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Original Studies |
Max-Planck-Institute of Psychiatry, Department of Endocrinology, Munich, Germany (J.S., U.R., U.H., U.P., G.K.S.); Division of Endocrinology, Department of Medicine, University of Essen, Germany (J.S.); Laboratory Fisiología y Biología Molecular, Department of Biology, Facultad de Ciencas Exactas y Naturales University of Buenos Aires and Consejo Nacional de Investigaciones Cientifìcas y Técnicas, Buenos Aires, Argentina (E.A.); Department of Neurosurgery, Klinikum Mannheim, University of Heidelberg, Mannheim, Germany (M.L.); Department of Neurosurgery (A.M.) and Department of Medicine (C.J.S.), University of Munich, Germany
Address all correspondence and requests for reprints to: G. K. Stalla Max Planck Institute of Psychiatry, Department of Endocrinology, Kraepelinstrasse 10, D-80804 Munich, Germany. E-mail: stalla{at}mpipsykl.mpg.de
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Abstract |
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Introduction |
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The IL-1ra constitutes a member of the IL-1 family that neutralizes the actions of IL-1 by binding to both types of IL-1 receptors during endotoxemia or inflammatory processes (reviewed in Ref. 12). Whereas the soluble form of IL-1ra is mainly expressed by activated monocytes and macrophages (reviewed in Ref. 12), the intracellular IL-1ra variant (13), is expressed in various tissues of epithelial or stromal origin (reviewed in Ref. 12). In addition, a second biologically active form of the intracellular IL-1ra containing an additional in-frame 63-bp sequence has been characterized and referred to as intracellular IL-1ra type II (14). Several studies indicate that IL-1 and IL-1ra are differentially regulated (15, 16, 17, 18, 19) and IL-1 has been shown to stimulate soluble IL-1ra in monocytes and synovial fibroblasts (16, 20) as well as intracellular IL-1ra in human retinal pigment epithelial cells (21).
IL-1 modulates hormone secretion at the level of hypothalamus, pituitary and peripheral glands (reviewed in Ref. 1). Concerning the effects of IL-1 on pituitary GH secretion however, both the in vitro and in vivo data are controversial and seem to depend on the experimental conditions used (22, 23, 24, 25, 26, 27, 28). On the other hand, it is established that GH augments the host’s response and resistance to infectious agents such as Salmonella typhimurium and Listeria monocytogenesis (29, 30) and enhances a number of immune responses that are important in providing protective immunity to the host (reviewed in Refs. 31, 32, 33). In addition to their effects on pituitary hormone secretion, there is increasing evidence that cytokines have growth-regulating properties at the level of the pituitary gland. We have previously demonstrated that IL-1, IL-2, and IL-6 inhibit the growth of normal rat anterior pituitary cells, whereas IL-1 does not regulate rat mammosomatotroph GH3 tumor cell growth (25), and IL-2 and IL-6 even stimulate the proliferation of GH3 cells (34). In line with this observation, it has recently been shown that IL-2 induces the growth of human somatotroph adenoma cells (35).
Because anterior pituitary function has been shown to be regulated by IL-1, and somatotrophs seem to be a preferential target for IL-1 actions on the pituitary, we investigated whether the IL-1ra protein and GH secretion are regulated by IL-1ß in human somatotroph adenomas cultured in vitro. Our data, showing that IL-1ß enhances intracellular IL-1ra content but not GH secretion, suggest the presence of an intrinsic counterregulatory pathway limiting the secretory response of anterior pituitary cells to IL-1.
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Methods |
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Pituitary adenoma tissue from nine patients with acromegaly
(numbered consecutively Tu 1 to Tu 9), obtained at transsphenoidal
microsurgery was studied. The diagnosis of acromegaly was confirmed by
dynamic endocrine testing, and the tumor visualized by magnetic
resonance imaging as previously described (36). The preoperative
clinical data and basal hormone levels of the patients with pituitary
adenomas are shown in Table 1
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Material and reagents, except where stated, were from Biochrom Seromed (Berlin, Germany), Gibco BRL Life Technologies (Eggenstein, Germany), Flow Labs. (Meckenheim, Germany), Falcon (Heidelberg, Germany), and Nunc (Wiesbaden, Germany). Pituitary cell culture was performed as previously described (10, 11, 25). In brief, the tissue was washed several times with preparation buffer (137 mmol/L NaCl, 5 mmol/L KCl, 0.7 mol/L Na2HPO4, 10 mmol/L glucose, 15 mmol/L HEPES, pH 7.3, 2.5 mg/L amphotericin B and 105 U/L penicillin/streptomycin). Sliced fragments were mechanically and enzymatically dispersed in preparation buffer containing 1000 U/mL collagenase (Worthington Biochemical, Freehold, NJ), 4 g/L BSA, 10 mg/L DNase II, 1 g/L soybean trypsin inhibitor, and 2 g/L hyaluronidase. Cells were centrifuged and resuspended in DMEM, pH 7.3, containing 10% FCS, 2.2 g/L NaHCO3, 10 mmol/L HEPES, 2 mmol/L glutamine, 10 mL/L nonessential amino acids, 10 mL/L MEM vitamins, 2.5 mg/L amphotericin B and 105 U/L penicillin/streptomycin, 5 mg/L insulin, 20 µg/L sodium selenite, 5 mg/L transferrin, and 30 pmol/L T3. Between 3 and 60 x 106 cells with a viability of at least 80% (acridine orange/ethidium bromide staining) were isolated, distributed (1 x 105 cells/well) in 48-well culture plates, and incubated (37 C, 5% CO2). The dispersed cells attached within 48–72 h. The pituitary cell cultures were free of contaminating leukocytes, determined as described previously (34). Forty-six days after plating, the cells were washed twice with DMEM and incubated with 1100 U/mL human recombinant IL-1ß (Boehringer, Mannheim, Germany) for the indicated times in 440 µL/well of stimulation medium (DMEM containing 2.2 g/L NaHCO3, 10 mmol/L Hepes, 2 mmol/L glutamine, 2.5 mg/L amphotericin B and 105 U/L penicillin/sptreptomycin, 0.1% BSA, and ascorbic acid). After the incubation periods, supernatants were removed for IL-1ra protein immunoassay. Cells were then rinsed twice with PBS and lysed by three freeze-thaw cycles in 440 µL PBS with 0.1% BSA and 400 kIU/mL aprotinin (Bayer, Leverkusen, Germany). Human peripheral monocytes from three normal subjects obtained from isolated peripheral blood mononuclear cells were purified by plastic adherence as previously described (37) and stimulated for 12, 24, and 48 h with 1100 U/mL IL-1ß in RPMI-1640 medium (440 µL/well). Supernatants and lysates of the monocytes were obtained as for the pituitary tumor cells. Conditioned media and cell lysates were centrifuged to remove particulate debris and stored at -20 C until assayed.
Determination of IL-1ra protein
IL-1ra protein in the supernatants and cell lysates was measured
with a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA)
for human IL-1ra (R&D Systems, Minneapolis, MN) used in previous
studies (37, 38). According to the manufacturer the ELISA shows no
cross-reactivity with other cytokines like IL-1
, IL-1ß, IL-6 or
tumor necrosis factor-, and other studies (39, 40) demonstrated that
the ELISA recognizes both forms of the IL-1ra. In control experiments,
no cross-reactivity with the human recombinant IL-1ß used, GH, or FCS
was detected. The least detectable concentration of IL-1ra was 8.5
pg/mL. The intraassay coefficient of variation was 5.7% and the
interassay coefficient of variation was 4.9%. Blank values of optical
density were automatically subtracted from the levels obtained for the
dilution series for standards, and from the levels obtained for the
samples. Standards and cell culture supernatants were diluted in DMEM
for the pituitary cells or RPMI-1640 medium for human monocytes, which
did not contain FCS, glutamine, or antibiotics.
Determination of human GH
GH levels in cell culture supernatants were determined using a
sensitive fluorescence immunometric assay, as described previously (36, 41) with minor modifications. In brief, antihuman GH monoclonal
antibody clone 8B11 was adsorbed to polystyrene microtiter plates (500
ng antibody/well) in 50 mmol/L phosphate buffer, pH 9.6. After 16
h incubation at 4 C, the plates were washed, and 50 µL sample
(diluted 1:20 to 1:50) or standard were added. Standards were prepared
by serial dilution of recombinant human GH (JRP 88/624, NIBSC, London,
U.K.) in DMEM. Then, 30 ng biotinylated antihuman GH monoclonal
antibody clone 7F8 in 150 µL assay buffer [50 mmol/L Tris-HCl, 150
mmol/L NaCl, 0.5% BSA, 0.05% (wt/vol) bovine 
-globulin, 0.05%
Tween-40, 0.05% NaN3 and 20 µmol/L
diethylenetriaminepentaacetic acid, pH 7.75], were added. After 2
h of incubation at room temperature, the plates were washed again, and
10 ng streptavidin-europium conjugate (Wallac Oy, Turku, Finland),
dissolved in assay buffer, were added to each well (final volume, 200
µL). After an incubation period of 30 min at room temperature and a
final wash step, 200 µL europium fluorescence enhancement solution
(Wallac Oy) were added, and the europium signal was measured in a
time-resolved fluorometer (DELFIA, Wallac Oy). The working range for
cell culture standards was 0.12–100 ng/mL, with an intraassay
coefficient of less than 10%.
Statistical analysis
Results are expressed as mean ± SD. Each experimental condition was performed in triplicate. Statistical analysis was made using the one-way ANOVA in combination with the Scheffe’s post hoc test. P < 0.05 was considered significant.
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Results |
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In the lysates of the cultured somatotroph tumor cells, we
observed a significant enhancement of IL-1ra concentrations by IL-1ß
in eight of the nine tumors studied. Figure 1 shows representatively the results of
tumor 4 when cells were incubated with or without 1100 U/mL IL-1ß for
24 h. The enhancement of the IL-1ra by IL-1ß in the cell lysates
was dose dependent, and there was a high interindividual variation of
IL-1ra enhancement by IL-1ß as shown in the inset of Fig. 1, which summarizes the percent enhancement of IL-1ra by IL-1ß of all
tumors at all incubation times studied.
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In contrast to the finding of an enhancement of IL-1ra in the
somatotroph adenoma cell lysates by IL-1ß (Figs. 1
and 2), there was no enhancement of IL-1ra by
IL-1ß in the cell culture supernatants of eight of the nine tumors.
Figure 2 shows the results of all tumors when incubated for 24 h
with 100 U/mL IL-1ß. Except for tumor 3, in which IL-1ß stimulated
supernatant IL-1ra, no change of IL-1ra values in the supernatants was
observed also at 12 and 48 h of incubation times, as well as when
the cells were incubated with 1 and 10 U/mL IL-1ß (data not shown).
There was no correlation of basal or IL-1ß-stimulated IL-1ra values
in lysates or supernatants to the clinical characteristics of the
patients presented in Table 1. Basal IL-1ra concentrations were
significantly higher in the lysates of the somatotroph tumor cells
compared with the corresponding supernatants (Fig. 2).
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In contrast to the somatotroph adenoma cell cultures, a different
pattern of IL-1ra regulation by IL-1ß was observed in cultured human
monocytes. In these cells, IL-1ß significantly enhanced IL-1ra
concentrations in the cell culture supernatants, whereas no change of
the IL-1ra was observed in the lysates of the cultured human monocytes.
Figure 3 shows the results for 24 h
of incubation with or without 100 U/L IL-1ß. Similar results were
obtained when the cells were incubated for 12 and 48 h with
different doses of IL-1ß (data not shown).
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We further evaluated the effect of IL-1ß incubation on GH
secretion in the cell culture supernatants of somatotroph adenomas
under our experimental conditions. Because of restricted cell numbers
obtained in some pituitary adenomas, secreted GH could only be measured
without and after incubation with 100 U/mL IL-1ß in four of the nine
somatotroph tumors at 24 and 48 h of incubation. No difference was
observed in the concentrations of GH in the supernatants following
stimulation of the cells with IL-1ß in any of the tumors studied
either at 24 h (Fig. 4) or 48 h
(data not shown) of incubation.
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Discussion |
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Previously, we demonstrated that human pituitary adenoma cells express intracellular IL-1ra mRNA (11) like other cells of epithelial or stromal origin (reviewed in Ref. 12). According to our previous observation of a colocalization of IL-1ra and human GH immunoreactivity in human somatotroph adenoma cells (11), we now demonstrate that human somatotroph adenoma cells already contain considerable amounts of IL-1ra protein, detectable by ELISA, under basal conditions. Furthermore, we show that IL-1ß enhances IL-1ra content in pituitary tumor cells. Recently, it was shown that in response to systemic inflammation the normal mouse anterior pituitary also synthesizes IL-1ra mRNA, showing a peak at 6 h following that of IL-1ß mRNA at 2 h (42). Because IL-1ra is induced in response to IL-1ß in various other cell systems (16, 20, 21), it has been proposed that IL-1ß originating from the pituitary anterior lobe together with IL-1ß synthesized in other sites may induce IL-1ra in the anterior lobe (42). In the present study we provide the first experimental evidence for this proposed action of IL-1ß on intrinsic pituitary IL-1ra synthesis.
The observation of higher basal IL-1ra concentrations as well as the enhancement of IL-1ra by IL-1ß in cellular lysates but not in supernatants of somatotroph adenoma cells points towards the majority of newly synthesized somatotroph IL-1ra remaining intracellularly, in contrast to the apparent secretion observed in cultured monocytes (15). A predominant expression of intracellular IL-1ra in somatotroph adenoma cells may be conveyed by the cell type-specific use of the two gene promoters on alternative first exons that control expression of IL-1ra from the same gene (43). It may also be related to the tumoral origin of the cells studied, taking into account that the normal mouse anterior pituitary has been shown to synthesize the secreted IL-1ra isoform (42).
Because mouse and rat pituitary possesses binding sites for IL-1 or express IL-1 receptors (57), and the predominant cell type of the normal mouse anterior pituitary gland expressing p80 types I and II IL-1 receptors corresponds to somatotrophs (7), our data indicate a direct effect of IL-1ß on somatotroph function resulting in an enhancement of IL-1ra content. Concerning the effects of IL-1 on pituitary GH secretion, however, rather controversial data were obtained previously. In nearly identical studies with rat anterior pituitary monolayer cultures it was demonstrated that IL-1ß does not modulate GH secretion (22, 25), whereas others showed an increase of GH secretion by IL-1ß (23, 24). In another study with human pituitary adenomas, the GH secretory response to IL-1 of three GH-secreting adenomas varied with stimulation in one culture, showed no significant effect in a second, and inhibition in the third (44). Moreover, it has been shown that the effects of IL-1 on pituitary GH secretion may also be caused by a modulation of hypothalamic SRIH or GHRH secretion (45, 46). In vivo, injection of IL-1 intracerebroventricularly or intravenously acutely suppresses plasma GH in rats (26) but not in sheep (47), and the administration of the IL-1ra into the third ventricle reverses the suppression of plasma GH induced by lipopolysaccharide intravenously in rats (27). In humans, a phase I trial administration of IL-1 to patients with advanced cancer has been shown to elevate circulating GH levels (28). On the basis of our present results, the controversial data on IL-1 regulation of pituitary GH secretion found in previous studies may at least in part be attributed to different qualitative or quantitative effects of IL-1 on intrinsic pituitary IL-1ra production depending on the experimental conditions and cellular milieu. The idea that the intracellularly expressed IL-1ra inhibits intracellular IL-1 activity (13) has been underscored by demonstrating that intracellular IL-1ra possesses an intrinsic antagonistic biological activity in altering IL-1-induced gene expression at a point downstream of the initial IL-1/IL-1 receptor interaction (48). Whereas there is no evidence that intracellular IL-1ra has a direct effect on membrane IL-1 receptor activation, it has been suggested that the previously observed IL-1 receptor desensitization of T lymphocytes to continuous IL-1 exposure does not result from changes in IL-1 receptor number or binding affinities (49), but may be cause by an interference of the intracellular IL-1ra with the receptor-proximal transducer (48, 49). Thus, intrinsic intracellular pituitary IL-1ra expression would neutralize the effects of IL-1 independently to the blockade of IL-1 receptors exerted by secreted IL-1ra during inflammatory responses or endotoxemia (50). Accordingly, this mechanism would explain the lack of effect of IL-1ß on GH secretion in vitro in the somatotroph adenomas studied.
Assuming that the inflammatory cytokine IL-1 augments circulating GH levels in man (28), enhancement of intrinsic IL-1ra by IL-1 could be of relevance for pituitary function by reducing or blocking secretion of GH, which together with IGF-1 is supposed to represent an immunostimulatory system (reviewed in Refs. 31, 32, 33). Because we have only examined adenoma cells, however, we cannot exclude that pituitary IL-1ra expression, and its regulation by IL-1ß in somatotrophs is exclusive for tumor cells. It may further be speculated that intrinsic expression and regulation of IL-1ra participates in pituitary tumorigenesis, because IL-1ra has been shown to antagonize the growth inhibition induced by IL-1 in normal rat pituitary cells (25) or human glioblastoma cells (51) and, on the other hand, IL-ra has been demonstrated in a human melanoma/nude mouse system to inhibit IL-1-induced augmentation of metastasis (52).
Our finding of a regulation of the IL-1ra in human somatotroph adenoma cells by IL-1ß point to the requirement for a tight control of the growth and hormone modulatory potential of this cytokine in somatotrophs during inflammation or the tumoral process. Although this study only addresses activity in human somatotroph adenomas, it is intriguing to speculate that the limitation of cellular IL-1 responsiveness caused by enhancement of intracellular IL-1ra also may be of potential significance in other normal or abnormal endocrine cell responses to IL-1.
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Footnotes |
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Received December 29, 1997.
Revised April 3, 1998.
Accepted April 10, 1998.
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References |
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and IL-1. Biochim Biophys Acta. 1175:302–305.[Medline]
, interleukin-1ß, and an interleukin-1 receptor
antagonist in human retinal pigment epithelial cells. Exp Eye Res. 55:325–335.[CrossRef][Medline]
. J Exp Med. 176:593–598. and interleukin-1ß stimulate adrenocorticotropin
secretion in the rat through a similar hypothalamic receptor(s):
effects of interleukin-1 receptor antagonist protein. Neuroendocrinology. 57:14–22.[Medline]
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