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Original Articles |
Department of Obstetrics and Gynecology, University of Wisconsin, and Wisconsin Regional Primate Research Center, Madison, Wisconsin 53792
Address all correspondence and requests for reprints to: Omid Khorram, M.D., Ph.D., Department of Obstetrics and Gynecology, Box 489, Harbor-University of California-Los Angeles Medical Center, 1000 West Carson Street, Torrance, California 90509. E-mail: Okhorram{at}rei.edu
Abstract
GHRH is a neuropeptide that has also been localized to the immune system. The physiological function of GHRH in the immune system has not been elucidated. This study was conducted to determine whether immune GHRH expression is altered in certain pathological states, such as immune cell tumors, and whether gender, aging, and alterations in the sex steroid milieu influence the expression of this peptide in immune cells.
Using double color flow cytometry, GHRH protein was found to be expressed in less than 2% of peripheral blood mononuclear cells (PBMC). Monocytes and B and T cells all expressed GHRH protein, although a greater percentage of T cells compared with B cells and monocytes expressed GHRH (5- to 7-fold). Semiquantitative RT-PCR was used to quantify GHRH messenger ribonucleic acid (mRNA) in PBMC and several immune cell-derived tumors. PBMC and granulocytes expressed low levels of GHRH mRNA with relatively higher levels of expression in monocytes. The tumor cell lines CEMX 174 (B/T cells), HUT 78 (T cells), WIL2-N (B cells), U937 (monocytes/macrophages), and JM 1 (pre-B cell lymphoma) all showed greater expression of GHRH mRNA relative to PBMC. However, two cell lines, CCRF-SB, a B lymphoblastoid cell line, and HL-60, a promyelocytic cell line, expressed GHRH mRNA at similar levels as PBMC.
A significant decrease in the percentage of lymphocytes (CD45+ cells) expressing GHRH protein was found in age-advanced men and women compared with young men and women. This decline was noted in B cells (CD20+) and monocytes (CD14+), but not in T cells (CD3+). GHRH mRNA expression in PBMC derived from postmenopausal women was lower than that from premenopausal women. However, no differences in PBMC GHRH mRNA expression were found in young and old men. Although in older men there were fewer peripheral lymphocytes that express GHRH protein, these cells secreted significantly more GHRH in vitro than cells from postmenopausal women with no hormone replacement therapy (HRT), but similar levels as cells from women receiving HRT. PBMC from women receiving HRT secreted more GHRH in vitro than cells from women receiving no hormone replacement.
This study demonstrates that the expression of immune GHRH is dynamic, and therefore likely to be regulated. Increased expression of GHRH in certain immune tumors suggests that GHRH may be mitogenic under certain conditions and therefore play a role in the pathogenesis of select immune cell tumors. Collectively, these results suggest a role for GHRH as a local immune modulator and in the pathophysiology of immunosenescence and immune cell tumors.
SUBSTANTIAL EVIDENCE indicative of bidirectional communication between the neuroendocrine and immune systems has accumulated, and many peptides thought to be of exclusive central nervous system origin have also been localized in the immune system (1). The somatotropic hormones represent one well characterized example, expressed in both the hypothalamic- pituitary axis and the immune system. GH, insulin-like growth factor I (IGF-I), and their receptors are expressed in lymphoid cells and organs, and most studies point to the stimulatory effect of these hormones on immune function (2, 3). Inhibitory effects of GH on immune function have also been reported. In these studies GH-deficient children were treated with GH, with a resultant decline in B cell number (4) and circulating Ig (5). Most recently in a mouse model with genetic deficiency of PRL, GH, and IGF-I, humoral and cell-mediated immunities were unaffected (6).
Immunoreactive GHRH, its cognate messenger ribonucleic acid (mRNA), and GHRH-binding sites have been reported in rodent lymphocytes (7, 8, 9). Human lymphocytes were shown to synthesize and secrete a biologically active GHRH peptide capable of stimulating GH release from both the pituitary gland and from lymphocytes (10). Two GHRH transcripts, one similar to the 0.75-kb hypothalamic species and a larger transcript of approximately 10 kb, were identified in human lymphocytes (10). The physiological function of immune-derived GHRH is unknown, although supraphysiological concentrations significantly inhibited the chemotactic response of human peripheral lymphocytes (11) and natural killer cell activity in vitro (12). GHRH(-1–29), but not GHRH-(1–44) at low concentrations stimulated, whereas high concentrations inhibited phytohemagglutinin (PHA)-induced lymphoproliferation, interleukin-2 secretion, and interleukin-2 receptor expression on activated human peripheral lymphocytes (13).
Few studies have investigated the in vivo effects of GHRH in the immune system. Kiess et al. reported that short-term treatment of GH-deficient children with GHRH did not restore their decreased NK cell activity (14). Recently, we reported that the administration of GHRH to age-advanced men and women resulted in potent activation of T lymphocyte, monocyte, and B cell function within 1 month of treatment. It was unclear from our study whether the effects of GHRH were mediated through the GH/IGF-I axis of immune origin or independently of this axis. Based on our previous findings we proposed that GHRH by virtue of its immune-enhancing effects may have potential therapeutic benefits in immunodeficient states (15). The purpose of this study was to characterize the expression of GHRH in immune cells and determine the influence of aging and exogenous sex hormones on the expression of this peptide.
Subjects and Methods
Subjects
Subjects were healthy nonsmoking men and women who were taking no medications that could influence immune function or the somatotropic axis. The subjects taking HRT were on a continuous daily regimen of 0.625 mg Premarin (Wyeth-Ayerst Laboratories, Inc., Philadelphia, PA) and 2.5 mg Provera (Upjohn, Kalamazoo, MI). The protocol was approved by the human subjects committee at the University of Wisconsin.
Cell isolation and culture
Peripheral blood mononuclear cells (PBMC) were isolated from freshly drawn blood by Ficoll-Hypaque (Sigma, St. Louis, MO) centrifugation and washed with PBS buffer before use (16). Granulocytes were isolated by layering 3–5 mL heparinized blood onto MONO-Poly Resolving Medium (ICN Biomedicals, Inc., Aurora, OH) and were centrifuged at 300 x g for 30 min. The second fraction containing the granulocytes was removed and washed as described for PBMC. Monocytes were isolated by selective panning, where PBMC were incubated at 37 C in 5% CO2 for 2 h, after which the supernatant containing T and B cells was decanted. Cells adherent to the dish bottom containing the monocytes were then washed in PBS. All cell lines were grown in culture according to instructions from the supplier (American Type Culture Collection, Manassas, MD).
For the cell culture experiments, PBMC (1 x 106 cells) were cultured in RPMI 1640 with 10% FCS and 1% penicillin-streptomycin at 37 C and 5% CO2. After 48 h of incubation the well contents were centrifuged, and the supernatants were frozen at -70 C until assayed for GHRH. GHRH was measured using an RIA kit purchased from Peninsula Laboratories, Inc. (Belmont, CA).
Flow cytometry
Freshly isolated PBMC were prepared for double color flow cytometry by adding 0.2 mL 2% paraformaldehyde to 25 µL isolated lymphocytes at a concentration of 107 cells/ml at room temperature for 10 min. Cells were then permeabilized by the addition of 200 µL 0.05% saponin in PBS at room temperature for 10 min, followed by a wash with PBS containing 0.01% saponin. Fifty microliters of GHRH primary antibody (hg56, courtesy of Dr. Wylie Vale, The Salk Institute, La Jolla, CA) of a 1:200 solution were added to each tube at 4 C for 30 min. After a wash in PBS, goat antirabbit phycoerthyrin- or fluorescein isothiocyanate-conjugated monoclonal antibodies (Becton Dickinson, San Jose, CA) against various cell surface antigens was added and incubated at 4 C for 30 min. The cells were then washed and suspended in 200 µL PBS for analysis in an EPICS analyzer (Coulter Corp., Hialeah, FL). Controls run in every analysis included samples with no antibody, fluorescein isothiocyanate IgG, and phycoerthyrin IgG.
GHRH RT-PCR
Total RNA was isolated from tissues using RNA STAT-60 (Tel-Test, Friendswood, TX) and quantitated by UV spectrophotometry. RT was performed using the GeneAmp RNA PCR Core Kit (Perkin-Elmer Corp., Foster City, CA) with oligo(deoxythymidine)16 primers according to the manufacturer’s instructions. PCR was carried out using 12.5 pmol of each primer, 2.5 mmol/L of each deoxy-NTP, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 0.01% (wt/vol) gelatin, and 5 µL complementary DNA template in a final volume of 50 µL. The primer sequences (5' to 3') used were: glyceraldehyde-3-phosphate dehydrogenase (G3PDH) up, TGA AGG TCG GAG TCA ACG GAT TTG GT; G3PDH down, CAT GTG GGC CAT GAG GTC CAC CAC; GHRH up, TTC TTT GTG ATC CTC ACC CTC AGC; and GHRH down, ATC TTC ATC CCT GGG AGT TCC TGC. The G3PDH up primer maps to the exon 2/3 junction, and the down primer maps to exon 9. The GHRH up primer maps to exon 2, and the down primer maps to exon 5. Reactions were amplified as follows: for G3PDH, 94 C for 30 s, 55 C for 30 s, and 72 C for 45 s for 35 cycles; and for GHRH, 94 C for 30 s, 65 C for 30 s, and 72 C for 45 s for 40 cycles. Reaction products (10 µL G3PDH and 20 µL GHRH) were electrophoresed simultaneously on 2.5% agarose gels, visualized by SYBR Gold (Molecular Probes, Inc., Eugene, OR), and analyzed with a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Semiquantitative analysis of mRNA expression was accomplished by scanning densitometry using ImageQuant software (Molecular Dynamics, Inc.) to calculate the ratio of GHRH/G3PDH. A PCR reaction for GHRH was serially diluted at a 1:1 ratio, and 1 µL of each dilution was reamplified via the PCR at the same reaction conditions as previously described. These reactions were electrophoresed and analyzed by scanning densitometry to produce a standard curve that determined the linear range of quantifiable reaction products.
PCR products from CEMX-174 cells were run on a 3% NuSieve gel (FMC Bioproducts, Rockland, ME) and isolated using GeneClean (BIO 101, Vista, CA). The isolated PCR products were cloned using the TA Cloning Kit (Invitrogen, Carlsbad, CA) and sequenced on an ABI Prism 377 DNA sequencer (PE Applied Biosystems, Foster City, CA). The sequence was determined to be identical to that of the published sequence for human GHRH (17).
Statistics
For multiple comparisons, ANOVA was used to determine significance among groups. Two-group comparisons were made using Student’s t test. Significance was established at P < 0.05.
Results
The semiquantitative nature of the GHRH RT-PCR assay is shown in
Fig. 1
. As shown in this figure a
dose-response relationship between the amount of PCR target (first
round amplification product) and the intensity of GHRH band detected by
fluorescence imaging was found. The amount of second round PCR product
obtained produced a linear standard curve for relative
quantitation.
|
. This experiment was replicated three
times with identical data obtained. PBMC, monocytes, and granulocytes
all expressed GHRH mRNA, although monocytes expressed relatively more
GHRH than PBMC and granulocytes. All of the immune cell lines tested
with the exception of CCRF-SB, which is a B lymphoblastoid cell line
derived from human peripheral blood, and HL-60, which is a human
promyelocytic cell line, expressed more GHRH mRNA relative to PBMC. The
U937 cell line, which is of monocyte/macrophage origin, expressed
2-fold higher GHRH mRNA than fresh peripheral monocytes.
|
. The distribution of GHRH in lymphocyte
subtypes from these subjects is also shown in Fig. 3. T and B
lymphocytes and monocytes all expressed the GHRH protein, with
relatively more T cells expressing GHRH. Aging resulted in a selective
reduction of GHRH in the B cells (P < 0.001) and
monocytes (P < 0.001), but not in T cells.
|
. In this experiment the
mean age of premenopausal women was 32 ± 1.8 yr, and that of the
postmenopausal group was 53 ± 0.58 yr. The mean age of young men
was 26.8 ± 1.2 yr, and that of old men was 71.8 ± 3.4 yr.
As shown, the GHRH mRNA in the PBMC of postmenopausal women was
significantly (P < 0.05) lower than that in
premenopausal women and was not different in the PBMC of young men
compared with old men. Furthermore, PBMC obtained from old men
expressed significantly (P < 0.05) more GHRH mRNA than
that from postmenopausal women.
|
) obtained from postmenopausal women
(66 ± 2 yr) not receiving HRT (n = 4) was significantly less
(P < 0.05) than those in PBMC from women receiving HRT
(n = 5) and old men (mean age, 67 ± 1.5 yr; n = 6;
P < 0.01). GHRH secretion from PBMC of the latter two
groups under basal conditions was not different (Fig. 5).
|
The current study confirms and extends previous reports on the expression of GHRH in peripheral immune cells. Using an RT-PCR assay, we demonstrated for the first time that human GHRH mRNA is readily detected in both peripheral mononuclear cells as well as lymphoid- and myeloid-derived cell lines composed exclusively of B cells, T cells, and monocytes. An earlier study (9) showed GHRH mRNA expression in rat peripheral lymphocytes, with no further characterization of the subtypes involved. Stephanou et al. (10) reported two GHRH mRNA transcripts by Northern analysis. We detected a single PCR product identical to the hypothalamic species, as did Weigent et al. in rat leukocytes (4). This suggests that differences that give rise to the larger transcript lie outside of the required targets for RT-PCR.
GHRH has been previously localized to a number of extrahypothalamic sites. Interestingly, the presence of GHRH has been consistently associated with oncogenic transformation, and in fact, GHRH was originally isolated from a pancreatic islet cell tumor (18). More recently, endometrial, ovarian, and breast cancer have been shown to express GHRH mRNA (19, 20). Furthermore, GHRH antagonists inhibited the growth of various tumors xenografted into nude mice (21). A potential involvement of GHRH in the formation of immune cell tumors comes from studies in transgenic mice overexpressing human GHRH. These transgenic animals had higher numbers of splenic progenitor cells (22), suggesting accelerated lymphocyte proliferation. Our study shows that compared with PBMC, most immune cell lines tested expressed higher levels of GHRH mRNA than PBMC. U-937, a monocyte/macrophage cell line, expressed more GHRH than monocytes from the peripheral circulation, and efforts are underway to obtain enough T and B cells from the peripheral circulation to make similar comparisons with specific cell lines. The CEMX-174 cell line, which is a transformed B/T tumor line, and WIL2-N, which is of B cell origin, expressed the highest level of GHRH mRNA relative to PBMC. These results suggest that dysregulation of GHRH gene transcription may contribute to the pathogenesis of tumors in select types of immune cells.
We also demonstrated here differential GHRH mRNA and protein expression in aged and young men and women. The aging process is associated with a significant reduction in GH/IGF-I secretion (23) and a concomitant decline in immune function (24). A cause and effect relation between these events has not been established. In our previous study in age-advanced humans, daily administration of GHRH resulted in increased circulating GH and IGF-I and sustained immune activation with GHRH treatment (15). The current study now documents that a decline in the percentage of peripheral lymphocytes expressing GHRH occurs in both men and women. This decline is cell specific, being restricted to B cells and monocytes, but not T cells. It remains to be determined whether a similar decrease in immune GH and IGF-I expression occurs. The decreased immune GHRH expression associated with aging may potentially be a significant contributing factor to immunosenescence. In addition, using the technique of double color flow cytometry we showed that a small percentage (<2%) of peripheral lymphocytes express GHRH. GHRH protein is found in all lymphocyte subtypes, with T cells expressing relatively more GHRH protein than B cells and monocytes. The relatively small number of cells that express GHRH raises questions about the sensitivity of flow cytometry to assess GHRH expression. This could be especially significant when differences between experimental groups are small. However, the data clearly indicate differences among the cell types regardless of absolute numbers, and in all cases GHRH fluorescence intensity was clearly above the nonspecific control value. Differences in immune localization of the somatotropic hormones raise the possibility that GHRH secreted by one immune cell type might influence the secretion of GH/IGF-I by another cell type similar to the hypothalamic-pituitary axis.
The age-related changes in GHRH mRNA expression is also influenced by gender. Although there was lower expression of mRNA in postmenopausal women compared with premenopausal women, there were no changes detected in old men compared with young men. The deficiency of estrogen and progesterone in postmenopausal women may potentially account for the lower GHRH mRNA expression in this group. This is evidenced by our finding that PBMC derived from women receiving HRT secreted more GHRH in vitro than cells from women receiving no hormonal therapy. We propose that an aging-induced decrease in immune GHRH could result in decreased GH and IGF-I expression, thereby leading to impaired T cell proliferation and activation, and immune deficiency. Rejuvenating interventions such as HRT could potentially reverse these changes through direct effects on immune cell GHRH.
In summary, GHRH is expressed in a small percentage of peripheral human lymphocytes. Various factors influence the expression of this neuropeptide in the immune system. The tumorigenic process may lead to up-regulation of GHRH gene transcription, whereas aging is associated with a decline in GHRH expression. Cell types affected by this process are B cells and monocytes, but not T cells. Interventions such as HRT reverse the age-induced changes in lymphocyte GHRH secretion. Collectively, our findings suggest multifactorial regulation of GHRH expression in the peripheral immune system and a significant role as a local immune modulator. Additional studies are needed to potentially unravel the regulatory factors involved and the physiological role of the immune somatotropic axis.
Acknowledgments
The kind gift of GHRH antibody by Dr. W. Vale (The Salk Institute, La Jolla, CA) is greatly appreciated. We thank Dr. Igor Sluvkin, Maureen Durning, and Richard Grendell for assistance with establishing the RT-PCR assay for GHRH, and Cynthia Tigelaar for manuscript preparation.
Footnotes
1 This work was supported in part by a Berlex Scholar Award (to O.K.)
and Primate Center Base Grant RR-00167 from the NIH. 
Received April 8, 2000.
Revised June 8, 2000.
Revised December 20, 2000.
Accepted March 13, 2001.
References
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