help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Borg, S. A.
Right arrow Articles by Jones, T. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Borg, S. A.
Right arrow Articles by Jones, T. H.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 10 4938-4944
Copyright © 2003 by The Endocrine Society

Expression of Interleukin-6 and Its Effects on Growth of HP75 Human Pituitary Tumor Cells

S. A. Borg, K. E. Kerry, L. Baxter, J. A. Royds and T. H. Jones

Endocrine Heart and Pituitary Group, Academic Unit of Endocrinology (S.A.B., K.E.K., T.H.J.), Academic Unit of Pathology (L.B.), Division of Genomic Medicine, University of Sheffield Medical School, Sheffield, United Kingdom S10 2RX; Center for Diabetes and Endocrinology, Barnsley District General Hospital (T.H.J.), Barnsley, S75 2EP United Kingdom; and Department of Pathology, University of Otago (J.A.R.), Otago, New Zealand

Address all correspondence and requests for reprints to: Dr. T. H. Jones, Endocrine Heart and Pituitary Group, Academic Unit of Endocrinology, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield, United Kingdom S10 2RX. E-mail: hugh.jones{at}bdgh-tr.trent.nhs.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of IL-6 in the pathogenesis of pituitary adenomas is unclear, as tumor biology is difficult to study in primary culture. We have shown here that the human pituitary cell line HP75 synthesizes IL-6 mRNA and expresses and secretes IL-6 (6167 ± 56 pg/ml/72 h for 30,000 cells). IL-6 receptor (IL-6R) mRNA was identified by in situ hybridization and RT-PCR. Exogenous IL-6 in low dose (1 ng/ml) stimulated, whereas higher doses (100 ng/ml) inhibited, growth. This diverse effect occurs in other cell types as a result of receptor down-regulation. Cell growth was inhibited by IL-6-blocking antibody (76 ± 6.5% inhibition; P < 0.0001). This demonstrates that IL-6 is an important growth regulator in HP75 cells, having an autocrine growth stimulatory effect under basal conditions. IL-1{alpha} and dibutyryl cAMP stimulated and dexamethasone inhibited IL-6 secretion; however, bacterial lipopolysaccharide, forskolin, and cholera toxin had no effect. This implies that there is a defect in the control of IL-6 secretion. Soluble IL-6R was not detected, but soluble gp130 receptor was present in the conditioned medium. Stimulation of cleavage of soluble IL-6R from the membrane-bound IL-6R could not be induced by phorbol ester or dexamethasone. Whether IL-6 has a similar effect in human pituitary adenomas requires further investigation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE AND OTHERS have shown that IL-6 is synthesized and released by the majority of human pituitary adenomas removed at routine surgical operations (1, 2). The role of IL-6, if any, in the pathogenesis of human pituitary tumors has proven difficult to elucidate, as cultured cells from most of the tumors do not display significant growth to be studied. Furthermore, the number of cells attained from surgical excision is small, the population of cells is mixed, and, in particular, fibroblasts usually become the predominant cells in the culture.

The human pituitary cell line HP75 has been recently developed by simian virus 40 transfection of tumor cells derived from a clinically nonfunctioning adenoma (silent gonadotropinoma) (3). This cell line retains a number of differentiated functions of the pituitary adenoma, including chromogranin A expression, and stains positively for LH (3). Jin et al. (4) have shown HP75 cells express leptin and its receptor in both long (OB-Rb) and short (OB-Ra) forms, and an inhibitory effect of leptin on the growth of HP75 has been reported (4). These cells have constitutively activated signal transducer and activator of transcription 3 protein expression (5). The advent of this cell line provides us with a unique opportunity to investigate the effects of IL-6.

Conflicting effects of IL-6 on cell growth have been found in different cell types, including pituitary cells. High doses of IL-6 added to a small number of human pituitary adenoma cells in culture stimulated [3H]thymidine incorporation (6). A stimulatory effect on cell growth has also been reported in GH3 rat pituitary tumor cells (7) and mouse nonfunctioning MtT/E (8) and mouse TtT/GF folliculostellate (9) cell lines. IL-6, however, inhibits cell growth of normal rat anterior pituitary cells (7). The conflicting effects of IL-6 have been further demonstrated by investigation of the expression of c-fos in human pituitary adenoma explants. Páez Pereda et al. (10) demonstrated that IL-6 could both induce and inhibit the expression of the c-fos gene, which encodes for a protein that forms part of the activating protein-1 nuclear transcription factor necessary for the induction of growth and mitosis. In human pituitary adenomas, IL-6 mRNA expression correlates with tumor invasiveness, but not with the expression Ki67, a cell cycle marker of tumor growth activity (11, 12).

The expression of the IL-6 gene is controlled at its promoter by a stimulatory multiresponse element that responds to IL-1, TNF{alpha}, bacterial lipopolysaccharide (LPS), and serum; a stimulatory cAMP response element; and two inhibitory glucocorticoid elements (13). IL-1{alpha} stimulation of IL-6 release by human pituitary adenomas has been demonstrated; however, IL-1{alpha} was unable to induce measurable secretion in those tumors that did not secrete IL-6 under basal conditions (14). Jones et al. (15) demonstrated that non-IL-6-secreting pituitary adenomas do not have elevated phosphoinositide metabolism or a response to kinins, but those showing kinin-responsive tumors were all IL-6 secretors. IL-6 exerts its actions on specific target cells by acting through a receptor complex consisting of an IL-6-specific receptor (IL-6R{alpha}) and a common signal-transducing subunit, gp130. Binding of IL-6 to its receptor leads to homodimerization of two gp130 molecules, which initiates activation of the intracellular Janus kinase/signal transducer and activator of transcription pathway (16). The presence of a specific binding site in the anterior pituitary gland was first identified by Ohmichi et al. (17), and the presence of the receptor in adenoma tissue has been further identified in four studies, with occurrence ranging from 32–100%, and has been observed in the MtT/E cell line and normal rat anterior pituitary (18, 19, 20, 21). The IL-6R may also occur in a soluble form through proteolytic cleavage of the membrane-bound form or as a result of alternative mRNA splicing, resulting in a transcript encoding only the extracellular domain portion with a modified C-terminal sequence. These modified forms of the IL-6R{alpha} retain the same binding affinity for IL-6 and the ability to complex with membrane-bound gp130 (22). The IL-6/soluble IL-6R (sIL-6R{alpha}) complex is an agonist, rendering cells expressing gp130, even in the absence of membrane-bound IL-6R{alpha}, responsive to IL-6 (23). The soluble form of gp130 does not act in a similar agonistic manner to the sIL-6R{alpha}, acting as an antagonistic to the IL-6/(s)IL-6R{alpha} complex. It is able to bind the ligand receptor complex, but is unable to fulfill its role as initiator of the downstream signaling pathway (22).

We have investigated whether IL-6 is produced and secreted by HP75 cells and have looked for the expression of IL-6R and its components. We have also studied the effect of IL-6 on growth of the cell line and control of IL-6 secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

HP75 cells were routinely cultured in DMEM (BioWhittaker, Cambrex Corp., Nottingham, UK) containing 15% horse serum (TCS Cellworks, Buckingham, UK) and 2.5% fetal calf serum (Life Technologies, Inc., Invitrogen Ltd., Paisley, UK). For growth and IL-6 stimulatory experiments, HP75 cells were plated in 24-well plates at 30,000/well in DMEM containing 2% fetal calf serum. After 24 h, medium was changed, and test substances, where required, were added. At 1, 3, or 6 d, conditioned medium was removed and stored at -80 C. All experiments were conducted in triplicate wells and were repeated a minimum of four times. A growth curve was constructed by seeding 0.3 x 105 HP75 cells into 6-well plates containing complete medium. Cell were refed, harvested, and counted using a hemocytometer at intervals of up to 14 d (Fig. 1Go).



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 1. Growth curve of HP75 cells in complete medium. The data shown are the mean cell count ± SEM for triplicate wells.

 
Cell growth was measured using AlamarBlue (Serotec Ltd., Oxford, UK). AlamarBlue was added to equal 10% of the culture volume and was incubated for a additional 5.5 h at 37 C. Absorbance was measured at 570 and 600 nm (Fig. 2Go). AlamarBlue is a growth indicator based on detection of cell metabolic activity, which changes color in response to chemical reduction of growth medium resulting from cell growth. We have observed a linear relationship between HP75 cell number and OD (Fig. 1Go).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Linear relationship between cell number and AlamarBlue reading (OD; r2 = 0.99).

 
For IL-6R in situ hybridization (ISH) and IL-6 immunocytochemistry, HP75 cells were seeded onto glass coverslips and grown to confluence. Medium was removed and replaced with ice-cold acetone for 10 min at room temperature. Acetone was removed, and cells were allowed to air-dry. Cells were stored at -20 C for up to 4 wk until required.

Dexamethasone, forskolin, dibutyryl cAMP (dbcAMP), LPS, cholera toxin, phorbol-12–13-dibutyrate (PDBu), dexamethasone, and recombinant human IL-6 and IL-1{alpha} were all obtained from Sigma-Aldrich Corp. (Dorset, UK). For neutralizing experiments, rabbit anti-IL-6 polyclonal human antibody (Calbiochem CN Biosciences Inc., Darmstadt, Germany) was used.

IL-6 and IL-6R ISH

For IL-6 detection, 5-µm sections from formalin-fixed, paraffin-embedded cell clots were mounted onto aminoproopioethanolamine silane-coated slides. After dewaxing and rehydration, sections were digested with pepsin at 500 µg/ml diluted 1:40 in 0.2 M HCl at 37 C for 20 min and incubated with prehybridization buffer for 60 min at 37 C. For IL-6R detection, acetone-fixed HP75 cells were incubated with prehybridization buffer for 4 h at 37 C. The IL-6 antisense probe used was a cocktail of four digoxygenin-labeled oligonucleotide strands (R&D Systems Europe Ltd., Abingdon, UK) used at a dilution of 1:60. IL-6R probes used were double-fluorescein isothiocyanate-labeled, custom-made oligonucleotides (Biognostik, Gottingen, Germany). Three probes each of sense and antisense strands were constructed and combined at a concentration of 30 U/ml each to create two oligonucleotide cocktails. Antisense sequences were complementary to bases 599–624, 699–728, and 1181–1210 of the GenBank sequence X12830. All sections were hybridized overnight at 37 C, followed by posthybridization washing in standard saline citrate at 37 C. After preincubation with 20% normal sheep serum to block nonspecific staining, specifically bound probe was detected using antidigoxygenin-alkaline phosphatase Fab or anti-fluorescein isothiocyanate-alkaline phosphatase Fab (Roche Diagnostics Ltd., East Sussex, UK) as appropriate. After further washing, staining was visualized using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Vector Laboratories Ltd., Peterborough, UK), and cells were counterstained with methyl green. For IL-6 detection, inflamed appendix was used as a positive control, and omission of probe was used as a negative control. For IL-6R detection, the corresponding IL-6R sense strand sequences and omission of probe were used as negative controls; {alpha}-tubulin, ß-actin, poly(deoxythymidine) probes, and LNCaP cells were all used as technical and positive controls.

IL-6 and IL-6R immunocytochemistry

The avidin-biotin-peroxidase system (Vector Laboratories Ltd.) was used on acetone-fixed HP75 cells and formalin-fixed, paraffin wax-embedded cell clots. Antigen retrieval was performed in 0.01 M trisodium citrate and microwaved for 8 min on high power. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide in methanol; after rinsing, nonspecific binding sites were blocked with normal goat serum. Cells and sections were incubated overnight at 4 C with a polyclonal anti-IL-6 rabbit antibody (Serotec Ltd., Oxford, UK) diluted 1:200 or polyclonal anti IL-6R antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:50. After addition of species-specific biotinylated secondary antibody and avidin-biotin-peroxidase complex, binding sites were visualized using diaminobenzidene and counterstained with hematoxylin.

RNA extraction and RT-PCR for IL-6R

Total RNA from HP75 and RPMI8226 cells was extracted using the SV Total RNA Isolation System according to manufacturer’s protocol (Promega, Southampton, UK). Briefly, freshly pelleted cells were incubated with lysis buffer. After ethanol washes and deoxyribonuclease treatment, RNA was recovered by centrifugation and eluted into water. RT-PCR was performed using the Enhanced Avian HS RT-PCR kit (Sigma-Aldrich Corp.). The RT reaction was performed using a reaction mixture containing 500 µM each of deoxy (d)-ATP, dTTP, dGTP, and dCTP (Sigma-Aldrich Corp.) and 0.6 µM 3' antisense oligonucleotide primer incubated with 5 µg total RNA at 70 C for 10 min. The reaction was completed by the addition of ribonuclease inhibitor (1 U/µl), enhanced avian myeloblastosis reverse transcriptase (1 U/µl), and PCR buffer to a total volume of 20 µl. Samples were incubated without RT enzyme for negative controls. Five-microliter aliquots from the RT reaction were used for the subsequent PCR reaction in the presence of 0.6 µM each of 3' and 5' primers (Sigma-Genosys, Pampisford, UK); reaction buffer containing 1.5 mM MgCl2; 200 µM each of dATP, dTTP, dGTP, and dCTP; and Jumpstart AccuTaq LA DNA polymerase (2.5 U/µl). Amplification was carried out for 40 cycles using the following program: 1) denaturation step of 94 C for 5 min; 2) 40 reaction cycles of 94, 58, and 72 C each for 1 min; and 3) a single primer extension step of 72 C for 7 min. The following primer sets were used: IL-6R, 5'-GAGACAGCGTGACTCTGACCT and 3'-CCGGACTGTTCTGAAACTTCC; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-GTCATCATCTCTGCCCCCTCTGCT and 3'-GACGCCTGCTTCACCACCT-TCTTG. The PCR products thus generated are 353 and 423 bp, respectively. The PCR products and 100-bp ladder (Sigma-Aldrich Corp.) were visualized on a 2% agarose gel containing ethidium bromide. To confirm the quality of the cDNA sample, oligonucleotide primers for the constitutively expressed GAPDH were also used with each cDNA sample. RPMI8226 cells, a human myeloma cell line that is known to constituitively express the IL-6R, were used in addition to HP75 cells to act as a positive control.

ELISA

IL-6 was measured in HP75 cell culture supernatants by ELISA using the IL-6 Eli-Pair antibody kit (IDS Ltd., Tyne and Wear, UK) according to the manufacture’s instructions. Briefly, 96-well plates were coated with capture antibody and allowed to incubate overnight at 4 C; after washing, nonspecific binding site-blocked samples and standards (appropriately diluted) were added and then coincubated with biotinylated anti-IL-6 antibody for 1 h at room temperature. After further washing, plates were incubated with horseradish peroxidase-strepavidin, followed by TMB for between 8–10 min. The reaction was stopped by the addition of 1 M H2SO4, and absorbance was read at 450 and 630 nm. The sensitivity ranged between 6.25–200 pg/ml. The interassay coefficient of variation for the IL-6 ELISA assay is 5.4%, and the intraassay coefficient of variation is 2.8%.

Soluble gp130 and sIL-6R were measured using commercially available ELISA kits (IDS Ltd., Tyne and Wear, UK) according to the manufacturer’s instructions. Detectable ranges were between 57–1800 pg/ml and 32–1000 pg/ml, respectively.

The value for the average basal IL-6 secretion by HP75 cells was measured from a sample of 241.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Positive staining for IL-6 mRNA was identified by ISH and observed in all HP75 cells (Fig. 3AGo). Figure 3BGo shows a negative control displaying only the green counterstain of the nuclei.



View larger version (61K):
[in this window]
[in a new window]
  		FIG. 3. ISH for IL-6 mRNA in HP75 cells. A, With IL-6 antisense probe; B, negative control by omission of probe, showing only methyl green counterstain. Magnification, x25.

 
IL-6 protein was visualized by immunocytochemistry, and positive cytoplasmic specific staining was identified in all HP75 cells (Fig. 4AGo). Figure 4BGo shows the negative control with only hematoxylin counterstain present.



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 4. Immunocytochemistry for IL-6 protein in HP75 cells. A, With IL-6 antibody; B, negative control by omission of antibody, showing only the counterstain of hematoxylin blued in Scott’s tap water. Magnification, x40.

 
The average level of IL-6 protein secreted by HP75 cells was measured by ELISA and was 6,167 ± 56 pg/ml (mean ± SEM) over 72 h. Basal secretion from these cells was variable ranging from 1,596–25,840 pg/ml.

IL-6R mRNA was identified by ISH. Positive cytoplasmic and nuclear staining was seen in some HP75 cells (Fig. 5AGo). Counterstain was only seen in the negative control (Fig. 5BGo). We also positively detected IL-6R by RT-PCR (Fig 6Go). We were unable to positively detect IL-6R protein by immunocytochemistry due to high levels of nonspecific nuclear and cytoplasmic staining.



View larger version (56K):
[in this window]
[in a new window]
  		FIG. 5. ISH of IL-6R mRNA in HP75 cells. A, With IL-6R antisense strand probe cocktail; B, negative control with IL-6R sense strand probe cocktail showing only methyl green counter stain. Magnification, x40.

 


View larger version (76K):
[in this window]
[in a new window]
  		FIG. 6. RT-PCR showing the presence of IL-6R in HP75 cells. A 2% agarose gel was stained with ethidium bromide and illuminated by UV irradiation. Lane A, DNA 100-bp ladder; lane B, HP75 IL-6R; lane C, RPMI8226 IL-6R; lane D, positive control; lane E, HP75 GAPDH; lane F, RPMI8226 GAPDH; lane G, negative control; lane H, DNA 100-bp ladder.

 
The sIL-6R was not detectable by ELISA in the conditioned medium of unstimulated HP75 cells, nor did PDBu or dexamethasone, agents known to stimulate cleavage of the sIL-6R, induce cleavage of the sIL-6R from HP75 cells at 24 or 72 h. Basal secretion of soluble gp130 was detected at a mean level of 311 pg/ml/72 h.

HP75 cells proliferate rapidly in medium containing serum (Fig. 1Go) and have previously been shown to display only minimal proliferation in serum-depleted medium (3). HP75 cells have linear growth, with no lag phase seen, and a doubling time of approximately 2 d during their most rapid growth phase.

We observed significant dose-dependent inhibition of cell growth on addition of neutralizing anti-IL-6 antibody to HP75 cell in culture incubated over 6 d (Fig. 7Go). The quantity of antibody used was calculated for the highest dose (300 µg/ml) to sufficiently neutralize 6000 pg/ml IL-6, approximately the total amount of IL-6 basally secreted by HP75 cells. Using the maximal antibody concentration, cell growth was inhibited by 76 ± 6.5% (P < 0.0001) compared with basal, whereas inhibiting approximately only one quarter of basal IL-6 production reduced growth by only 24% (P < 0.05). There was no effect on growth after 3-d incubation of IL-6-neutralizing Ab (basal, 100 ± 3.2%; IL-6-neutralizing Ab: 300 µg/ml, 101 ± 2.6%; 150 µg/ml, 92.7 ± 3.3%; 75 µg/ml, 98.4 ± 2.7%; P > 0.05).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 7. Effect on growth of HP75 on addition of neutralizing IL-6 antibody to cultures over 6 d. *, P < 0.05; **, P < 0.0005.

 
Addition of exogenous IL-6 (1 ng/ml) to cultures resulted in significant growth stimulation (148 ± 8%; P < 0.01). At 10 ng/ml IL-6, no effect on growth was observed, and 100 ng/ml produced inhibition of 27 ± 8% (P < 0.01) over 6 d of growth (Fig. 8Go). After 3 d of culture, growth inhibition of 18 ± 5% occurred using only the highest dose of 100 ng/ml.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 8. Effect on growth of HP75 on addition of exogenous IL-6 (1–100 ng/ml) to cultures over 3 and 6 d. *, P < 0.05, significant stimulation of growth at 6 d with 1 ng/ml; {dagger}, P < 0.05, significant growth inhibition at both 3 and 6 d after addition of 100 ng/ml.

 
IL-1{alpha} (100 U/ml) and dbcAMP (10 mM) significantly increased IL-6 secretion from HP75 cells (596% and 263%, respectively). A concentration of 10 nM dexamethasone maximally inhibited basal IL-6 production by 79%. The same dose efficiently inhibited IL-1{alpha}-stimulated IL-6 production by 40%. Neither forskolin, cholera toxin, nor LPS had any effect on IL-6 production (Fig. 9Go).



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 9. Control of IL-6 secretion by activators/inhibitors of the IL-6 gene promoter showing significant stimulation of IL-6 secretion by 100 U/ml IL-1{alpha} and 10 mM dbcAMP (*, P < 0.0005), significant inhibition of IL-6 secretion by 10 nM dexamethasone ({dagger}, P < 0.0005), and significant inhibition of IL-1{alpha}-stimulated IL-6 release (§, P < 0.0005). All cells were stimulated for 72 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of IL-6 and its receptor have previously been reported in human pituitary adenomas (1, 2). The existence of both ligand and receptor in the same cell suggests that IL-6 may provide an autocrine mechanism for growth of these tumor cells.

We have detected the presence of IL-6 mRNA by ISH in HP75 cells; staining was predominantly nuclear, with some cytoplasmic expression. Immunocytochemistry identified IL-6 protein, and this was observed in all HP75 cells. Staining was strong and specific throughout the cytoplasm; no staining was observed in the nuclei of any cells. Levels of IL-6 secreted by HP75 cells mirrored this strong cytoplasmic expression. Previous reports detected IL-6 in 53–93% of human pituitary adenomas (1, 2, 24, 25).

The mRNA for the IL-6R is present and was identified by ISH. The mRNA was not expressed by the majority of cells; those that stained positively showed strong reactivity, with staining clearly present in the cytoplasm and nucleus. We also detected the IL-6R by RT-PCR; we were, however, unable to detect the IL-6R by immunocytochemistry. It may be that immunocytochemistry did not work due to the lack of a suitable antibody, or the protein levels of IL-6R expressed by the cells may be too low to be detected by this method. More likely, the antibody used may not have been optimal for use on formalin-fixed, paraffin wax-embedded sections. However, we demonstrated the presence of IL-6R by the more sensitive immunocytochemical assay flow cytometry (submitted for publication).

The sIL-6R was not detectable in the conditioned medium of HP75 cells. The sIL-6R is produced either as an mRNA splice variant or as a result of cleavage of the membrane receptor. With an agonistic function it is able to initiate signaling when associated with membrane-bound gp130 (26). As the soluble form of the IL-6R functions in the same manner as the membrane-bound form, shedding of this receptor allows trans-signaling to occur. This is a phenomenon proposed by Rose-John and Heinrich (27); a cell that expresses gp130 but no membrane-bound IL-6R would not be able to respond to IL-6, but a second cell thath releases a soluble receptor will render the first cell type responsive to the cytokine. The mechanism of shedding is not fully understood, but is believed to be mediated by metalloproteases (28, 29). Dexamethasone and phorbol esters (protein kinase C activators) are potent agents known to enhance shedding (29, 30, 31, 32). We were unable to induce cleavage of the IL-6R from HP75 cells using either dexamethasone or the phorbol ester PDBu. We do not know whether the original tumor from which HP75 is derived produced sIL-6R in vivo. The soluble form may be produced to act upon another cell type in response to a trans-activating signal that is not present in the in vitro immortalized state, in which the tumor type is the only cell type present. It is possible that the tumor cells need another cell type to facilitate the production of the soluble form. It may also be that we were unable to induce shedding of the receptor due to a functional or structural abnormality in the IL-6R in these cells.

Soluble gp130 is an antagonist of IL-6 actions; although it is able to bind the IL-6/(s)IL-6R complex, it possesses no intracellular or membrane portion and is therefore unable to fulfill its downstream signaling role. It is present in measurable amounts in the basal secretions from HP75 cells; unless the amounts measured are insignificant compared with the levels of membrane-bound gp130 and IL-6R, the overall activity induced by IL-6 may be diminished in this cell line’s basal state. We have not evaluated the levels of membrane-bound gp130, and we therefore do not know the proportion present compared with the soluble form. It is unclear how production of the soluble form is regulated in vivo or in vitro, and it is known that its production cannot be stimulated in a similar fashion to that of the IL-6R (32). The soluble form of this subunit, through its antagonistic signaling effect, may provide further regulation of IL-6 activity by modulating signaling activity through its receptor to dampen the effects of this cytokine.

The finding that the IL-6-neutralizing antibody almost completely inhibits growth demonstrates that HP75 cells are dependent on the autocrine production of IL-6. Although we did not see total inhibition of growth using the antibody at doses up to 300 µg/ml, which was calculated to counter maximal secretion, this may have been due to a lack of neutralization of all of the basally secreted IL-6. HP75 cells show small variations in the levels of IL-6 secreted, and the quantity of antibody used was based on an estimation of IL-6 secretion by HP75 based on previous measurements. The lack of the complete inhibition of cell growth may also be due to the possibility that not cells are able to respond to IL-6, as we observed by ISH that the IL-6R was present in some, but not all, HP75 cells.

Addition of exogenous IL-6 at low doses stimulated proliferation; however, at high doses of more than 10 ng/ml, IL-6 inhibited growth. It has been demonstrated in other cells that supraoptimal doses of cytokines lead to diminished responses due to down-regulation of their surface receptors (33). This can occur by persistent internalization and degradation of the receptor, leading to desensitization of the cells to protect the cell against overstimulation (34, 35). Therefore, low levels of IL-6, either basal production by HP75 acting as an autocrine growth factor or exogenous addition resulting in an increase in proliferation, show a positive effect on HP75 growth. However, the addition of high doses of the cytokine leads to a desensitization of HP75 cells to IL-6, thus leading to an inhibition of their growth.

The expression of IL-6 is controlled by the gene at its promoter (13). There are two inhibitory glucocorticoid response elements, a multiresponse element (activated by IL-1{alpha}, TNF{alpha}, IL-6, bacterial LPS, and serum) and a cAMP response element. We observed a 596% increase in IL-6 secretion in response to addition of IL-1{alpha} in HP75 cultures, but no response to LPS; the reason for this is unclear. Increasing intracellular cAMP levels using an exogenous agent, dbcAMP, showed a significant increase in IL-6 production compared with basal. However, attempts to stimulate endogenous levels of cAMP using cholera toxin (Gs protein activator) and forskolin (direct stimulator) to stimulate adenylate cyclase activity resulted in no stimulation of IL-6 production. This may be due to basal intracellular cAMP levels being already at maximal due to maximum activation of all adenylate cyclase present or a defect in this enzyme.

The role of IL-6 secreted by pituitary adenomas may not only be through its direct effects on tumor cell proliferation, but also to act on surrounding tissue to facilitate invasion and stimulate angiogenesis. IL-6 is known to influence bone resorption (36, 37), and the presence of IL-6 is associated with pituitary tumor invasiveness and size (12). We have previously observed a positive correlation between the levels of IL-6 and vascular endothelial growth factor basally secreted by human pituitary adenomas in vitro, suggesting that increased expression of IL-6 may stimulate angiogenesis, leading to an increase in tumor volume (38).

In conclusion, these findings further support the role of IL-6 in the pathogenesis of human pituitary tumors. The dose-responsive effect of IL-6 on growth suggests that it is an important factor in the growth of these tumors.


   Acknowledgments
 
We thank Prof. Ricardo Lloyd, Mayo Clinic, for the kind donation of the HP75 cell line.


   Footnotes
 
This work was supported by Yorkshire Cancer Research (to Y.C.R.) and the Barnsley District General Hospital Endocrinology Research Fund.

Abbreviations: d, Deoxy; dbcAMP, dibutyryl cAMP; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-6R, IL-6 receptor; ISH, in situ hybridization; LPS, lipopolysaccharide; PDBu, phorbol-12–13-dibutyrate; sIL-6R, soluble IL-6R.

Received December 27, 2002.

Accepted June 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jones TH, Daniels M, James RA, Justice SK, McCorkle R, Price A, Kendall-Taylor P, Weetman AP 1994 Production of bioactive and immunoreactive interleukin-6 (IL-6) and expression of IL-6 messenger ribonucleic acid by human pituitary adenomas. J Clin Endocrinol Metab 78:180–187[Abstract]
  2. Jones TH, Justice S, Price A, Chapman K 1991 Interleukin-6 secreting human pituitary adenomas. J Clin Endocrinol Metab 73:207–209[Abstract/Free Full Text]
  3. Jin L, Kulig E, Qian X, Scheithauer BW, Eberhadt NL, Lloyd RV 1998 A human pituitary adenoma cell line proliferates and maintains some differentiated functions following expression of SV40 large T-antigen. Endocr Pathol 9:169–184
  4. Jin L, Burguera G, Couce ME, Scheithauer BW, Lamsan J, Eberhardt NL, Kulig E, Lloyd RV 1999 Leptin and leptin receptor expression in normal and neoplastic human pituitary: evidence of a regulatory role for leptin on pituitary cell proliferation. J Clin Endocrinol Metab 84:2903–2911[Abstract/Free Full Text]
  5. Tsumanuma I, Jin L, Zhang S, Bayliss JM, Scheithauer BW, Lloyd RV 2000 Leptin signal transduction in the HP75 cell line. Pituitary 3:211–220[CrossRef][Medline]
  6. Jones TH, Justice S 1995 Effect of interleukin-6 on human pituitary tumour cell growth. J Endocrinol 144:P292
  7. Arzt E, Buric R, Steltzer G, Stalla J, Sauer J, Renner U, Stalla GK 1993 Interleukin involvement in anterior pituitary cell growth regulation: effects of IL-2 and IL-6. Endocrinology 132:459–467[Abstract/Free Full Text]
  8. Sawada T, Koike K, Kanda Y, Ikegami H, Jikihara H, Maeda T, Osako Y, Hirota K, Miyake A 1995 Interleukin-6 stimulates cell proliferation of rat pituitary clonal cell lines in vitro. J Endocrinol Invest 18:83–90[Medline]
  9. Renner U, Gloddek J, Arzt E, Inoue K, Stalla GK 1997 Interleukin-6 is an autocrine growth factor for folliculostellate-like TtT/GF mouse pituitary tumour cells. Exp Clin Endocrinol Diabetes 105:345–352[Medline]
  10. Paez Pereda M, Goldberg V, Chervin A, Carrizo G, Molina A, Andrada J, Sauer J, Renner U, Stalla GK, Arzt E 1996 Interleukin-2 (IL-2) and IL-6 regulate c-fos protooncogene expression in human pituitary adenoma explants. Mol Cell Endocrinol 124:33–42[CrossRef][Medline]
  11. Gandour-Edwards R, Kapadia SB, Janecka IP, Martinez AJ, Barnes L 1995 Biological markers of invasive pituitary adenomas involving the sphenoid sinus. Mod Pathol 8:160–164[Medline]
  12. Suliman M, Borg S, Kerry K, Royds JA, Powell T, Cullen DR, Jones TH 2001 IL-6 is associated with pituitary tumour invasiveness. Endocr Pathol 12:244–245
  13. Kishimoto T 1989 The biology of interleukin-6. Blood 74:1–10[Free Full Text]
  14. Jones TH, Kennedy RL, Justice S, Price A 1993 Interleukin-1 stimulates the release of interleukin-6 from cultured human pituitary adenoma cells. Acta Endocrinol (Copenh) 128:405–410[Abstract/Free Full Text]
  15. Jones TH, Kennedy RL, Justice SK, Price A 1993 Pituitary adenomas with high and low basal inositol phospholipid turnover; the stimulatory effect of kinins amd an association with interleukin-6 secretion. Clin Endocrinol (Oxf) 39:433–439[Medline]
  16. Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L 1998 Interleukin-6-type cytokine signalling through the gp 130/Jak/STAT pathway. Biochem J 334:297–314
  17. Ohmichi M, Hirota K, Koike K, Kurachi H, Ohtsuka S, Matsuzaki N, Yamaguchi M, Miyake A, Tanizawa O 1992 Binding sites for interleukin-6 in the anterior pituitary gland. Neuroendocrinology 55:199–203[Medline]
  18. Velkeniers B, Vergani P, Trouillas J, D’Haens J, Hooghe RJ, Hooghe-Peters EL 1994 Expression of IL-6 mRNA in normal rat and human pituitaries and in human pituitary adenomas. J Histochem Cytochem 42:67–76[Abstract]
  19. Hanisch A, Dieterich D, Dietzmann K, Ludecke K, Buchfelder M, Fahlbusch R, Lehnert H 2000 Expression of members of the interleukin-6 family of cytokines and their receptors in human pituitary and pituitary adenomas. J Clin Endocrinol Metab 85:4411–4414[Abstract/Free Full Text]
  20. Kurotani R, Yasuda M, Oyama K, Egashira N, Sugaya M, Teramoto A, Osamura RY 2001 Expression of interleukin-6, interleukin-6 receptor (gp80), and the receptor’s signal transducing subunit (gp130) in human normal pituitary glands and pituitary adenomas. Mod Pathol 14:791–797[CrossRef][Medline]
  21. Rezai AR, Rezai A, Martinez-Maza O, Vander-Meyden M, Weiss MH 1994 Interleukin-6 and interleukin-6 receptor gene expression in pituitary tumors. J Neurooncol 19:131–135[CrossRef][Medline]
  22. Montero-Julian FA 2001 The soluble IL-6 receptors: serum levels and biological function. Cell Mol Biol 47:583–597[Medline]
  23. Peters M, Muller AM, Rose-John S 1998 Interleukin-6 and soluble interleukin-6 receptor: direct stimulation of gp130 and hematopoiesis. Blood 92:3495–3504[Free Full Text]
  24. Green VL, Atkin SL, Speirs V, Jeffreys RV, Landolt AM, Mathew B, Hipkins L, White MC 1996 Cytokine expression in human anterior pituitary adenomas. Clin Endocrinol (Oxf) 45:179–185[CrossRef][Medline]
  25. Tsagarakis S, Kontogeorgos G, Giannou P, Thalassinos N, Woolley J, Besser GM, Grossman A 1992 Interleukin-6, a growth promoting cytokine, is present in human pituitary adenomas: an immunocytochemical study. Clin Endocrinol (Oxf) 37:163–167[Medline]
  26. Jones SA, Horiuchi S, Topley N, Yamamoto N, Fuller GM 2001 The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J 15:43–58[Abstract/Free Full Text]
  27. Rose-John S, Heinrich PC 1994 Soluble receptors for cytokines and growth factors: generation and biological function. Biochem J 300:281–290
  28. Mullberg J, Durie FH, Ottenevans C, Alderson MR, Rosejohn S, Cosman D, Black RA, Mohler KM 1995 A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J Immunol 155:5198–5205[Abstract]
  29. Althoff K, Reddy P, Voltz N, Rose-John S, Mullberg J 2000 Shedding of interleukin-6 receptor and tumour necrosis factor {alpha}. Eur J Biochem 267:2624–2631[Medline]
  30. Paysant J, Blanque R, VAsse M, Soria C, Gardner CR 2000 Factors influencing the effect of the soluble IL-6 receptor on IL-6 responses in HEpG2 hepatocytes. Cytokine 12:774–779[CrossRef][Medline]
  31. Mori S, Murakami-Mori K, Bonavida B 1998 Dexamethasone enhances expression of membrane and soluble interleukin-6 receptors by prostate carcinoma cell lines. Anticancer Res 18:4403–4408[Medline]
  32. Rose-John S, Ehlers M, Grotzinger J, Mullberg J 1995 The soluble interleukin-6 receptor. Ann NY Acad Sci 762:207–221[CrossRef][Medline]
  33. Dohlman HG, Thorner J, Caron MG, Lefkowitz RJ 1991 Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem 60:653–688[CrossRef][Medline]
  34. Zohlnhofer D, Graeve L, Rose-John S, Schooltink H, Dittrich E, Heinrich PC 1992 The hepatic interleukin-6 receptor. Down regulation of the interleukin-6 binding subunit (gp80) by its ligand. FEBS Lett 306:219–222[CrossRef][Medline]
  35. Rose-John S, Hipp E, Lenz D, Legres LG, Korr H, Hirano T, Kishimoto T, Heinrich PC 1991 Structural and functional studies on the human interleukin-6 receptor. J Biol Chem 266:3841–3846[Abstract/Free Full Text]
  36. Rahman S, Bunning RA, Dobson PRM, Evans DB, Chapman K, Jones TH, Brown BL, Russel RGG 1992 Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells. Biochim Biophys Acta 1135:97–102[Medline]
  37. Moonga BS, Adebanjo OA, Wang H-J, Li S, Wu XB, Troen B, Inzerillo A, Abe E, Minkin C, Huang CL-H, Zaidi M 2002 Differential effects of interleukin-6 receptor activation on intracellular signaling and bone resorption by isolated rat osteoclasts. J Endocrinol 173:395–405[Abstract]
  38. Borg SA, Kerry KE, Jones TH 2002 Positive correlation between IL-6 and VEGF secretion by human pituitary adenoma cells. Exp Clin Endocrinol Diabetes 110:P107



This article has been cited by other articles:


Home page
 Exp PhysiolHome page
J. Gerez, J. Bonfiglio, S. Sosa, D. Giacomini, M. Acuna, A. Carbia Nagashima, M. J. Perone, S. Silberstein, U. Renner, G. K. Stalla, et al.
Molecular transduction mechanisms of cytokine-hormone interactions: role of gp130 cytokines
Exp Physiol, September 1, 2007; 92(5): 801 - 806.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
S A Borg, K E Kerry, J A Royds, R D Battersby, and T H Jones
Correlation of VEGF production with IL1{alpha} and IL6 secretion by human pituitary adenoma cells
Eur. J. Endocrinol., February 1, 2005; 152(2): 293 - 300.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Borg, S. A.
Right arrow Articles by Jones, T. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Borg, S. A.
Right arrow Articles by Jones, T. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals