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Department of Pharmacology, Kansai Medical University, Osaka 570-8506, Japan
Address all correspondence and requests for reprints to: Naoki Hattori, M.D., Department of Pharmacology, Kansai Medical University, Fumizono-cho 10-15, Moriguchi-City, Osaka 570-8506, Japan.
Abstract
We examined GH and GH receptor expression in human leukemic cell lines and leukocytes of normal subjects to elucidate the cell types expressing GH and GH receptor, the individual variations of their expressions, their correlation and the relationships with serum IgG and IGF-I concentrations. In addition, the expression of GH secretagogue receptor, which enhances GH secretion from the anterior pituitary by synthetic GH secretagogues and that of its endogenous ligand, ghrelin, were also examined in these immune cells.
GH expression in human leukemic cell lines was observed mainly in B cell lines at both the mRNA and protein level [3.8 ± 0.2 pg/106 cells in Raji and 19.9 ± 3.3 pg/106 cells in Daudi vs. negligible in T cell lines (Jurkat and Hut-78) and in myeloid cell lines (K-562 and HL-60)]. B cells in normal subjects were also found to be the major immune cells expressing GH mRNA, with significant individual variation. GH receptor mRNA expression was detectable in all human leukemic cell lines, although the expression level varied widely among the cell lines and was weaker than that in the liver. On the other hand, GH receptor mRNA expression was mainly found in B cells, with marked individual variation in normal subjects. There was a positive correlation between the mRNA expressions of GH and GH receptor in B cells of normal subjects (r = 0.89; P < 0.001). Single cell RT-PCR revealed that some B cells expressed both GH and GH receptor transcripts, and others expressed only GH. GH/GH receptor expression levels in B cells did not show any correlation with serum IgG and IGF-I levels in normal subjects. Expression of GH secretagogue receptor and ghrelin was detectable in all immune cells regardless of the maturity and cell types with great individual variations. In summary, GH secreted from B cells may act locally on their own receptors, and their variable expressions may be related to individual immune functions. Widespread distribution of ghrelin and GH secretagogue receptor in human immune cells may indicate unknown biological functions other than enhancing GH secretion in the immune system.
IT HAS BEEN reported that lymphocytes synthesize and secrete GH that is identical to pituitary GH (1, 2, 3, 4, 5, 6, 7, 8). As lymphocytes possess GH receptors (GHR) (9, 10), lymphocyte-derived GH may act on their own receptors in an autocrine/paracrine fashion. GH plays an important role in the development and function of the immune system: increasing natural killer cell activity (11), erythropoiesis (12), lymphopoiesis (13), granulopoiesis (14), and the production of superoxide anions from neutrophils and macrophages (15). To further elucidate the biological significance of GH in the immune system, it is important to know which cell types in the immune system produce GH and bear GHR. Expression of GH and GHR in the immune system has been analyzed mainly by a RT-PCR technique for the detection of their mRNA, but measurement of GH at the protein level requires a highly sensitive assay. Expression of GH and GHR has been separately described in human lymphocytes, especially B cells, but there have been no studies analyzing both GH and GHR expression in the same subjects. In addition, it is not clear whether the same B cells express GH and GHR, and the relationship between GH/GHR mRNA levels in immune cells and individual immune status or the systemic GH-IGF-I axis. To answer these questions, we examined GH and GHR expression in various human leukemic cell lines and in the immune cells of normal subjects and the relationship between GH and GHR expression in the same subjects using RT-PCR and a highly sensitive immunodetection system with the sensitivity of 0.3 pg/ml (16). Single cell RT-PCR technique (17) was employed to check the co expression of GH and GHR in a single B cell. We also examined the relationship of GH/GHR expression levels in B cells of normal subjects with serum IgG (IgG) levels, one of the parameters of individual immune status, and with serum insulin-like growth factor-I (IGF-I) concentrations.
GH secretion from the anterior pituitary gland is stimulated by GHRH via its receptor, both of which are reportedly expressed in lymphocytes (18, 19). GH release is also enhanced by a class of synthetic molecules, termed GH secretagogues (GHS), and the receptor (GHS-R) exists in the anterior pituitary gland, hypothalamus and various neuroendocrine tumors (20, 21). Recently, an endogenous ligand for GHS-R has been cloned and termed ghrelin (22), whose expression is exclusively observed in stomach and hypothalamus. In the present study we also examined the expression of ghrelin and GHS-R in immune cells to elucidate the regulation of GH secretion in the immune system.
Materials and Methods
Culture of human leukemic cells
Human leukemic Raji and Daudi cell lines were purchased from Human Science Research Resource Bank (Osaka, Japan); Jurkat, K-562, and HL-60 were obtained from RIKEN Cell Bank (Tsukuba, Japan); and Hut-78 was provided by the Fujisaki Cell Center (Okayama, Japan). The cells were cultured in RPMI 1640 supplemented with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. The cells were cultured in a humidified incubator with a CO2 concentration of 5% at 37 C.
Separation of T cells, B cells, and neutrophils from the blood of normal subjects
Venous blood (10–20 ml) was taken from 22 normal adult volunteers (12 men and 10 women, 20–42 yr old), and peripheral blood mononuclear cells and neutrophils were separated by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) as described previously (6). Peripheral blood mononuclear cells were further separated into T cells and B cells by anti-CD2 (T cell surface marker) and anti-CD19 (B cell surface marker) antibody-coated magnetic beads (Dynal, Oslo, Norway). Approximately 5 x 106 T cells, 2 x 106 B cells, and 7 x 106 neutrophils were obtained from 10-ml blood samples.
RNA isolation and RT-PCR
Total RNA was isolated from human leukemic cells and T cells, B cells, and neutrophils of normal subjects using a monophasic solution of phenol and guanidine isothiocyanate (TRIzol reagent, Life Technologies, Inc., Gaithersburg, MD), followed by extraction and precipitation with isopropyl alcohol. Total RNA was also extracted from the normal liver tissue surrounding the resected hepatocellular carcinoma from a patient after receiving informed consent. The amounts of RNA were quantitated using UV spectroscopy (absorbance at 260 nm). RT-PCR was performed with a commercially available RT-PCR system (Promega Corp., Madison, WI) in 50 µl 10 mmol/liter Tris-HCl buffer containing 1 µmol/liter each of sense and antisense primers, 0.1 U/ml avian myeloblastosis virus reverse transcriptase, 0.1 U/ml Thermus flavus DNA polymerase, 0.2 mmol/liter deoxy (d)-NTP, 1.2 mmol/liter MgSO4, and 0.25–0.5 µg RNA, which was pretreated with deoxyribonuclease I (Life Technologies, Inc.) to remove trace amounts of genomic DNA. cDNA was produced by the reaction at 48 C for 45 min. Then, 20–38 cycles of PCR were carried out in a GeneAmp PCR System 9600 (Perkin-Elmer Corp., Foster City, CA) under the following conditions: denaturing at 94 C for 30 sec, annealing at 58 C (GH and ghrelin), 56 C (GHR and ß-actin), or 52 C (GHS-R) for 1 min, and extension at 68 C for 2 min. The primers used for GH were 5'-CAGGCTTTTTGACAACGCTATG-3' (sense) and 5'-GTTCTTGAGTAGTGCGTCATCGTT-3' (antisense) flanking exons 2, 3, 4, and 5, and the expected PCR product of GH was 457 bp in length. The primers used for GHR were 5'-AGAGACTTTTTCATGCCACT-3' (sense) and 5'-GGTTGCACTATTTCATCAAC-3' (antisense), flanking exons 4, 5, and 6, and the expected length of the PCR product was 264 bp. The primers used for GHS-R were 5'-AGCGCTACTTCGCCATC-3' (sense) and 5'-CCGATGAGACTGTAGAG-3' (antisense), and the expected length of the PCR product was 288 bp. The primers used for ghrelin were 5'-TGGAATCAAGCTGTCAGG-3' (sense) and 5'-ACAGTCGTGGGAGTTGC-3' (antisense) on exons separated by an intron 809 bp in length, and the expected size of the PCR product was 196 bp. ß-Actin was included as an internal control (sense primer, 5'-TTGTAACCAACTGGGACGATATGG-3'; antisense primer, 5'-CCGCTCATTGCCGATAGTGATGA-3'), resulting in a PCR product 539 bp in length. To control for the integrity of the cDNA templates and to rule out DNA contamination of the samples, the primers were designed to span the introns, except for the primers of GHS-R, in which PCR without RT was included as a control. In addition, as we do not have RNA samples used for positive controls of RT-PCR for GHS-R (human pituitary gland) and ghrelin (human stomach), the primers for these two peptides were chosen from the sequence similar to that of rat GHS-R cDNA (100% identical in sense and antisense primers) and rat ghrelin cDNA (one mismatch in sense and three mismatches in antisense primers), so that rat RNA samples can be used as positive controls. The linear range of the PCR amplification was determined by carrying out the RT-PCR for a varying number of cycles on a fixed quantity of RNA, so that the expression levels could be evaluated semiquantitatively. The optimal numbers of cycles for GH, GHR, GHS-R, ghrelin, and ß-actin were 34, 34, 36, 36, and 24, respectively.
Single cell RT-PCR for GH and GHR
Single cell RT-PCR was performed as previously described (17). B cells isolated by anti-CD19 antibody-coated magnetic beads from a normal subject were incubated overnight in poly-L-lysine-coated 35-mm dishes (Sumilon, Sumitomo Bakelite, Tokyo, Japan) filled with 3 ml RPMI 1640 containing 10% FCS and antibiotics. Under a phase contrast microscope (IMT-2, Olympus Corp., Tokyo, Japan), a single B cell was sucked into the pipette, which was filled with 3 µl reaction buffer for RT-PCR and 3 U ribonuclease inhibitor (Roche, Mannheim, Germany). The pipette tip was broken into the reaction tube, and a single cell was extruded with the reaction buffer. The reaction tube was placed immediately in liquid nitrogen and then kept at -80 C until the assay. The content was divided into two aliquots for examining the expression of GH and GHR in a single B cell. RT-PCR was performed in the same way as the usual RT-PCR, except for the primers and the cycles (50 cycles) of PCR. To facilitate the amplification for a longer period, other primer sets for GH and GHR were designed to produce shorter fragments: 5'-CAGGCTTTTTGACAACGCTATG-3' (sense) and 5'-TTGGAGGGTGTCGGAATAGACT -3' (antisense), flanking exons 2 and 3 with the expected 168-bp fragment for GH, and 5'-GTTCACCTGAGCGAGAGACT-3' (sense) and 5'-AGGTATCCAGATGGAGGT-3' (antisense), flanking exons 4 and 5 with the expected 203-bp fragment for GHR.
Cloning and sequencing
The PCR products were electrophoresed in 2% agarose gels, and ethidium bromide-stained bands of the PCR fragments were excised from the gel, followed by purification using Gene Clean spin (BIO 101, Vista, CA). To avoid the effects of byproducts that make the readable sequence very short, DNA sequencing was not performed directly on the PCR product, but, rather, on the plasmid in which cDNA fragments were inserted. PCR fragments were ligated to a plasmid vector (pCR 2.1) by T4 DNA ligase and transfected into competent cells using a TA cloning kit (Invitrogen, Carlsbad, CA). The plasmid DNA was purified by an alkaline lysis procedure, and the sequencing of the cDNA fragments was performed using a Big Dye Termination Cycle Sequencing Ready Reactions Kit (PE Applied Biosystems, Foster City, CA) in a GeneAmp PCR System 9600.
Image analysis and calculation of relative RNA levels
The PCR products were electrophoresed in 2% agarose gel, and the bands of PCR products were stained with Vistra green (Amersham Pharmacia Biotech, Little Chalfont, UK) for 20 min. The fluorescence intensity of each band was measured with a FluorImager 595 (Molecular Dynamics, Inc., Sunnyvale, CA), which is an optical scanner that detects light emitted from fluorescent samples and produces a digital image. The wavelengths of excitation and emission were 488 and 530 nm, respectively. The data were then analyzed by ImageQuant, a software program from Molecular Dynamics, Inc.. The ratio of the fluorescence intensity of each band [(GH - background)/(ß-actin - background), or (GHR - background)/(ß-actin - background)] was determined.
Northern blot analysis
cDNA probe for GH was labeled by PCR with digoxigenin-11-dUTP using a dNTP mixture containing the labeled dUTP (DIG DNA labeling mixture, Roche Molecular Biochemicals). Digoxigenin-11-dUTP was incorporated into the PCR-amplified fragment using the target cDNA, which was ligated to a plasmid vector. The PCR mixture for labeling (50 µl) contained 1 µmol/liter of each primer set; 1 ng target cDNA; 0.2 mmol/liter of each dATP, dGTP, dCTP, and dTTP plus labeled dUTP; and 2.5 U Taq polymerase in 50 mmol/liter Tris-HCl (pH 8.0), 50 mmol/liter KCl, 1.5 mmol/liter MgCl2, and 0.015% gelatin. Amplification was carried out for 40 cycles of 1 min at 94 C, 2 min at 58 C, and 2 min at 72 C. The amplified fragments were purified by Sephadex G-50 column (Quick Spin Column, Roche Molecular Biochemicals). Denatured total RNA (20 µg/lane) was separated by electrophoresis on 1% agarose-formaldehyde gel. The gel was washed in 20 x SSC (3 mol/liter NaCl and 0.3 mol/liter sodium citrate, pH 7.0), and the RNA was transferred onto a positively charged nylon membrane (Roche Molecular Biochemicals) by capillary blotting with 20 x SSC for 17 h. The RNA was fixed by UV cross-linking at 254 nm (0.16 J/cm2). The membrane was prehybridized at 42 C for 2 h in hybridization solution [50% formamide, 5 x SSC, 50 mmol/liter sodium phosphate (pH 7.4), 2% blocking reagent (Roche Molecular Biochemicals), 50 µg/ml salmon testes DNA, and 0.5% SDS]. Denatured digoxigenin-labeled probe and dextran sulfate were added to the hybridization solution at final concentrations of 12.5 ng/ml and 5%, respectively, and incubation was continued for 16 h. The blot was rinsed briefly and washed twice at 65 C for 20 min each time in 1 x SSC/5% SDS. The hybridized probe was detected using alkaline phosphatase-conjugated antidigoxigenin Fab (Roche Molecular Biochemicals) diluted 1:10,000 and 0.26 mmol/liter disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]-decan}-4-yl)phenyl phosphate (Tropix, Bedford, MA), a chemiluminescent substrate, according to the manufacturer’s instructions. The membrane was exposed to Kodak OMAT AR film(Eastman Kodak, Rochester, NY). To normalize the amount of total RNA, the membrane was reprobed with a ß-actin cDNA probe after stripping twice with 1x SSC/1% SDS at 95 C for 10 min each.
Enzyme immunoassay (EIA) of GH
Human leukemic cells were cultured at a cell density of 2 x 105 cells/ml in 2 ml RPMI 1640 containing FCS and the antibiotics for 3 d starting from the change of medium. The culture medium was collected and centrifuged, and GH concentrations in the supernatant were measured by a highly sensitive EIA, as described previously (16). Anti-GH IgG-coated polystyrene balls were incubated with GH standards (HS2243E, NPH) or samples (0.1 ml) in duplicate in a total volume of 0.15 ml at 37 C for 6 h with continuous shaking. After removal of the supernatant, the balls were washed twice with 150 mmol/liter saline and incubated with affinity-purified anti-GH Fab'-horseradish peroxidase conjugate at 4 C for 16 h without shaking and at 20 C for 6 h with continuous shaking. After removal of the supernatant, the balls were washed three times with 150 mmol/liter saline, and the peroxidase activity bound to the balls was assayed by an enzyme reaction using 3-(p-hydroxyphenyl)propionic acid (Aldrich Chemical Co., Inc., Milwaukee, WI) as a substrate at 30 C for 90 min. Fluorescence intensity was measured by a spectrofluorophotometer (F-2000, Hitachi, Ibaragi, Japan). The minimal detectable quantity of GH by this assay was 0.3 pg/ml. The intra- and interassay coefficients of variations were 6.0% and 8.6%, respectively.
Gel chromatography
The culture medium of Raji and Daudi cell lines (1–2 ml) was applied to an Ultrogel AcA44 column (1 x 70 cm) and eluted with 0.01 mol/liter sodium phosphate buffer (pH 7.0) containing 0.1 mol/liter NaCl, 0.1% NaN3, and 0.1% BSA. Fractions of 1 ml were collected, lyophilized, and reconstituted by water to be 0.25 ml (4-fold concentrations). The GH concentration in each fraction was measured by EIA.
Measurements of IgG and IGF-I
Serum IgG and IGF-I concentrations in normal subjects were measured by a commercially available ELISA kit (Bethyl Laboratory, Inc., Montgomery, TX) and an immunoradiometric assay kit (Bayel, Tokyo, Japan), respectively.
Statistical analysis
The quantitative values are shown as the mean ± SEM. The statistical analyses were performed using t test, and correlation coefficients of variation were determined by linear regression analysis.
Results
GH mRNA expression in various kinds of human leukemic cells is
shown in Fig. 1a
. PCR products
with lengths corresponding to the GH cDNA fragment (457 bp), which was
verified by DNA sequencing, were observed mainly in Raji and Daudi cell
lines, both of which are of B cell origin. Slight expression of GH was
observed in one of the T cell type leukemias (Hut-78), but almost no
expression of GH was seen in myeloid cell lines. Northern blot analysis
revealed that a GH signal of about 1 kb was only detectable in B cell
lines, with the expression level greater in Daudi than in Raji cells
(Fig. 1b).
|
shows GH mRNA expression in T
cells, B cells, and neutrophils from the blood of five representative
normal subjects. Although there was variation in the expression of GH
among individuals, the level of GH mRNA expression was significantly
greater in B cells (ratio of the fluorescence intensity to ß-actin,
0.46 ± 0.10; n = 22; P < 0.001) than T
cells (0.13 ± 0.04) or neutrophils (0.01 ± 0.01). In
addition to the major band, another band with a length of 666 bp was
detected in B cells, the intensity of which seemed to parallel that of
the main band. This higher band was also detectable in human leukemic B
cell lines (Fig. 1a
). DNA sequencing of this higher band disclosed a
splicing variant of GH in which an intron (209 bp) between exons 2 and
3 was retained. Then, the GH secretion from human leukemic cell lines
was quantitatively examined using a highly sensitive enzyme
immunoassay. Starting the culture of human leukemic cells (2 x
105/ml) in RPMI 1640 with 10% FCS, these cells
grew exponentially and reached a plateau approximately 5 d later.
Therefore, the cells and the culture medium were collected during the
exponentially growing phase (3 d after the start of culture). The
number of cells increased 6-fold, on the average, during these 3
d. GH secretion was found exclusively in Raji (3.8 ± 0.2
pg/106 cells) and Daudi (19.9 ± 3.3
pg/106 cells) cells and was negligible in the
other human leukemic cell lines (Fig. 3),
in agreement with the results of RT-PCR and Northern blotting. Gel
chromatographic analysis revealed that GH secreted from Raji and Daudi
cell lines had the same molecular mass as 22-kDa pituitary GH. Large
molecular mass forms of GH were not observed (Fig. 4).
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|
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, PCR products with lengths
corresponding to GHR cDNA fragments (264 bp), verified by DNA
sequencing, were obtained in all cell lines, with great difference in
the expression levels, i.e. strongest in K-562 and very weak
in Jurkat and HL-60 cell lines, although the GHR expression levels in
leukemic cell lines were much less than that in the liver. Figure 6 shows the GHR mRNA expression in T
cells, B cells, and neutrophils from the blood of five representative
normal subjects. Although there was variation in the expression of GHR
among the individuals, the level of GHR mRNA expression was
significantly greater in B cells (ratio of the fluorescence intensity
to ß-actin, 0.39 ± 0.08; n = 22; P <
0.001) than T cells (0.04 ± 0.02) or neutrophils (0.01 ±
0.01). The relationship between GH and GHR expression levels in B cells
was examined in 22 normal subjects (Fig. 7). There was a significant positive
correlation between the expression levels of GH and GHR (r = 0.89;
P < 0.001).
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|
|
shows the results for seven individual
B cells obtained from a normal subject. GH was expressed in all B
cells, whereas GHR expression varied widely among individual B
cells.
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Expression levels of GHS-R and its endogenous ligand (ghrelin) were
examined by RT-PCR in human leukemic cell lines and immune cells from
normal subjects. Human leukemic cell lines were all positive for GHS-R
expression (288 bp), which was verified by DNA sequencing (Fig. 9a). PCR performed directly on RNA
preparations without prior RT failed to yield visible amplified bands,
excluding the possibility of the amplification of DNA. Figure 9b shows
that GHS-R expression was detectable in T cells, B cells, and
neutrophils in normal subjects, but the expression levels varied widely
among individuals. Human leukemic cell lines were also all positive for
ghrelin expression (196 bp), which was verified by DNA sequencing (Fig. 10a). Ghrelin expression was also
detected in all immune cells from normal subjects, with great
individual variations in the expression levels (Fig. 10b).
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Expression of GH mRNA (1, 2) as well as production and secretion of GH (3, 4, 5, 6, 7, 8) have been reported in rat and human lymphocytes. Among the rat lymphocytes, B cells reportedly predominate over T cells in the expression of GH at both mRNA and protein levels (4). In human lymphocytes, expression of GH mRNA has been observed predominantly in B cell-enriched areas in immune organs (2) and in peripheral B cells rather than T cells (2, 23). Furthermore, a human B cell line, IM-9, has been shown to express GH by immunofluorescence and reverse hemolytic plaque assay, although GH secretion was not quantitatively detected (7). In the present study GH secretion from human leukemic cell lines was first demonstrated quantitatively using a highly sensitive EIA. GH with the same mol wt as pituitary GH was secreted exclusively from B cell lines, with the predominant expression of GH mRNA. In B cells isolated from normal subjects, GH secretion could not be quantitatively measured despite the predominant expression of GH mRNA. GH in the medium of B cells from normal subjects was apparently higher than that in the medium background, but it was close to the detection limit (0.3 pg/ml). The discrepant results between GH mRNA and protein expression levels in normal subjects can be explained as follows. RNA for examining GH mRNA expression was extracted just after the isolation of B cells, whereas the culture medium for examining secreted GH was collected 3 d later. In contrast to the 100% viability of leukemic cells that were actively growing, the viability of B cells isolated from normal subjects declined gradually during the 3-d period, reaching 20% on the third day, probably because of the influence of insult during the separation procedure. Therefore, it is likely that GH is actively produced and secreted from B cell lines, whereas the amount of GH from B cells in normal subjects is very small during the incubation period despite the almost equal expression of GH transcript at the start of incubation.
We found another GH transcript with a retained intron between exons 2 and 3 in human B cells. To our knowledge, there have been no reports describing this type of GH variant expression in the pituitary and immune cells. We previously reported that human lymphocytes expressed 20-kDa GH, which was generated by alternative splicing within exon 3 resulting in a 45-bp deletion, another variant spliced also within exon 3, but at a different site, resulting in a 73-bp deletion and an exon 3 and 4 deleted form (6). Taken together, human B cells express various kinds of splicing variants, although the biological significance of these variants remains to be elucidated.
Human GHR reportedly exists on cultured human lymphocytes of the B cell lineage (9). GHR on circulating human peripheral mononuclear cells was recently detected using a flow cytometric assay, showing that B cells and monocytes predominantly bear GHR (24, 25). In the present study GHR mRNA was found to be expressed in all human leukemic cell lines and in human peripheral B cells from normal subjects, with significant variation among cell lines and individuals, although the expression level was low compared with that in the liver. It is of note that GHR was expressed not only in B cell lines but also in T cell and myeloid cell lines, whereas GHR was almost exclusively expressed in B cells from normal subjects, suggesting that GHR expression can be altered by cell differentiation. In addition, this marked variation in expression may be attributed to the finding that receptors are down- or up-regulated by the environment (26). We examined the expression of both GH and GHR in individuals to clarify the relationship between them. Interestingly, a positive correlation was observed between the expression levels of GH and GHR in B cells. The role of GH in the regulation of its own receptors is controversial. Adipocytes isolated from hypophysectomized rats have low GH binding, and treatment with GH resulted in an increase in receptor levels (27). However, homologous down-regulation of GHR by GH has been shown in IM-9 lymphocytes (28). Although the precise mechanism is unclear, GH secreted by B cells may act in an autocrine/paracrine fashion and possibly up-regulate GHR expression in B lymphocytes. It is unlikely that the systemic GH/IGF-I axis can influence GH/GHR expression in B cells or vice versa, because we did not find any correlation between serum IGF-I levels and GH/GHR expression in B cells.
Single cell RT-PCR (17) allowed us to examine whether the same B cell coexpresses both GH and GHR or whether different B cells express GH or GHR separately. Although this is preliminary, seven B cells examined all expressed GH mRNA, whereas the expression of GHR mRNA varied widely among B cells. In other words, some B cells express both GH and GHR, and others express only GH even in the same individual. It can be said that GHR expression in B cells varied widely not only among individuals but also among B cells in an individual.
It has been reported that exogenous GH administration enhances IgG production by B cells (29). We, therefore, examined the relationship between GH/GHR expression levels in B cells and serum IgG concentrations. There was no correlation between GH/GHR expression levels and systemic IgG levels, suggesting that GH/GHR in B cells may act locally on IgG production.
There have been contradictory reports about the regulation of GH secretion from lymphocytes; some investigators have reported that lymphocytes possess GHRH and their receptors, and exogenous administration of GHRH increases GH secretion (18, 19), whereas we did not find any stimulatory effect of GHRH on GH secretion from lymphocytes (5). In the present study we first examined the expression of another receptor that stimulates GH secretion from the anterior pituitary, GHS-R and its endogenous ligand, ghrelin, in immune cells. GHS-R expression has been reported in the anterior pituitary, hypothalamus, and a variety of neuroendocrine tumors (20, 21), and ghrelin expression has been reported exclusively in stomach and hypothalamus (22), although expression levels of these peptides in the immune cells have not been examined. It is of interest that both GHS-R and ghrelin were widely expressed in human immune cells regardless of the maturity and cell types, and that there was a great difference in their expression in normal subjects. Our finding that GHS-R and ghrelin were expressed not only in B cells but also in T cells and neutrophils that did not express substantial GH transcripts suggests that ghrelin/GHS-R has unknown biological functions other than enhancing GH secretion in the immune system. The present finding that the expression levels of GH, GHR, ghrelin, and GHS-R varied widely among individuals may reflect the variations in GH-related immune functions among individuals.
Acknowledgments
We thank the National Hormone and Pituitary Program (U.S.) for supplying human GH standards, and Kansai Medical University Laboratory Center for providing experimental instruments.
Footnotes
This work was supported by the Institute of Growth Science of Japan.
Abbreviations: dNTP, Deoxy-NTP; EIA, enzyme immunoassay; GHR, GH receptor; GHS, GH secretagogue; GHS-R, GH secretagogue receptor.
Received September 26, 2000.
Accepted May 31, 2001.
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 K. Nakahara, M. Nakagawa, Y. Baba, M. Sato, K. Toshinai, Y. Date, M. Nakazato, M. Kojima, M. Miyazato, H. Kaiya, et al. Maternal Ghrelin Plays an Important Role in Rat Fetal Development during Pregnancy Endocrinology, March 1, 2006; 147(3): 1333 - 1342. [Abstract] [Full Text] [PDF]
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 R. Wu, W. Dong, M. Zhou, X. Cui, H. Hank Simms, and P. Wang Ghrelin improves tissue perfusion in severe sepsis via downregulation of endothelin-1 Cardiovasc Res, November 1, 2005; 68(2): 318 - 326. [Abstract] [Full Text] [PDF]
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 S. Stanley, K. Wynne, B. McGowan, and S. Bloom Hormonal Regulation of Food Intake Physiol Rev, October 1, 2005; 85(4): 1131 - 1158. [Abstract] [Full Text] [PDF]
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 D. Panidis, D. Farmakiotis, G. Koliakos, D. Rousso, A. Kourtis, I. Katsikis, C. Asteriadis, V. Karayannis, and E. Diamanti-Kandarakis Comparative study of plasma ghrelin levels in women with polycystic ovary syndrome, in hyperandrogenic women and in normal controls Hum. Reprod., August 1, 2005; 20(8): 2127 - 2132. [Abstract] [Full Text] [PDF]
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M. Kojima and K. Kangawa Ghrelin: Structure and Function Physiol Rev, April 1, 2005; 85(2): 495 - 522. [Abstract] [Full Text] [PDF]
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C. De Vriese, F. Gregoire, P. De Neef, P. Robberecht, and C. Delporte Ghrelin Is Produced by the Human Erythroleukemic HEL Cell Line and Involved in an Autocrine Pathway Leading to Cell Proliferation Endocrinology, March 1, 2005; 146(3): 1514 - 1522. [Abstract] [Full Text] [PDF]
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M. Sohmiya, I. Kanazawa, and Y. Kato Effect of recombinant human GH on circulating granulocyte colony-stimulating factor and neutrophils in patients with adult GH deficiency Eur. J. Endocrinol., February 1, 2005; 152(2): 211 - 215. [Abstract] [Full Text] [PDF]
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K. Wynne, S. Stanley, B. McGowan, and S. Bloom Appetite control J. Endocrinol., February 1, 2005; 184(2): 291 - 318. [Abstract] [Full Text] [PDF]
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 G. Rindi, A. Torsello, V. Locatelli, and E. Solcia Ghrelin Expression and Actions: A Novel Peptide for an Old Cell Type of the Diffuse Endocrine System Experimental Biology and Medicine, November 1, 2004; 229(10): 1007 - 1016. [Abstract] [Full Text] [PDF]
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 C. Gauna, F. M. Meyler, J. A. M. J. L. Janssen, P. J. D. Delhanty, T. Abribat, P. van Koetsveld, L. J. Hofland, F. Broglio, E. Ghigo, and A. J. van der Lely Administration of Acylated Ghrelin Reduces Insulin Sensitivity, Whereas the Combination of Acylated Plus Unacylated Ghrelin Strongly Improves Insulin Sensitivity J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5035 - 5042. [Abstract] [Full Text] [PDF]
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G. Rindi, M. Civallero, M. E. Candusso, A. Marchetti, C. Klersy, R. Nano, and A. B. Leiter Sudden Onset of Colitis After Ablation of Secretin-Expressing Lymphocytes in Transgenic Mice Experimental Biology and Medicine, September 1, 2004; 229(8): 826 - 834. [Abstract] [Full Text] [PDF]
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 R. Wu, M. Zhou, X. Cui, H. H. Simms, and P. Wang Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1296 - H1302. [Abstract] [Full Text] [PDF]
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 A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]
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 A. INUI, A. ASAKAWA, C. Y. BOWERS, G. MANTOVANI, A. LAVIANO, M. M. MEGUID, and M. FUJIMIYA Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ FASEB J, March 1, 2004; 18(3): 439 - 456. [Abstract] [Full Text] [PDF]
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 K. L.J. Ellacott and R. D. Cone The Central Melanocortin System and the Integration of Short- and Long-term Regulators of Energy Homeostasis Recent Prog. Horm. Res., January 1, 2004; 59(1): 395 - 408. [Abstract] [Full Text]
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M. M. Malagon, R. M. Luque, E. Ruiz-Guerrero, F. Rodriguez-Pacheco, S. Garcia-Navarro, F. F. Casanueva, F. Gracia-Navarro, and J. P. Castano Intracellular Signaling Mechanisms Mediating Ghrelin-Stimulated Growth Hormone Release in Somatotropes Endocrinology, December 1, 2003; 144(12): 5372 - 5380. [Abstract] [Full Text] [PDF]
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V. D. Dixit, M. Mielenz, D. D. Taub, and N. Parvizi Leptin Induces Growth Hormone Secretion from Peripheral Blood Mononuclear Cells via a Protein Kinase C- and Nitric Oxide-Dependent Mechanism Endocrinology, December 1, 2003; 144(12): 5595 - 5603. [Abstract] [Full Text] [PDF]
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K. Kawamura, N. Sato, J. Fukuda, H. Kodama, J. Kumagai, H. Tanikawa, A. Nakamura, Y. Honda, T. Sato, and T. Tanaka Ghrelin Inhibits the Development of Mouse Preimplantation Embryos in Vitro Endocrinology, June 1, 2003; 144(6): 2623 - 2633. [Abstract] [Full Text] [PDF]
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N. Moller, J. Nygren, T. K. Hansen, H. Orskov, J. Frystyk, and K. S. Nair Splanchnic Release of Ghrelin in Humans J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 850 - 852. [Abstract] [Full Text] [PDF]
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 L. A. Welniak, R. Sun, and W. J. Murphy The role of growth hormone in T-cell development and reconstitution J. Leukoc. Biol., March 1, 2002; 71(3): 381 - 387. [Abstract] [Full Text] [PDF]
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