Growth Hormone Stimulates the Proliferation of
Activated Mouse T Lymphocytes*
MARIE-CATHERINE POSTEL-VINAY, VALERIA DE MELLO COELHO†,
MARIE-CLAUDE GAGNERAULT, AND MIREILLE DARDENNE
INSERM U-344, Endocrinologie Mole´culaire (M.-C.P.-V., V.d.M.C.), and CNRS URA 1461 (V.d.M.C., M.-C.G., M.D.), Universite´ Paris V, Hoˆpital Necker, Paris, France
ABSTRACT
A modulatory role for GH on immune function has been suggested, but hormonal effects have been difficult to demonstrate with isolated cells. We have recently shown that GH receptors are present in mu-rine hematopoietic tissues, with a lower receptor number in T lym-phocytes than in B cells or macrophages. The binding of bovine GH (bGH) to murine splenocytes is increased after T cell activation with either concanavalin A or anti-CD3 antibody. In the present study, we show that bGH is able to stimulate the proliferation of activated murine T cells. Splenocytes were stimulated with either Con A or anti-CD3 antibody; addition of the mitogen resulted in increased [3H]thymidine uptake. When added together with the mitogen to the
culture medium, bGH was able to further stimulate thymidine up-take. A bell-shaped dose-response curve was observed. bGH was able to increase cell proliferation by 2.5-fold over the effect of anti-CD3 alone. The amplitude of the bGH response was greater in
unfraction-ated splenocytes than in purified T lymphocytes or thymocytes. Splenocytes were also stimulated by lipopolysaccharide, a B cell-specific mitogen; no change in the level of bGH binding was observed during activation of B cells, and no effect of bGH on the proliferative response of splenocytes to lipopolysaccharide was detected. The GH proliferative effect on T lymphocytes is probably direct and not through locally produced like growth factor I, because insulin-like growth factor I did not affect the cell proliferation when added at concentrations ranging from 1029-1027M. Ovine PRL was also able
to stimulate [3H]thymidine uptake in splenocytes and thymocytes,
and a synergistic effect was observed when bGH and ovine PRL were added together at 1028M. Our findings support the biological
signif-icance of the GH receptors identified in murine T lymphocytes and confirm the role of GH in the regulation of immune functions. (Endocrinology 138: 1816 –1820, 1997)
I
NCREASING evidence shows that GH interacts with the lymphohematopoietic system on the basis of the follow-ing arguments. 1) Positive effects of GH on T cell develop-ment, particularly in the thymus, have been observed in hypophysectomized or GH-deficient animals (1–3) and in aging animals implanted with GH-secreting tumors (4, 5) or treated with GH (6); in the latter case, these effects include increased cytokine production and proliferative responses to lectins. 2) Constitutive overexpression of GH, as observed in bovine GH (bGH) transgenic mice, leads to a marked in-crease in the absolute number of hematopoietic progenitor cells, especially those localized in the spleen (7). 3) Con-versely, lymphoproliferation is blocked by specific antibod-ies to GH (8) or by antisense oligonucleotide to GH messen-ger RNA (9). Together, these findings lead to the suggestion that GH can influence, directly or indirectly, lymphocyte proliferation. However, conflicting results have been re-ported regarding the in vitro effects of GH on lymphopro-liferation. Both positive (10 –13) and negative (14 –17) find-ings have been reported using human or murine resting or activated peripheral lymphocytes and thymocytes.Recently, using biotinylated bGH and flow cytofluorom-etry, we demonstrated the presence of receptors for GH in murine hematopoietic tissues and the up-regulation of their expression on splenocytes and thymocytes after mitogen-induced T cell activation (18). In the present study, we show that bGH and PRL are able to stimulate the in vitro prolif-eration of lectin- or anti-CD3-activated murine T lympho-cytes, confirming the biological significance of the receptors present on these cells.
Materials and Methods Animals
Six- to 8-week-old male and female C57BL/6 mice were bred in our animal facilities, under specific pathogen-free conditions and according to the regulations of the European Community for the care and use of laboratory animals (19). Mice were fed regular pellets and water ad libitum and were maintained at 2261 C on a 12-h dark, 12-h light cycle, with lights on from 0700 –1900 h.
Reagents and antibodies
Recombinant bGH was generously provided by Dr. William Baum-bach (American Cyanamid Co., Princeton, NJ). Biosynthetic insulin-like growth factor I (IGF-I) was obtained from Euromedex (Souffelweyer-sheim, France), and ovine PRL (oPRL-16) was obtained from the Na-tional Hormone Pituitary Program, NIDDK (Baltimore, MD). Anti-IGF-I and anti-IGF-I receptor antibodies (clones 82–9A and anti-IR3, respec-tively) were obtained from Genzyme (Paris, France). Lipopolysaccha-ride (LPS) from Salmonella typhimurium was obtained from Difco (De-troit, MI), and concanavalin A was provided by Pharmacia (St. Quentin-en-Yvelines, France). Anti-CD3 mAb (clone 145.2C11, hamster IgG), provided by Dr. Lucienne Chatenoud (INSERM U-25), was purified from ascites by protein G affinity column chromatography. The anti-PRL Received November 14, 1996.
Address all correspondence and requests for reprints to: Marie-Catherine Postel-Vinay, INSERM U-344, Endocrinologie Mole´culaire, Faculte´ de Me´decine Necker Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
* Presented in part at the 10th International Congress of Endocrinol-ogy, San Francisco, CA, June 1996. This work was supported by CNRS and INSERM.
† Recipient of a grant from Conselho Nacional de Desenvolvimento Cientifico e tecnolo´gico.
Copyright © 1997 by The Endocrine Society
receptor mAb (clone T1, mouse IgG1), kindly provided by Dr. Paul Kelly, was conjugated to biotin as previously described (19). The fol-lowing mAbs used for cytofluorometric analysis (as ascitic fluids) were obtained from Caltag (Tebu, Le Perray en Yvelines, France) as phyco-erythrin (PE) or fluorescein isothiocyanate (FITC) conjugates: anti-CD4 (clone GK 1.5, rat IgG2b) and anti-CD8 (clone 53– 6.7, rat IgG2a) for classical T cell markers, anti-B220 (clone RA3-gB2, rat IgG2a) for a pan B cell marker, and anti-Mac 1 (clone M1/70.15, rat IgG2b) for specific labeling of monocytic/macrophage cells. Unrelated mouse IgG1 and rat IgG2a and IgG2b (Caltag) were used as isotype-matched control anti-bodies in immunofluorescence studies and blocking experiments. Biotin labeling of recombinant bGH
bGH was conjugated to biotin according to a technique previously described (18). Positive labeling with biotinylated bGH was revealed with streptavidin-PE (SAV-PE; Caltag).
Cell preparation and culture
Spleens and thymuses were removed from exsanguinated mice. Sin-gle cell suspensions of splenocytes and thymocytes were prepared in MEM using a homogenizer. After one wash, cell viability was deter-mined by trypan blue exclusion. Six to 10 donors of the same age were used for each experimental point.
Depletion of splenic B cells and monocytes was accomplished by double panning on goat anti-mouse Ig- and anti-Mac 1-coated petri dishes (90-min incubation at 4 C). The efficiency of cell depletion was analyzed by flow cytometry after labeling with anti-B 220 and anti-Mac 1 antibodies; T cell-enriched splenocytes contained more than 97% T cells.
Unfractionated splenocytes, purified T splenocytes, or thymocytes from young C57BL/6 mice were diluted in RPMI 1640 culture medium supplemented with 0.2% BSA, 2 mm l-glutmine, 1000 U/ml penicillin-streptomycin, 10 mmHEPES buffer (Life Technologies, Cergy Pontoise, France), and 50mm2-mercaptoethanol (Sigma Immunochemicals, La Verpilliere, France).
Proliferation assays
Cells (23105in 200ml) were seeded into 96-well flat microtiter plates and incubated at 37 C in 5% CO2with or without LPS (1mg/ml) or anti-CD3 (1mg/ml). Anti-CD3, immobilized to tissue culture surfaces (2.5mg/ml), was incubated overnight at 4 C, followed by two washes to remove excess antibody. In addition, cells were incubated in the presence or absence of increasing concentrations of recombinant bGH (10214–1025m). At the indicated times, cells were exposed to 1mCi/well [methyl-3H]thymidine (5.0 Ci/mmol; Amersham, Les Ulis, France). Cells were then harvested, and the radioactivity incorporated into DNA was quantitated in a Betaplate counter (LKB Wallac, St. Quentin en Yvelines, France). All proliferation assays designed to test exogenously added GH were performed under serum-free culture conditions.
For surface antigen detection, splenocytes, distributed in 50-ml tissue culture flasks (13106cells/ml), were incubated at 37 C in 5% CO
2with or without mitogens (anti-CD3 or LPS at the concentrations indicated above) in a final volume of 10 ml/flask. At appropriate time points, cells were harvested and processed for immunofluorescence staining. Immunofluorescence labeling and flow cytometry
Indirect labeling of cells was performed in microtiter plates. Briefly, 13 106cells, resuspended in PBS containing 0.2% BSA and 0.01m sodium azide, were incubated with biotinylated bGH (2.5mg) for 120 min at 4 C. After washing with staining medium, cells were incubated for 30 min with 10ml SAV-PE and optimal dilutions of FITC-conjugated anti-CD4, anti-CD8, anti-B220, or isotype-matched control antibodies. The cells were then washed twice and resuspended in PBS containing 1% formaldehyde before analysis. Controls included staining with one reagent (FITC or PE) alone or with biotinylated BSA. Flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA). Propidium iodide was used systematically for the exclusion of dead cells. At least 104lymphoid cells were acquired in each run, and the results were analyzed using Lysis II software.
Statistics
All values were expressed as the mean6semof triplicate determi-nations. Differences between means were evaluated using unpaired Student’s t test.
Results
Effect of bGH on anti-CD3-induced T cell proliferation Experiments were designed to investigate the effect of bGH on the in vitro proliferation of activated murine T splenocytes. Unfractionated T cells were stimulated via their antigen-specific TCR complex (TCR-CD3) using a soluble mAb to CD3 in the presence or absence of increasing con-centrations (10214–1025m) of bGH. As shown in Fig. 1, anti-CD3 alone, over a 2-day-culture period, allowed the prolif-eration of T splenocytes, as assessed by [3H]thymidine uptake. The addition of bGH at the beginning of the culture yielded a significant increase in [3H]thymidine uptake com-pared to that of cultures containing anti-CD3 alone. A bell-shaped dose-response curve was observed, with an effect of bGH at concentrations ranging from 10212–1026 m. In six experiments, the response observed with bGH (1028 m) reached a 2.560.2-fold (mean6sem) increase over the effect of anti-CD3 alone. The bGH effect was consistently lost at a very high hormone concentration (1025mbGH). bGH alone never induced any stimulatory effect on the proliferation of murine T lymphocytes in the absence of anti-CD3 antibody (Fig. 1). In all experiments, [3H]thymidine uptake remained below 5000 cpm when the cells were not treated with a mitogen.
The effect of bGH on anti-CD3-induced proliferation was also detected with purified T splenocytes, with a lower re-sponse than with unfractionated lymphocytes; bGH in-creased the proliferation of purified T cells by 5363% (P, 0.01) at 1028mand by 3462% (P,0.02) at 10210m.
In addition, significant stimulation of cell proliferation was observed when bGH was added to murine activated thymocytes; thymidine uptake was increased by 786 4% (P , 0.01) at 1028 m bGH. Taken together, these results
FIG. 1. Effect of bGH on the proliferation of anti-CD3-activated mu-rine splenocytes. Splenocytes (53105/200ml) were stimulated with
soluble anti-CD3 (F; 0.1mg/ml) in the presence of increasing concen-trations of bGH (10214–1025M) for 48 h at 37 C in 5% CO
2. Control
cultures (E) contained bGH alone. The data shown are representative of one set of six experiments. Cells were exposed to [3H]thymidine for
suggest that GH is able to directly stimulate both mature and immature T cells.
In five different experiments, IGF-I, at concentrations ranging from 1029–1027m, did not stimulate the proliferation of resting or activated splenocytes (Table 1). At these con-centrations, IGF-I has previously been shown to stimulate thymulin production and thymic epithelial cell proliferation (20). Moreover, the addition of either anti-IGF-I antibody or anti-IGF-I receptor antibody to the culture medium did not alter the bGH-induced proliferation of unfractionated splenocytes or purified T lymphocytes (Table 1).
Effects of bGH and oPRL on anti-CD3-induced T cell proliferation
Splenocytes were cultured under standard conditions with anti-CD3 (0.1 mg/ml) and exposed to bGH (1028 m) alone, PRL (1028m) alone, or both hormones. As shown in Fig. 2, a significant enhancement of [3H]thymidine uptake was observed in cells stimulated by anti-CD3 in the presence of either bGH or oPRL alone. The addition of both hormones resulted in a further increase in T cell proliferation. Lack of effect of bGH on the proliferation of murine B cells
To test the ability of bGH to modulate the proliferation of murine B lymphocytes, we stimulated splenocytes with sub-optimal concentrations of LPS, a polyclonal B cell-specific mitogen, in the presence or absence of bGH in serum-free medium. bGH did not affect the proliferative response of splenocytes to LPS (Table 2).
Expression of GH receptors (GHR) in activated murine splenocytes
Expression of GHR on both CD41and CD81 subpopula-tions of T cells during activation by anti-CD3 was analyzed by FACS, using biotinylated bGH. As shown in Fig. 3 (upper
panel), the percentages of CD4 and CD8 cells expressing GHR
were not significantly different at the initiation of the culture. As early as 2 h after the addition of anti-CD3, several hours before the rise in the proliferative rate, a 2-fold increase in the proportion of GHR1cells was detected. However, the
num-ber of GHR per cell remained unchanged, as assessed by fluorescence intensity. The pattern of expression of GHR1 cells was very similar for CD41 and CD81 subsets. The highest GHR expression was observed at 24 h, in parallel with the increased proliferation of anti-CD3-treated cells (Fig. 3, lower panel).
Using B220 as a pan-B cell marker, we also analyzed the pattern of expression of GHR on B cells after activation of splenocytes by LPS. As shown in Fig. 4 (lower panel), LPS induced a significant increase in cellular proliferation. How-ever, the proportion of B cells expressing GHR, as assessed by double staining with anti-B220, was not influenced by LPS stimulation (upper panel). In addition, the density of GHR per cell, evaluated by the relative fluorescence intensity, re-mained unchanged during the activation process.
Discussion
Our results demonstrate that GH is able to stimulate the proliferation of activated murine T lymphocytes and support the physiological importance of the GHR that we have pre-viously identified in murine cells (18).
In vitro studies regarding the lymphoproliferative effect of
GH on human or rat lymphocytes have yielded contradictory
TABLE 1. Effect of IGF-I on lymphocyte proliferation
Culture conditions [3H]Thymidine uptake (cpm)
Splenocytes alone 2,7256119 1IGF-I (1028M) 2,9026217 Splenocytes1anti-CD3 68,13061,981 1IGF-I (1028M) 74,93762,495 1Anti-IGF-I 68,77862,194 1GH (1028M) 115,45361,305 1GH (1028M)1anti-IGF-I 104,91063,036
Purified T splenocytes alone 2,6276244
1IGF-I (1028M) 2,9506254
Purified T splenocytes1anti-CD3 83,10563,225
1IGF-I (1028M) 94,66963,165
1anti-IGF-I 89,88864,789
Unfractionated or purified T splenocytes (53 1025cells) were
cultured in serum-free medium containing 0.2% BSA and the indi-cated addition. Cells were exposed to [3H]thymidine (1mCi/well) for
16 h before harvest. Data shown (mean6SEMof triplicate determi-nations) are representative of three separate experiments.
FIG. 2. Effects of oPRL and bGH on anti-CD3 T cell-induced prolif-eration. Splenocytes (53105) were cultured for 48 h with anti-CD3
(0.1mg/ml) alone or in the presence of bGH (1028M), PRL (1028M),
or both hormones. Cells were exposed to [3H]thymidine for 16 h before
harvest for proliferation assay. The results of one experiment, per-formed in triplicate, are shown. Three separate experiments gave similar results. *, P,0.02; **, P,0.01; ***, P,0.001.
TABLE 2. Effect of bGH on LPS-induced proliferation of B
splenocytes
Culture conditions [3H]Thymidine uptake (cpm)
Splenocytes alone 3,603687 Splenocytes1LPS (1mg/ml) 47,91863,058 1bGH (10212M) 51,23061,135 1bGH (10210M) 50,73961,388 1bGH (1028M) 47,37461,666 1bGH (1026M) 48,4216422
Splenocytes (531025cells) were cultured in serum-free medium
containing 0.2% BSA with the indicated additions. Cells were exposed to [3H]thymidine (1 mCi/well) for 16 h before harvest. Data shown
(mean6SEMof triplicate determinations) are representative of three separate experiments.
results. Although it has been reported that human GH can induce blastogenesis and increase thymidine uptake in un-stimulated peripheral blood lymphocytes (11, 12, 15) or thy-mocytes (13), our attempt to demonstrate a proliferative re-sponse to GH in unstimulated murine lymphocytes was unsuccessful. Our results are in agreement with those re-ported by Schimpff (14) and Kooijman et al. (16), who dem-onstrated hormonal effects on activated human peripheral blood lymphocytes using various culture conditions.
One major reason for the difficult demonstration of the GH effect could be GH production by the cells; expression of the GH messenger RNA and production of GH have been shown in human peripheral mononuclear lymphocytes (8, 21, 22), thymocytes (13), or B cell lines (23, 24) and in rat splenocytes, primarily in B cells (25). Moreover, a marked rise in GH production has been observed upon stimulation of the cells with concanavalin A (21, 22). Weigent et al. (9) showed that antisense GH oligodeoxynucleotide-mediated inhibition of GH production resulted in a decrease in lymphocyte prolif-eration. GH production by murine splenocytes and thymo-cytes remains to be demonstrated; in the present study, if there is hormone production by the cells, it is not sufficient to prevent the exogenous GH-mediated response.
The proliferative effect of bGH is shown exclusively on
activated T lymphocytes. To obtain the hormonal response, the cells have to be activated either by a T cell mitogen, such as concanavalin A, or via their antigen-specific TCR complex (TCR/CD3) using an anti-CD3 antibody. As previously shown (18), the number of GHR on T lymphocytes increases during the activation process. From the cytofluorometric data, this enhanced GH binding is related to a higher number of cells expressing GHR rather than an increased number of receptors per cell. Interestingly, the increased expression of GHR occurs very early after stimulation, before the onset of T cell proliferation, and is distributed within both subpopu-lations of activated T cells, suggesting a role for GH in reg-ulating various T cell effector functions.
No effect of bGH on the proliferation of B lymphocytes could be detected, even though GHR have been shown to be more widely expressed on B cells than on T cells of the mouse. Upon treatment with the B cell mitogen LPS, the high level of GH binding is not further enhanced concurrent with the absence of response of the cells to bGH. These findings are in contrast with those of Yoshida et al. (26), who could
FIG. 3. Binding of bGH to CD41(E) and CD81(F) splenocytes as a function of time, during T cell activation by anti-CD3 mAb. Upper panel, Cells (13106) cultured at 37 C in RPMI medium with 0.5%
BSA with or without anti-CD3 mAb (0.1mg/ml) were labeled at ap-propriate time points with biotinylated bGH followed by SAV-PE as the second stage reagent, and FITC-labeled anti-CD4 or anti-CD8 antibody. Dual color immunofluorescence analysis was performed by cytofluorometry. Lower panel, Proliferative response of splenocytes to anti-CD3. Cells (53105) in 200ml were cultured at 37 C with (
F) or without (E) anti-CD3 (0.1mg/ml). At the indicated times, cells were exposed to 1mCi/ml [3H]thymidine for 16 h, and the radioactivity was
determined by liquid scintillation counting. Results are expressed as the mean6SEMof three separate experiments.
FIG. 4. GHR expression on B2201cells from LPS-stimulated spleno-cytes. Upper panel, Splenocytes in RPMI containing 0.2% BSA in the presence (o) or the absence (M) of LPS (1mg/ml) were harvested at different time points for immunofluorescence analysis with biotinyl-ated bGH and with FITC-conjugbiotinyl-ated anti-B220, as described in Fig. 1. Parallel cultures in microtiter plates were exposed to [3
H]thymi-dine (1mCi/well) for 16 h before harvest for proliferation assays at the same time points. The upper panel represents the percentages of B cells expressing GHR 4 and 24 h after LPS addition. The lower panel indicates the proliferative response of splenocytes to LPS 4 and 24 h after the beginning of the culture. The data shown are the mean6SEM of one experiment performed in triplicate and are representative of three separate experiments.
demonstrate a small enhancing effect of hGH on thymidine uptake in various human B cell lines. GH could also affect processes other than proliferation in B cells, and indeed, GH has been shown to stimulate immunoglobulin synthesis (26 –28).
The stimulation of cell proliferation was observed with bGH concentrations as low as 10212m. The effect was lost at a very high hormone concentration (1025m). The bell-shaped dose-response curve is consistent with the sequential for-mation of an active hormone receptor-dimer complex. The formation of a homodimer consisting of one molecule of GH and two receptors has been shown to be a crucial step in GH signaling (29).
Both direct and indirect effects of GH on immunocompe-tent cells have been reported (30); indirect effects are medi-ated by paracrine/autocrine production of IGF-I. Our data support the hypothesis that GH directly stimulates T cell mitogenesis: 1) exogenous IGF-I had no effect on the prolif-eration of either resting or activated lymphocytes, 2) anti-IGF-I or anti-anti-IGF-I receptor antibodies did not inhibit the GH proliferative effect observed with unfractionated spleno-cytes, and 3) the cellular proliferative effect of GH is also observed with purified, monocyte-depleted, T cell popula-tions and thymocytes, which favors this hypothesis, as it is known that macrophages are the most abundant source of IGF-I (30). However, a recent report by Sabharwal suggests an indirect role of GH, via locally synthesized IGF-I, on the proliferation of human thymic cells (13).
We observed a greater GH proliferative effect in unfrac-tionated splenocytes than in purified T lymphocytes. IGF-I does not appear to be responsible for the GH-induced pro-liferation. A possible explanation for our data is that GH stimulates the production of cytokines by monocytes present in the mixed cell population.
By using bGH, which binds only to GHR and does not interact with the PRL receptor, we show a hormonal effect via the GHR. However, PRL is also able to stimulate the prolif-eration of murine T lymphocytes, and a greater effect is observed when the two hormones are added together.
The signaling mechanisms by which GH exerts its effect on lymphocyte proliferation is not defined. The hormone could also act by stimulating cells to enter the cell cycle, or it could act indirectly on cell replication, through lymphokines.
Acknowledgments
The expert secretarial assistance of C. Slama and C. Coridun is grate-fully acknowledged. We thank M. Netter for her help with the art work.
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