antigen (data not shown).
Secretion of hGHfrom Th clones Because GHBP inhibited the cell proliferation of Thl cells (Fig. 2), it was suggested that hGH was secreted from Thl cells. In order to clarify this notion we measured hGH in the supematant of culture media of Th clones. Thl cells did not secrete detectable hGH in the absence of mite antigen, whereas hGH release was stimulated in the presence of mite antigen (Table 1). Mean value of hGH secreted from mite antigen-stimulated Thl cells was 39.0 f 6.87 pg/m1/24hr/106 cells (mean f SE)(n=8). hGH was not detected in the supematant of mite antigen-stimulated Th2 cells. In ThO cells hGH was detectable only in 1. batch, but was undetectable in other 3 batches. TABLE 1 ThO
Thl
Th2
38 49 74 42 14 13 41 41
t
GH secreted from Th clones (pg/m1/24hr/106 cells)
Discussion The results of the present study revealed that both 20K and 22K hGH stimulated the proliferation of Th2 and ThO cells in the presence of mite antigen. Although the proliferation of Thl cells was not stimulated by hGH, the experiments of GHBP and hormone assay revealed that hGH was secreted from Thl cells. It has been reported hGH is produced by human peripheral blood (7). However, this is the first report that hGH is mainly produced by Thl cells. Thl cells participate in the cellular immunity, and Th2 cells, in humoral immunity. ThO cells are intermediate between Thl and Th2 cells (13). ThO cells change their characteristics to those of Thl or Th2 cells by various cytokines (18). In the present study ThO cells showed similar characteristics to Th2 cells in the case of cell proliferation. However, hGH secretion pattern of ThO cells was different from that of Th2 cells and in one batch hGH was dectected similar to the case of Thl cells. Thus ThO cells possessed intermediate characteristics of both Thl and Th2 cells in the present results. hGH significantly stimulated the proliferation of Th2 cells at the concentrations of 1 and 10 ng/ml. In the case of ThO cells statistical significance was obtained only at 1 ng/ml hGH probably because the stimulatory effect of hGH on Th2 cells was greater than that of ThO cells. The finding that the addition of GHBP abolished the effect of hGH indicates the specific action of hGH, which was mediated through its specific receptor. The majority of circulating hGH is 22K hGH. The 20K hGH is formed by alternative splicing of mRNA from pituitary-expressed hGH-N gene and consequently lacks residue 32-46 of 22K hGH (19,20). This deleted region is involved in the interface with both the GH receptor and the
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pro&tin receptor in 22K hGH (21). Thus, 20K hGH poorly binds to prolactin receptors, Three mechanisms have although 22K hGH binds to both hGH and prolactin receptors (22). The first is been postulated in concerning with the effects of hGH on immune system (1). through the effects of prolactin receptors, the second is through insulin-like growth factor-l (IGFAlthough prolactin also augments T cell 1) and the third is the direct effects of hGH. proliferation (l), the effects on Th cell clone proliferation were not different between 20K and 22K The effects via IGF-1 and hGH. Therefore, the effects through prolactin receptors are unlikely. the direct effects of hGH are probable. Although many effects of GH on immune systems have been reported through the effects of IGF-1 (1,10,23), there exist direct effects of GH (24,25). Administration of hGH at the time of The precise mechanism should be fUrther investigated. human immuno-deficiency virus envelope protein (HIV-1-gp120) provokes a change in the In mice a cytokine production pattern with a preferential switch from Th2 to Thl cells (26). conversion of Th cell populations from Thl to Th2 occurs after burn and administration of GH can reverse this change (27). Thus GH appears to control Th cell responses in other systems.
1. 2. 3.
4. 5. 6. 7. 8. 9. 10. Il. 12. 13. ’ 14.
15. 16. 17.
18. 19. 20.
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