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Growth hormone restores glucocorticoid-induced T cell suppression¹

HIROAKI DOBASHI^*,1, MAKOTO SATO^*, TERUKAZU TANAKA^*,, MICHIAKI TOKUDA^* and TOSHIHIKO ISHIDA^*

^* First Department of Internal Medicine, School of Medicine and
Department of Environmental Health Sciences, Kagawa Medical University, Kagawa, Japan

²Correspondence: First Department of Internal Medicine, Kagawa Medical University, 1750–1 Ikenobe, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan. E-mail: hdobashi{at}kms.ac.jp

SPECIFIC AIMS

Growth hormone (GH) has been extended to the improvement of metabolic imbalance in many diseases, including autoimmune disease treated with glucocorticoids (GCs), but it is unclear whether GH can be used safely in autoimmune disorders that are treated with GC. Here we focused on the effect of GH on peripheral T lymphocytes under GC excess.

PRINCIPAL FINDINGS

Expression of GHR mRNA and tyrosine phosphorylation of JAK2 and STAT5b in human peripheral T lymphocytes
We investigated the expression of GHR mRNA in human peripheral T lymphocytes (HPTL) and peripheral mononuclear cells (PBMC). GHR mRNA was detected in both cell fractions by RT-PCR Southern blotting using a pair of primers set on exons 4 and 6 (Fig. 1A ). The expression of GHR mRNA was down-regulated by treatment with recombinant human GH (rhGH) (data not shown). To determine whether GH can stimulate tyrosine phosphorylation of cellular proteins in HPTL, the cells were treated with rhGH. Recombinant hGH stimulated the tyrosine phosphorylation of several different cellular proteins (date not shown). To further test whether rhGH can enhance the tyrosine phosphorylation of JAK2 and STAT5b, which are representative molecules in GHR-mediated signaling pathways, we assessed tyrosine phosphorylation of these two molecules immunoprecipitated with the relevant specific antibodies. Recombinant hGH enhanced the tyrosine phosphorylation of JAK2 and STAT5b without changing the amount of these proteins (Fig. 1B ). These findings indicate that rhGH activates HPTL, at least in part, through the GHR-JAK2/STAT5b cascade.

View larger version (38K): [in this window] [in a new window]   Figure 1. A) Expression of GHR mRNA in human PBMC and HPTL. The upper photograph shows RT-PCR Southern blots of GHR. Lanes: 1, positive control; 2, purified HPTL; 3, purified HPTL from another healthy volunteer; 4, PBMC. B) Effects of rhGH treatment on tyrosine phosphorylation of JAK2 and STAT5b in purified HPTL. Purified HPTL were incubated with 200 ng/ml rhGH. Tyrosine-phosphorylated JAK2 and STAT5b were immunoblotted using anti-phosphotyrosine MoAb and peroxidase-conjugated second antibodies, followed by development with ECL. C) Effects of rhGH and IGF-I on the proliferation of activated HPTL with or without GC treatment. Purified HPTL (2x105) were cultured for 48 h with immobilized anti-CD3 and anti-CD28 in the presence or absence of rhGH (GH1; 0.1 µg/ml, GH2; 1 µg/ml) or IGF-I (0.5 µg/ml) or Dex (1 µM). Values are representatives of 5 independent experiments. Numbers in parentheses indicate the number of samples in each group. *P < 0.05 (vs. control), **P < 0.01 (vs. control). D) Analysis of rhGH effect on Bcl-2 expression in human peripheral CD4+ and CD8+ T lymphocytes. Bcl-2 expression was detected by staining the permeabilized cells with FITC-conjugated anti-Bcl-2 MoAb. All analyses were performed by an EPICScan. Numbers in parentheses indicate the number of samples in each group. *P < 0.05 (vs. CD4+ HPTL without rhGH). E) Effect of rhGH on Bcl-2 expression in purified human peripheral CD4+ (A) and CD8+ T lymphocytes (B). Purified CD4+ (3x106) or CD8+ (1x106) HPTL were treated with (+) or without (-) rhGH at 37°C for 48 h. Cells were lysed and subjected to Western blotting analysis. The blots represent the 26 kDa Bcl-2 protein detected by anti-Bcl-2 MoAb.

Effects of rhGH and IGF-I on the proliferation of HPTL
We next determined whether rhGH affects the proliferation of HPTL. HPTL in culture were costimulated with immobilized anti-CD3 and anti-CD28 monoclonal antibodies (MoAbs) for 48 h. The addition of Dex at the beginning of the culture yielded a significant decrease in [³H]thymidine incorporation into HPTL compared with controls without Dex (Fig. 1C ). Recombinant hGH and IGF-I, however, partially alleviated the Dex-induced inhibition of [³H]thymidine uptake and the effect was more prominent in IGF-I than in rhGH. In the absence of Dex, IGF-I stimulated the [³H] thymidine uptake, but rhGH had no effect on the HPTL proliferation (Fig. 1C ). These findings indicate that rhGH and IGF-I can both alleviate the Dex-induced suppression of DNA synthesis in activated HPTL.

Enhancement of Bcl-2 expression and alleviation of Dex-induced apoptosis in CD4-positive (CD4⁺) HPTL by rhGH treatment
Recombinant hGH might rescue lymphocytes from Dex-induced apoptosis. Since Bcl-2 is a key molecule that prevents cells from apoptosis, we analyzed Bcl-2 expression in HPTL by flow cytometry. The mean fluorescence intensity of Bcl-2 in CD4⁺ HPTL was significantly increased by rhGH (Fig. 1D ). In contrast, Bcl-2 expression in CD8⁺ HPTL was not altered by rhGH treatment. We next examined the effect of rhGH on Bcl-2 expression by Western blotting analysis. The expression of Bcl-2 was enhanced by rhGH in CD4⁺ HPTL. In CD8⁺ HPTL, however, Bcl-2 expression was decreased after rhGH treatment (Fig. 1E ). These results were consistent with the findings by flow cytometric analysis. Bcl-2 mRNA expression was also increased after treatment with rhGH for 6 and 12 h in CD4⁺ HPTL, but not in CD8⁺ HPTL (data not shown). The enhancement of Bcl-2 expression induced by rhGH may have rescued CD4⁺ HPTL, but not CD8⁺ HPTL, from the apoptosis induced by GC. Recombinant hGH alleviated apoptosis in CD4⁺ HPTL (% apoptosis; Dex 39.2±1.0 vs. Dex+rhGH 32.4±1.0, P<0.01). Recombinant hGH enhanced apoptosis in CD8⁺ HPTL (% apoptosis; Dex 53.6±0.2 vs. Dex+rhGH 57.5±0.9, P<0.05).

Alteration of the CD4⁺/CD8⁺ ratio of T lymphocyte subsets treated with rhGH and/or GC
We first examined the effect of rhGH on T lymphocyte subsets in vitro by flow cytometric analysis. Recombinant hGH tended to raise the ratio of CD4⁺/CD8⁺ in the presence of Dex, but its effect was not significant (data not shown). We next tested whether rhGH alters the ratio of T lymphocyte subsets in vivo. Rats were treated with Dex in combination with rhGH for 4 wk. The results of flow cytometric analysis of T lymphocyte subsets are shown in Fig. 2 . The CD4⁺/CD8⁺ ratio of T lymphocytes was not altered by Dex treatment, although the total number of lymphocytes was dramatically decreased by the treatment. Recombinant hGH administration significantly raised the CD4⁺/CD8⁺ ratio of T lymphocytes in Dex-treated rats. This effect was not found in control rats treated with saline alone.

View larger version (12K): [in this window] [in a new window]   Figure 2. The CD4+/CD8+ ratio of splenic lymphocytes assessed by flow cytometry. **P < 0.01 (vs. saline+saline). Numbers in parentheses indicate the number of rats in each group.

DISCUSSION

The cross talk between neuroendocrine and immune systems had been proposed in 1930s, before the discovery of the pituitary hormones. It is now well established that pituitary hormones, including GH, affect immune responses. GH stimulates DNA synthesis in human T lymphocytes and enhances the cytotoxic activity of human natural killer (NK) cells. GH also stimulates the proliferation of normal and leukemic human T lymphocytes. In the present study, rhGH had no significant effect on HPTL proliferation. This discrepancy may be due to the difference in the preparation of T lymphocytes and the dose of rhGH used. However, in this series of experiments, the most important observations are that rhGH partially alleviated the GC-induced suppression of DNA synthesis in HPTL.

Attention has been directed to the clinical use of GH, which has been widely expanded into therapy for many wasting diseases including various autoimmune diseases and AIDS. In Europe, several clinical trials are attempting to rescue catabolic side effects along with disease treatment. Supraphysiological doses of GH have been used for the treatment of children with uremic growth failure, juvenile rheumatoid arthritis (RA), and bronchial asthma to restore the growth velocity. GH has also been used in adult patients with RA and other autoimmune diseases to improve the catabolic conditions. More recently, GH was used to improve wasting syndromes observed in AIDS patients. In most of these studies, GH therapy was combined with GC treatment. The present findings regarding anti-GC effects of rhGH on T lymphocytes enforce the precaution of the clinical application of GH. GH therapy may change the disease activity in GC-treated autoimmune diseases in which T lymphocytes play a major role in controlling immune functions. Indeed, one study has indicated that GH therapy raised the activity of autoimmune diseases, including SLE treated with GC.

We have shown that HPTL express GHR mRNA as reported previously. We have also indicated that rhGH stimulated the tyrosine phosphorylation of JAK2 and STAT5b in HPTL. These observations agree with previous studies showing that the JAK2/STAT5b cascade was the most important signaling pathway mediated by GH in hepatocytes and other nonhematopoietic cells. The activation of this signaling pathway appears to promote the proliferation of target cells. In the present study, rhGH alleviated the GC-induced suppression of DNA synthesis in HPTL. Since GC induces apoptosis in lymphocytes and Bcl-2 is a key molecule for preventing apoptosis, we focused on Bcl-2 expression. It was previously reported that GH increased Bcl-2 expression in human monocytic cells. The present observations that rhGH enhances Bcl-2 expression selectively in CD4⁺ T lymphocytes can clearly explain why rhGH raised the CD4⁺/CD8⁺ ratio of T lymphocytes in GC-treated rats. GC appears to induce apoptosis in both CD4⁺ and CD8⁺ T lymphocytes. CD4⁺ T lymphocytes could be rescued from GC-induced apoptosis due to the rhGH-enhanced Bcl-2 expression in them. Indeed, GH alleviated GC-induced apoptosis selectively in CD4⁺ HPTL in the present study.

The present findings suggest that rhGH might interfere with the immunosuppressive effect of GC by changing the sensitivity of T lymphocytes to apoptotic effects of GC. In many diseases that were previously treated with GC, especially in T lymphocytes-mediated autoimmune diseases, once the T lymphocytes have been activated, removal of the activating agents may not easily reverse the disease activity unless the immunosuppressive therapy is intensified. This must be considered before recommending GH therapy for many complications due to GC excess. Additional studies are required to conclude that GH can be safely used for these patients.

View larger version (12K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized GH effects on GC-induced apoptosis of T lymphocytes. GH enhances Bcl-2 expression selectively in CD4+ T lymphocytes but down-regulates Bcl-2 expression in CD8+ T lymphocytes. GC induces apoptosis in both CD4+ and CD8+ T lymphocytes. In the combination of GH with GC, the apoptosis of CD4+ T lymphocytes could be restored, but that of CD8+ T lymphocytes could be slightly enhanced. The ratio of CD4+ /CD8+ T lymphocytes is then increased.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0702fje ; to cite this article, use FASEB J. (June 27, 2001) 10.1096/fj.00-0702fje

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