0013-7227/90/1261-0582$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Production of Interleukin-6 by Anterior Pituitary Cells in Vitro* BRYAN L. SPANGELO, ROBERT M. M A C L E O D , AND PETER C. ISAKSON Departments of Medicine and Pharmacology (P.C.I.), University of Virginia Health Sciences Center, Charlottesuille, Virginia 22908
(10-100 nM) approximately 2-fold and by lipopolysaccharide (0.001-10.0 jig/ml) 4-fold during 4-h incubations. In contrast, the cytokine recombinant human IL-la had no effect on IL-6 release by cultured pituitary cells. Freshly dissected hemipituitary tissue also secreted more than 3000 U/ml IL-6 during a 4h incubation. This secretion was enhanced 3-fold by 10 Mg/ml lipopolysaccharide. Our results suggest that the anterior pituitary may produce IL-6 in situ, where it may function as an intrapituitary releasing factor. (Endocrinology 126: 582-586, 1990)
ABSTRACT. We recently reported that the cytokine interleukin-6 (IL-6) is a potent stimulator of anterior pituitary hormone release in vitro. Since IL-6 is not normally detectable in the blood, we hypothesized that IL-6 may be produced by the anterior pituitary in situ and thereby affect hormone secretion through paracrine or autocrine mechanisms. The present study demonstrates that cultured anterior pituitary cells spontaneously secrete large quantities of IL-6 in vitro. IL-6 was detectable in the incubation medium within 2 h, and by 8 h of culture had attained concentrations of 2000-4000 U/ml-4 x 105 cells. IL-6 production was stimulated by phorbol myristate acetate
I
NTERLEUKIN-6 (IL-6) is a cytokine produced by Tcells, fibroblasts, monocytes, and endothelial cells that exerts diverse biological effects, including plasmacytoma growth factor activity (1), induction of acute phase protein production (2), stimulation of T-cells (3), hematopoietic growth factor activity (4), and terminal differentiation of B-cells to immunoglobulin-secreting plasma cells (5). We recently found that IL-6 stimulates anterior pituitary hormone release in concentrations that also promote phytohemagglutinin (PHA)-induced spleen cell mitogenesis (6). This suggests that IL-6 in the peripheral circulation may act to regulate anterior pituitary hormone secretion. In fact, Naitoh et al. (7) have reported that iv injection of IL-6 results in increased plasma ACTH in the conscious rat. However, the concentration of IL-6 is normally below 10 U/ml in human serum (8, 9). Since picomolar concentrations of this cytokine stimulate anterior pituitary hormone release (6), we hypothesized that one source of this protein in vivo could be the anterior pituitary gland. We report here that cultured dispersed anterior pituitary cells and primary pituitary explants spontaneously secrete large amounts of IL-6, Received August 11,1989. Address all correspondence and requests for reprints to: Dr. Robert M. MacLeod, Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. * This work was supported by NCI Grant CA-07535 and a research grant from Fidia Pharmaceutical Corp. (to R.M.M.) and a new initiative grant from the University of Virginia DERC (AM-22125; to P.C.I.).
and that this secretion can be markedly enhanced by low concentrations of lipopolysaccharide (LPS) or the phorbol ester 4/3-phorbol 12/?-myristate 13a-acetate (PMA). Materials and Methods Animals BALB/c mice (6-12 weeks of age) were obtained from Cumberland Farms (Clinton, TN). Female Sprague-Dawley rats (225-250 g) were purchased from Dominion Laboratories (Dublin, VA). Reagents Salmonella typhosa LPS was purchased from Difco Laboratories, Inc. (Detroit, MI). PMA (Sigma, St. Louis, MO) was dissolved in ethanol (1 mM) and stored at -20 C until diluted with medium for use in an experiment. Cytokines Recombinant mouse IL-6 (rmIL-6; 3 X 106 U/ml) was generously provided by Dr. J. Van Snick, Ludwig Institute (Brussels, Belgium). Recombinant human IL-la (rhIL-la) was provided by Dr. P. Lomedico, Hoffman LaRoche, Inc. (Nutley, NJ. Anterior pituitary cell culture After decapitation and removal of pituitaries from female Sprague-Dawley rats, anterior pituitary tissue was quickly separated from the intermediate and posterior lobes and placed into complete medium (RPMI-1640 containing 2.5% fetal calf
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS serum, 7.5% horse serum, 100 fj.g/m\ penicillin-G, and 3.8 ng/ ml fungizone; Gibco, Grand Island, NY). The anterior lobes were dissected into halves, each of which was further diced into 1- to 2-mm3 pieces. Dispersed cells were obtained by exposing the pieces to trypsin (2 mg/ml in serum-free medium, 190-210 U/mg; Cooper Biomedical, Freehold, NJ) for 30 min at 37 C, with gentle mixing every 5 min. The tissue was then exposed to grade II pancreatin (2.5 mg/ml in serum-free RPMI; Sigma) for 10 min and resuspended in SMEM containing 10% horse serum for 30 min. The tissue was dispersed to single cells in serum-free SMEM by mild shearing through plastic pipette tips of decreasing orifice. Cells were seeded into 24-well tissue culture plates (Falcon, Oxnard, CA) at a density of 0.4 x 106 viable cells/well in 1.5 ml complete medium and allowed 4 days to attach to the wells in a humidified atmosphere of 5% CO295% air at 37 C before an experiment was performed. Anterior pituitary cell-conditioned medium was obtained by removing spent culture medium and incubating the cells for various time periods with fresh complete medium (1 ml/well) alone or medium containing rhIL-1/3, LPS, or PMA. To generate hemipituitary tissue-conditioned medium, the 1- to 2-mm3 pieces were immediately transferred into 24-well plates containing 1 ml complete medium (one-half anterior pituitary per well). IL-6 assays The concentration of IL-6 that accumulated in the incubation medium of cultured anterior pituitary cells was quantitated using the IL-6-dependent hybridoma 7TD1 (10). Hybridomas such as 7TD1 are exquisitely sensitive to IL-6; concentrations of less than 10 U IL-6/ml (—10 pg/ml) result in half-maximal growth. In the absence of IL-6, 7TD1 cells fail to grow in response to any known cytokines or hormones, including IL-1, -2, -3, -4, or -5; interferon-a and -/3; lymphotoxin; tumor necrosis factor; colony-stimulating factors; PRL; or GH (11) (our unpublished observations). Aliquots of the pituitary incubation medium were serially diluted in microtiter plates (in duplicate) starting from 1:50 (i.e. 4 n\ conditioned medium in 200 n\); usually six dilutions of each specimen were included in each bioassay. After 3 days of incubation, proliferation of the 7TD1 cells was determined colorimetrically (12). The IL-6 concentration in samples was computed by comparison to the linear portion of a standard curve obtained with rmIL-6 (generously provided by Dr. J. Van Snick). The mean value for two or three dilutions of each sample was usually determined. The SE of the mean was approximately 20%, an acceptable value for a bioassay. All specimens from the same experiment were assayed at the same time. Using these conditions, the response curve was linear between 1-10 U rmIL-6/ml. A typical standard curve is shown in Fig. 1; neither LPS (10 Mg/ml) nor PMA (10 nM) affected the proliferation of 7TD1 cells in the absence or presence of rmIIL-6 (data not shown). T-Cell assays Spleen cells (2 X 105/ml) or thymocytes (2 x 106/ml) obtained from BALB/c mice were cultured (200 /ul/well) with a suboptimal concentration (1:100) of PHA (Gibco) in RPMI-1640
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IL-6 (units/ml) FlG. 1. IL-6 hybridoma growth factor assay. The response of the IL6-dependent hybidoma 7TD1 to increasing concentrations of IL-6 is shown. All specimens were diluted so that the concentrations of IL-6 were within the linear portion of the standard curve (1-10 U/ml), as described in Materials and Methods. Shown are the mean ± SD of triplicate wells. containing 5% fetal calf serum and 450 /*M 2-mercaptoethanol. After 3 days of incubation, 1 fiCi [3H]thymidine was added, and the cells were harvested 16 h later. These conditions are similar to those described previously for assay of IL-6 (13). The concentration of IL-6 required to elicit responses from T-cells is 100-fold greater than that needed to maintain the growth of hybridomas (3). Results Anterior pituitary cell production of IL-6: effects of LPS and PMA We first ascertained IL-6 production by anterior pituitary cells that had been dispersed and cultured for 4 days. As shown in Fig. 2, these cells produced detectable amounts of IL-6 within 2-4 h of incubation in fresh complete RPMI-1640 medium. Spontaneous release of IL-6 was approximately linear for 8 h, reaching levels of 2000-4000 U/ml, which are equivalent to 80-160 pM IL6. In four experiments, basal release of IL-6 ranged from 263-1600 U/ml-4 h. Bacterial LPS, a potent stimulator of macrophage IL-6 production (14), was tested for its ability to stimulate IL-6 release by these pituitary cells. In the experiment illustrated in Fig. 2 (upper panel), LPS (10 ixg/m\) stimulated detectable increases in IL-6 secretion within 1 h; IL-6 concentrations were greater than 6000 U/ml at 8 h, about 3-fold higher than basal release. Treatment of anterior pituitary cultures with 10 nM PMA also resulted in enhanced IL-6 production (Fig. 2, lower panel). In four separate experiments, LPS (10 /xg/ml) and PMA (10 nM) increased IL-6 release 4.1 ± 0.4- and 2.2 ± 0.4-fold, respectively, during 4-h incubations of anterior pituitary cells in vitro. It is noteworthy
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS
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with LPS, and the contralateral half was incubated in control medium. In the experiment illustrated in Fig. 3, a substantial linear accumulation of IL-6 in the medium was observed after a lag period of approximately 2 h from basal hemipituitary tissue. In contrast, LPS (10 (xg/ ml) stimulated IL-6 release (a 5-fold increase during a 4h incubation). In four experiments, LPS stimulated IL6 release 2.6 ± 0.6-fold. The data in Fig. 4 are collected from five separate experiments and summarize the timedependent accumulation of IL-6 and the effects of LPS (10 fxg/ml) on this accumulation. At all time points examined, LPS stimulated IL-6 release. Extended incubation of hemipituitary glands for 20 h resulted in IL-6 concentrations exceeding 40,000 U/ml. Thus, freshly isolated hemipituitaries secreted substantial IL-6 within 4
PITUITARY CULTURES A
6000- •
A
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HOURS FIG. 2. Time courses of LPS- and PMA-induced productions of IL-6 from anterior pituitary cells in vitro. Anterior pituitary tissue was dispersed, as described in Materials and Methods, and the resultant single cells were cultured for 4 days. Spent culture medium was replaced with complete medium containing no additions, LPS (10 /xg/ml), or PMA (10 nM). At various intervals, the conditioned media were removed, and IL-6 concentrations were determined with the 7TD1 growth factor assay. Shown are the average concentrations of IL-6 from two wells of each treatment group at each time point in a representative experiment.
that very low concentrations of LPS are capable of stimulating the release of IL-6; 1 ng/ml LPS was sufficient to elicit IL-6 production from anterior pituitary cells (data not shown). Since IL-1 has been reported to stimulate IL-6 release from fibroblasts and endothelial cells (11, 15), we added rhIL-la (10-200 U/ml) to anterior pituitary cultures and assessed IL-6 production. No significant increases in IL6 release were noted in two separate experiments (data not shown).
FIG. 3. Production of IL-6 by hemianterior pituitary tissue. Hemipituitary tissue was cultured for 4 h with or without LPS (10 /ig/ml); 50fil aliquots were removed and replaced with fresh complete medium at 30-min intervals, and IL-6 concentrations were determined with the 7TD1 growth factor assay. Shown are the concentrations of IL-6 found in a representative experiment using a single anterior pituitary gland.
18000 15000
• BASAL (2)
ffi LPS (10 ug/ml)
0)
9000 6000 3000
1
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•-> 12000
(4) (4)
0
Hemipituitary cell production of IL-6: effects of LPS Given the possibility that the IL-6 release observed in anterior pituitary cultures was due to selective survival of a particular cell type that is not well represented in the intact pituitary or that the release was merely a response to enzymatic dispersion, we examined the response of freshly explanted hemipituitary tissue to LPS. One lobe of an anterior pituitary gland was incubated
1 2 4 6 INCUBATION PERIOD (HR) FIG. 4. Time-dependent accumulation of IL-6 from hemipituitary tissue: effects of LPS. Hemipituitary glands were incubated in complete RPMI-1640 medium for 1-6 h with or without LPS (10 fig/ml). Results from five separate experiments using eight anterior pituitary glands, showing both basal and LPS-induced IL-6 release, are illustrated. Numbers above each bar refer to the number of observations per group. Results are expressed as the mean ± SEM.
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS h of isolation, indicating that the IL-6 produced by cultured cells reflected the capacity of the intact gland. Effects of anterior pituitary IL-6 mitogenesis
on lymphocyte
In the absence of antibodies against rat IL-6, it was important to determine whether the material released by anterior pituitary cells had other established biological activities of IL-6. The very large amounts of IL-6 secreted by hemipituitaries allowed us to test this material in less sensitive bioassays. We, therefore, examined the incubation medium from hemipituitary glands for costimulation of thymocyte and spleen cell mitogenesis with PHA. Although IL-6 alone had little effect with PHA on thymocyte mitogenesis under these conditions, it synergized with IL-1 to stimulate these cells (13). Figure 5 (top panel) shows that the stimulative effect of IL-1 on thymocytes was greatly augmented by increasing the concentrations of IL-6. Neither hemipituitary-conditioned medium nor IL-6 alone stimulated thymocyte proliferation (Fig. 5, top panel), although both synergized with IL-1 to do so. Hemipituitary-conditioned medium and IL-6 also stimulated the proliferation of PHA-stimulated spleen cells (Fig. 5, bottom panel). It is noteworthy 12 10
O—OIL-6 A A PIT. CM (A) D DPIT. CM (B) •
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O A
OIL-6 A PIT. CM (A)
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x
E Q. O
LJ
0 1 500 IL-6(U/ml): 200 FIG. 5. Effects of hemipituitary tissue-conditioned (PIT. CM) medium on thymocyte and spleen cell mitogenesis. Hemipituitary-conditioned medium (20 h) was tested in the thymocyte costimulation and PHAspleen cell assays, as described in Materials and Methods. Shown are the results of the titrations of two separate conditioned media.
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that in both assays, the hemipituitary-conditioned medium increased T-cell mitogenesis at dilutions corresponding approximately to their IL-6 contents, as determined by the hybridoma growth factor assay.
Discussion The anterior pituitary is the site of the synthesis and release of numerous circulating hormones. We recently found that the inflammatory cytokine IL-6 is a potent stimulator of the release of GH, PRL, and LH from the pituitary in vitro (6). The studies reported here indicate that the pituitary also has the capacity to secrete large quantities of IL-6. Significant accumulations of IL-6 activity were detected in anterior pituitary cell-conditioned medium within 2-4 h of incubation using the monospecific 7TD1 growth factor assay. As further evidence that anterior pituitary cells produce IL-6, we found that hemipituitary-conditioned medium synergizes with IL-1 in the thymocyte costimulation assay and increases the proliferation of PHA-treated spleen cells. These data suggest that a cell population (s) in the anterior pituitary can produce IIL-6 constituitively, and that this production can be further increased by LPS or PMA. The present results showing anterior pituitary production of IL-6 complement recent studies by Vankelecom and co-workers (16), who reported that IL-6 was present in folliculo-stellate cells of rat and mouse pituitary glands. The folliculo-stellate cell is a poorly characterized macrophage-like cell that forms part of the pituitary matrix in which somatotrophs and other hormone-secreting cells are embedded. The close juxtaposition of folliculo-stellate cells and hormone-secreting cells has led to speculation that the former may be involved in regulating hormone secretion (17). The fact that IL-6 may be produced by the folliculo-stellate cells and acts as a potent pituitary secretagogue provides strong support for such a hypothesis. It is noteworthy that the concentrations of IL-6 we observed in short term cultures of primary pituitary explants (4 h) are similar to those that induce maximal hormone release from the pituitary (2,000-10,000 U/ml). IL-6 synthesis has been detected in a diverse array of tissues and cell types, including fibroblasts (15), macrophages (14), endothelial cells (11), and T cells (10). LPS is a potent stimulator of IL-6 production in fibroblasts and macrophages (14, 18). Our data, showing enhanced IL-6 release from cultured pituitary cells with 1 ng/ml LPS, are in accord with these previous reports. Administration of LPS in man results in increased GH, ACTH (19), and IL-6 (20) secretion, which suggests that in pathophysiological conditions, such as sepsis, circulating LPS may elicit local pituitary as well as peripheral production of IL-6, resulting in enhanced hormone release.
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS
However, since normal circulating IL-6 concentrations are rarely sufficient to stimulate pituitary hormone release (6, 8, 9), the intrapituitary production of IL-6 may be a mechanism by which this cytokine regulates anterior pituitary function. It has been previously suggested that angiotensin-II (21) and vasoactive intestinal peptide (22) may function as paracrine/autocrine factors for the regulation of hormone release. We suggest that IL-6 may act in a similar manner. Hormone-secreting cells in the anterior pituitary gland are not randomly distributed, and PRL-secreting cells are often physically associated with LHsecreting cells (23). GnRH treatment of anterior pituitary cell aggregates results in increased PRL production, possibly through the release of angiotensin-II from the gonadotroph (21, 24). Studies are underway to determine whether IL-6 production in the anterior pituitary may also be affected by hypothalamic neurohormones. Our results, demonstrating IL-6 stimulation of hormone release from the anterior pituitary as well as production of IL-6 by this gland, add to the growing list of activities and sources for this pleiotropic cytokine.
8. 9.
10.
11. 12. 13. 14.
15.
Acknowledgments
16.
We wish to acknowledge Jill A. Sfetko, M. Kathleen Borland, and John Schetz for their excellent technical assistance, and Jo Ann M. Eliason for her editorial expertise.
17.
References 1. Van Damme J, Opdenakker G, Simpson RJ, Rubira MR, Cayphas S, Vink A, Billiau A, Van Snick J 1987 Identification of the human 26-kD protein, interferon 02 (INF-02), as a B-cell hybridoma/ plasmacytoma growth factor induced by interleukin-1 and tumor necrosis factor. J Exp Med 165:914 2. Gauldie J, Richards C, Harnish D, Lansdorf P, Baumann H 1987 Interferon /32/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 84:7251 3. Uyttenhove C, Coulie PG, Van Snick J 1988 T-cell growth and differentiation induced by interleukin-HPl/IL-6, the murine hybridoma/ plasmacytoma growth factor. J Exp Med 167:1417 4. Ikebuchi K, Wong GG, Clark SC, Ihle JN, Hirai Y, Ogawa M 1987 Interleukin-6 enhancement of interleukin-3 dependent proliferation of multipotential hemopoietic progenitors. Proc Natl Acad Sci USA 84:9035 5. Kishimoto T, Hirano T 1988 Molecular regulation of B-lymphocyte response. Annu Rev Immunol 6:485 6. Spangelo BL, Judd AM, Isakson PC, MacLeod RM 1989 Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 125:575 7. Naitoh Y, Fukata J, Tominaga T, Nakai Y, Tamai S, Mori K,
18.
19.
20.
21.
22. 23. 24.
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Imura H (1988) Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 155:1459 Van Oers MHJ, Van Der Heyden AAPAM, Aarden LA 1988 Interleukin-6 (IL-6) in serum and urine of renal transplant recipients. Clin Exp Immunol 71:314 Houssiau FA, Bukasa K, Sindic CJM, Van Damme J, Van Snick J 1988 Elevated levels of the 26K human hybridoma growth factor (interleukin-6) in cerebrospinal fluid of patients with acute infection of the central nervous system. Clin Exp Immunol 71:320 Van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubira MR, Simpson RJ 1986 Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA 83:9679 Sironi M, Breviario F, Proserpio P, Biondi A, Vecchi A, Van Damme J, Dejana E, Mantovani A 1989 IL-I stimulates IL-6 production in endothelial cells. J Immunol 142:549 Mosmann T 1983 Rapid colorimetric assay for cellular growth and survival:, application to proliferation and cytotoxicity assays. J Immunol Methods 65:55 Helle M, Brakenhoff JPJ, De Groot ER, Aarden LA 1988 Interleukin 6 is involved in interleukin 1-induced activities. Eur J Immunol 18:957 Chiu C-P, Moulds C, Coffman RL, Rennick D, Lee F 1988 Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Natl Acad Sci USA 85:7099 Elias JA, Trinchieri G, Beck JM, Simon PL, Sehgal PB, May LT, Kern JA 1989 A synergistic interaction of IL-6 and IL-I mediates the thymocyte-stimulating activity produced by recombinant IL-Istimulated fibroblasts. J Immunol 142:509 Vankelecom H, Carmeliet P, Van Damme J, Billiau A, Denef C 1989 Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 49:102 Baes M, Allaerts W, Denef C 1987 Evidence for functional communicaiion between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary cell aggregates. Endocrinology 120:685 Helfgott DC, May ILT, Sthoeger Z, Tamm I, Sehgal PB 1987 Bacterial lipopolysaccharide (endotoxin) enhanced expression and secretion of /32 interferon by human fibroblasts. J Exp Med J 66:1300 Revhaug A, Michie HR, Manson IM, Watters JM, Dinarello CA, Wolff AM, Wilmore DW 1988 Inhibition of cyclooxygenase attenuates the metabolic response to endotoxin in humans. Arch Surg 123:162 Fong Y, Moldawer ILL, Marano M, Wei II, Tatter SB, Clarick RH, Santhanam U, Sherris D, May ILT, Sehgal PB, Lowry 1989 Endotoxemia elicits increased circulating /32-IFN/IL-6 in man. J Immunol 142:2321 Jones TH, Brown BIL, Dobson PRM 1988 Evidence that angiotensin II is a paracrine agent mediating gonadotrophin-releasing hormone-stimulated inositol phosphate production and prolactin secretion in the rat. J Endocrinol 116:367 Nagy G, Mulchahey JJ, Neill ID I 988 Autocrine control of prolactin seretion by vasoactive intestinal peptide. Endocrinology 122:364 Denef C 1986 Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab 15:1 Denef C, Andries M 1983 Evidence for paracrine interaction between gonadotrophs and lactotrophs in pituitary cell aggregates. Endocrinol J 12:813
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Vol. 126, No. 1 Printed in U.S.A.
Production of Interleukin-6 by Anterior Pituitary Cells in Vitro* BRYAN L. SPANGELO, ROBERT M. M A C L E O D , AND PETER C. ISAKSON Departments of Medicine and Pharmacology (P.C.I.), University of Virginia Health Sciences Center, Charlottesuille, Virginia 22908
(10-100 nM) approximately 2-fold and by lipopolysaccharide (0.001-10.0 jig/ml) 4-fold during 4-h incubations. In contrast, the cytokine recombinant human IL-la had no effect on IL-6 release by cultured pituitary cells. Freshly dissected hemipituitary tissue also secreted more than 3000 U/ml IL-6 during a 4h incubation. This secretion was enhanced 3-fold by 10 Mg/ml lipopolysaccharide. Our results suggest that the anterior pituitary may produce IL-6 in situ, where it may function as an intrapituitary releasing factor. (Endocrinology 126: 582-586, 1990)
ABSTRACT. We recently reported that the cytokine interleukin-6 (IL-6) is a potent stimulator of anterior pituitary hormone release in vitro. Since IL-6 is not normally detectable in the blood, we hypothesized that IL-6 may be produced by the anterior pituitary in situ and thereby affect hormone secretion through paracrine or autocrine mechanisms. The present study demonstrates that cultured anterior pituitary cells spontaneously secrete large quantities of IL-6 in vitro. IL-6 was detectable in the incubation medium within 2 h, and by 8 h of culture had attained concentrations of 2000-4000 U/ml-4 x 105 cells. IL-6 production was stimulated by phorbol myristate acetate
I
NTERLEUKIN-6 (IL-6) is a cytokine produced by Tcells, fibroblasts, monocytes, and endothelial cells that exerts diverse biological effects, including plasmacytoma growth factor activity (1), induction of acute phase protein production (2), stimulation of T-cells (3), hematopoietic growth factor activity (4), and terminal differentiation of B-cells to immunoglobulin-secreting plasma cells (5). We recently found that IL-6 stimulates anterior pituitary hormone release in concentrations that also promote phytohemagglutinin (PHA)-induced spleen cell mitogenesis (6). This suggests that IL-6 in the peripheral circulation may act to regulate anterior pituitary hormone secretion. In fact, Naitoh et al. (7) have reported that iv injection of IL-6 results in increased plasma ACTH in the conscious rat. However, the concentration of IL-6 is normally below 10 U/ml in human serum (8, 9). Since picomolar concentrations of this cytokine stimulate anterior pituitary hormone release (6), we hypothesized that one source of this protein in vivo could be the anterior pituitary gland. We report here that cultured dispersed anterior pituitary cells and primary pituitary explants spontaneously secrete large amounts of IL-6, Received August 11,1989. Address all correspondence and requests for reprints to: Dr. Robert M. MacLeod, Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. * This work was supported by NCI Grant CA-07535 and a research grant from Fidia Pharmaceutical Corp. (to R.M.M.) and a new initiative grant from the University of Virginia DERC (AM-22125; to P.C.I.).
and that this secretion can be markedly enhanced by low concentrations of lipopolysaccharide (LPS) or the phorbol ester 4/3-phorbol 12/?-myristate 13a-acetate (PMA). Materials and Methods Animals BALB/c mice (6-12 weeks of age) were obtained from Cumberland Farms (Clinton, TN). Female Sprague-Dawley rats (225-250 g) were purchased from Dominion Laboratories (Dublin, VA). Reagents Salmonella typhosa LPS was purchased from Difco Laboratories, Inc. (Detroit, MI). PMA (Sigma, St. Louis, MO) was dissolved in ethanol (1 mM) and stored at -20 C until diluted with medium for use in an experiment. Cytokines Recombinant mouse IL-6 (rmIL-6; 3 X 106 U/ml) was generously provided by Dr. J. Van Snick, Ludwig Institute (Brussels, Belgium). Recombinant human IL-la (rhIL-la) was provided by Dr. P. Lomedico, Hoffman LaRoche, Inc. (Nutley, NJ. Anterior pituitary cell culture After decapitation and removal of pituitaries from female Sprague-Dawley rats, anterior pituitary tissue was quickly separated from the intermediate and posterior lobes and placed into complete medium (RPMI-1640 containing 2.5% fetal calf
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS serum, 7.5% horse serum, 100 fj.g/m\ penicillin-G, and 3.8 ng/ ml fungizone; Gibco, Grand Island, NY). The anterior lobes were dissected into halves, each of which was further diced into 1- to 2-mm3 pieces. Dispersed cells were obtained by exposing the pieces to trypsin (2 mg/ml in serum-free medium, 190-210 U/mg; Cooper Biomedical, Freehold, NJ) for 30 min at 37 C, with gentle mixing every 5 min. The tissue was then exposed to grade II pancreatin (2.5 mg/ml in serum-free RPMI; Sigma) for 10 min and resuspended in SMEM containing 10% horse serum for 30 min. The tissue was dispersed to single cells in serum-free SMEM by mild shearing through plastic pipette tips of decreasing orifice. Cells were seeded into 24-well tissue culture plates (Falcon, Oxnard, CA) at a density of 0.4 x 106 viable cells/well in 1.5 ml complete medium and allowed 4 days to attach to the wells in a humidified atmosphere of 5% CO295% air at 37 C before an experiment was performed. Anterior pituitary cell-conditioned medium was obtained by removing spent culture medium and incubating the cells for various time periods with fresh complete medium (1 ml/well) alone or medium containing rhIL-1/3, LPS, or PMA. To generate hemipituitary tissue-conditioned medium, the 1- to 2-mm3 pieces were immediately transferred into 24-well plates containing 1 ml complete medium (one-half anterior pituitary per well). IL-6 assays The concentration of IL-6 that accumulated in the incubation medium of cultured anterior pituitary cells was quantitated using the IL-6-dependent hybridoma 7TD1 (10). Hybridomas such as 7TD1 are exquisitely sensitive to IL-6; concentrations of less than 10 U IL-6/ml (—10 pg/ml) result in half-maximal growth. In the absence of IL-6, 7TD1 cells fail to grow in response to any known cytokines or hormones, including IL-1, -2, -3, -4, or -5; interferon-a and -/3; lymphotoxin; tumor necrosis factor; colony-stimulating factors; PRL; or GH (11) (our unpublished observations). Aliquots of the pituitary incubation medium were serially diluted in microtiter plates (in duplicate) starting from 1:50 (i.e. 4 n\ conditioned medium in 200 n\); usually six dilutions of each specimen were included in each bioassay. After 3 days of incubation, proliferation of the 7TD1 cells was determined colorimetrically (12). The IL-6 concentration in samples was computed by comparison to the linear portion of a standard curve obtained with rmIL-6 (generously provided by Dr. J. Van Snick). The mean value for two or three dilutions of each sample was usually determined. The SE of the mean was approximately 20%, an acceptable value for a bioassay. All specimens from the same experiment were assayed at the same time. Using these conditions, the response curve was linear between 1-10 U rmIL-6/ml. A typical standard curve is shown in Fig. 1; neither LPS (10 Mg/ml) nor PMA (10 nM) affected the proliferation of 7TD1 cells in the absence or presence of rmIIL-6 (data not shown). T-Cell assays Spleen cells (2 X 105/ml) or thymocytes (2 x 106/ml) obtained from BALB/c mice were cultured (200 /ul/well) with a suboptimal concentration (1:100) of PHA (Gibco) in RPMI-1640
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IL-6 (units/ml) FlG. 1. IL-6 hybridoma growth factor assay. The response of the IL6-dependent hybidoma 7TD1 to increasing concentrations of IL-6 is shown. All specimens were diluted so that the concentrations of IL-6 were within the linear portion of the standard curve (1-10 U/ml), as described in Materials and Methods. Shown are the mean ± SD of triplicate wells. containing 5% fetal calf serum and 450 /*M 2-mercaptoethanol. After 3 days of incubation, 1 fiCi [3H]thymidine was added, and the cells were harvested 16 h later. These conditions are similar to those described previously for assay of IL-6 (13). The concentration of IL-6 required to elicit responses from T-cells is 100-fold greater than that needed to maintain the growth of hybridomas (3). Results Anterior pituitary cell production of IL-6: effects of LPS and PMA We first ascertained IL-6 production by anterior pituitary cells that had been dispersed and cultured for 4 days. As shown in Fig. 2, these cells produced detectable amounts of IL-6 within 2-4 h of incubation in fresh complete RPMI-1640 medium. Spontaneous release of IL-6 was approximately linear for 8 h, reaching levels of 2000-4000 U/ml, which are equivalent to 80-160 pM IL6. In four experiments, basal release of IL-6 ranged from 263-1600 U/ml-4 h. Bacterial LPS, a potent stimulator of macrophage IL-6 production (14), was tested for its ability to stimulate IL-6 release by these pituitary cells. In the experiment illustrated in Fig. 2 (upper panel), LPS (10 ixg/m\) stimulated detectable increases in IL-6 secretion within 1 h; IL-6 concentrations were greater than 6000 U/ml at 8 h, about 3-fold higher than basal release. Treatment of anterior pituitary cultures with 10 nM PMA also resulted in enhanced IL-6 production (Fig. 2, lower panel). In four separate experiments, LPS (10 /xg/ml) and PMA (10 nM) increased IL-6 release 4.1 ± 0.4- and 2.2 ± 0.4-fold, respectively, during 4-h incubations of anterior pituitary cells in vitro. It is noteworthy
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS
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with LPS, and the contralateral half was incubated in control medium. In the experiment illustrated in Fig. 3, a substantial linear accumulation of IL-6 in the medium was observed after a lag period of approximately 2 h from basal hemipituitary tissue. In contrast, LPS (10 (xg/ ml) stimulated IL-6 release (a 5-fold increase during a 4h incubation). In four experiments, LPS stimulated IL6 release 2.6 ± 0.6-fold. The data in Fig. 4 are collected from five separate experiments and summarize the timedependent accumulation of IL-6 and the effects of LPS (10 fxg/ml) on this accumulation. At all time points examined, LPS stimulated IL-6 release. Extended incubation of hemipituitary glands for 20 h resulted in IL-6 concentrations exceeding 40,000 U/ml. Thus, freshly isolated hemipituitaries secreted substantial IL-6 within 4
PITUITARY CULTURES A
6000- •
A
No Add
• + LPS
4000-
=
2000
§ 8000A—A
co
6000- •
Endo • 1990 Voll26«Nol
No Add • + PMA
HEMI-PITUITARIES
4000-
10000--
2000 -5000-
4
6
8
HOURS FIG. 2. Time courses of LPS- and PMA-induced productions of IL-6 from anterior pituitary cells in vitro. Anterior pituitary tissue was dispersed, as described in Materials and Methods, and the resultant single cells were cultured for 4 days. Spent culture medium was replaced with complete medium containing no additions, LPS (10 /xg/ml), or PMA (10 nM). At various intervals, the conditioned media were removed, and IL-6 concentrations were determined with the 7TD1 growth factor assay. Shown are the average concentrations of IL-6 from two wells of each treatment group at each time point in a representative experiment.
that very low concentrations of LPS are capable of stimulating the release of IL-6; 1 ng/ml LPS was sufficient to elicit IL-6 production from anterior pituitary cells (data not shown). Since IL-1 has been reported to stimulate IL-6 release from fibroblasts and endothelial cells (11, 15), we added rhIL-la (10-200 U/ml) to anterior pituitary cultures and assessed IL-6 production. No significant increases in IL6 release were noted in two separate experiments (data not shown).
FIG. 3. Production of IL-6 by hemianterior pituitary tissue. Hemipituitary tissue was cultured for 4 h with or without LPS (10 /ig/ml); 50fil aliquots were removed and replaced with fresh complete medium at 30-min intervals, and IL-6 concentrations were determined with the 7TD1 growth factor assay. Shown are the concentrations of IL-6 found in a representative experiment using a single anterior pituitary gland.
18000 15000
• BASAL (2)
ffi LPS (10 ug/ml)
0)
9000 6000 3000
1
(2)
•-> 12000
(4) (4)
0
Hemipituitary cell production of IL-6: effects of LPS Given the possibility that the IL-6 release observed in anterior pituitary cultures was due to selective survival of a particular cell type that is not well represented in the intact pituitary or that the release was merely a response to enzymatic dispersion, we examined the response of freshly explanted hemipituitary tissue to LPS. One lobe of an anterior pituitary gland was incubated
1 2 4 6 INCUBATION PERIOD (HR) FIG. 4. Time-dependent accumulation of IL-6 from hemipituitary tissue: effects of LPS. Hemipituitary glands were incubated in complete RPMI-1640 medium for 1-6 h with or without LPS (10 fig/ml). Results from five separate experiments using eight anterior pituitary glands, showing both basal and LPS-induced IL-6 release, are illustrated. Numbers above each bar refer to the number of observations per group. Results are expressed as the mean ± SEM.
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS h of isolation, indicating that the IL-6 produced by cultured cells reflected the capacity of the intact gland. Effects of anterior pituitary IL-6 mitogenesis
on lymphocyte
In the absence of antibodies against rat IL-6, it was important to determine whether the material released by anterior pituitary cells had other established biological activities of IL-6. The very large amounts of IL-6 secreted by hemipituitaries allowed us to test this material in less sensitive bioassays. We, therefore, examined the incubation medium from hemipituitary glands for costimulation of thymocyte and spleen cell mitogenesis with PHA. Although IL-6 alone had little effect with PHA on thymocyte mitogenesis under these conditions, it synergized with IL-1 to stimulate these cells (13). Figure 5 (top panel) shows that the stimulative effect of IL-1 on thymocytes was greatly augmented by increasing the concentrations of IL-6. Neither hemipituitary-conditioned medium nor IL-6 alone stimulated thymocyte proliferation (Fig. 5, top panel), although both synergized with IL-1 to do so. Hemipituitary-conditioned medium and IL-6 also stimulated the proliferation of PHA-stimulated spleen cells (Fig. 5, bottom panel). It is noteworthy 12 10
O—OIL-6 A A PIT. CM (A) D DPIT. CM (B) •
8
I o
THYMOCYTES + PHA
• I L - 6 + IL-1 CM (A) + IL-1
A
A PIT.
•
BPIT. CM (B) + IL-1
O A
OIL-6 A PIT. CM (A)
D
DPIT. CM (B)
6
x
E Q. O
LJ
0 1 500 IL-6(U/ml): 200 FIG. 5. Effects of hemipituitary tissue-conditioned (PIT. CM) medium on thymocyte and spleen cell mitogenesis. Hemipituitary-conditioned medium (20 h) was tested in the thymocyte costimulation and PHAspleen cell assays, as described in Materials and Methods. Shown are the results of the titrations of two separate conditioned media.
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that in both assays, the hemipituitary-conditioned medium increased T-cell mitogenesis at dilutions corresponding approximately to their IL-6 contents, as determined by the hybridoma growth factor assay.
Discussion The anterior pituitary is the site of the synthesis and release of numerous circulating hormones. We recently found that the inflammatory cytokine IL-6 is a potent stimulator of the release of GH, PRL, and LH from the pituitary in vitro (6). The studies reported here indicate that the pituitary also has the capacity to secrete large quantities of IL-6. Significant accumulations of IL-6 activity were detected in anterior pituitary cell-conditioned medium within 2-4 h of incubation using the monospecific 7TD1 growth factor assay. As further evidence that anterior pituitary cells produce IL-6, we found that hemipituitary-conditioned medium synergizes with IL-1 in the thymocyte costimulation assay and increases the proliferation of PHA-treated spleen cells. These data suggest that a cell population (s) in the anterior pituitary can produce IIL-6 constituitively, and that this production can be further increased by LPS or PMA. The present results showing anterior pituitary production of IL-6 complement recent studies by Vankelecom and co-workers (16), who reported that IL-6 was present in folliculo-stellate cells of rat and mouse pituitary glands. The folliculo-stellate cell is a poorly characterized macrophage-like cell that forms part of the pituitary matrix in which somatotrophs and other hormone-secreting cells are embedded. The close juxtaposition of folliculo-stellate cells and hormone-secreting cells has led to speculation that the former may be involved in regulating hormone secretion (17). The fact that IL-6 may be produced by the folliculo-stellate cells and acts as a potent pituitary secretagogue provides strong support for such a hypothesis. It is noteworthy that the concentrations of IL-6 we observed in short term cultures of primary pituitary explants (4 h) are similar to those that induce maximal hormone release from the pituitary (2,000-10,000 U/ml). IL-6 synthesis has been detected in a diverse array of tissues and cell types, including fibroblasts (15), macrophages (14), endothelial cells (11), and T cells (10). LPS is a potent stimulator of IL-6 production in fibroblasts and macrophages (14, 18). Our data, showing enhanced IL-6 release from cultured pituitary cells with 1 ng/ml LPS, are in accord with these previous reports. Administration of LPS in man results in increased GH, ACTH (19), and IL-6 (20) secretion, which suggests that in pathophysiological conditions, such as sepsis, circulating LPS may elicit local pituitary as well as peripheral production of IL-6, resulting in enhanced hormone release.
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IL-6 PRODUCTION BY ANTERIOR PITUITARY CELLS
However, since normal circulating IL-6 concentrations are rarely sufficient to stimulate pituitary hormone release (6, 8, 9), the intrapituitary production of IL-6 may be a mechanism by which this cytokine regulates anterior pituitary function. It has been previously suggested that angiotensin-II (21) and vasoactive intestinal peptide (22) may function as paracrine/autocrine factors for the regulation of hormone release. We suggest that IL-6 may act in a similar manner. Hormone-secreting cells in the anterior pituitary gland are not randomly distributed, and PRL-secreting cells are often physically associated with LHsecreting cells (23). GnRH treatment of anterior pituitary cell aggregates results in increased PRL production, possibly through the release of angiotensin-II from the gonadotroph (21, 24). Studies are underway to determine whether IL-6 production in the anterior pituitary may also be affected by hypothalamic neurohormones. Our results, demonstrating IL-6 stimulation of hormone release from the anterior pituitary as well as production of IL-6 by this gland, add to the growing list of activities and sources for this pleiotropic cytokine.
8. 9.
10.
11. 12. 13. 14.
15.
Acknowledgments
16.
We wish to acknowledge Jill A. Sfetko, M. Kathleen Borland, and John Schetz for their excellent technical assistance, and Jo Ann M. Eliason for her editorial expertise.
17.
References 1. Van Damme J, Opdenakker G, Simpson RJ, Rubira MR, Cayphas S, Vink A, Billiau A, Van Snick J 1987 Identification of the human 26-kD protein, interferon 02 (INF-02), as a B-cell hybridoma/ plasmacytoma growth factor induced by interleukin-1 and tumor necrosis factor. J Exp Med 165:914 2. Gauldie J, Richards C, Harnish D, Lansdorf P, Baumann H 1987 Interferon /32/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 84:7251 3. Uyttenhove C, Coulie PG, Van Snick J 1988 T-cell growth and differentiation induced by interleukin-HPl/IL-6, the murine hybridoma/ plasmacytoma growth factor. J Exp Med 167:1417 4. Ikebuchi K, Wong GG, Clark SC, Ihle JN, Hirai Y, Ogawa M 1987 Interleukin-6 enhancement of interleukin-3 dependent proliferation of multipotential hemopoietic progenitors. Proc Natl Acad Sci USA 84:9035 5. Kishimoto T, Hirano T 1988 Molecular regulation of B-lymphocyte response. Annu Rev Immunol 6:485 6. Spangelo BL, Judd AM, Isakson PC, MacLeod RM 1989 Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 125:575 7. Naitoh Y, Fukata J, Tominaga T, Nakai Y, Tamai S, Mori K,
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Imura H (1988) Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 155:1459 Van Oers MHJ, Van Der Heyden AAPAM, Aarden LA 1988 Interleukin-6 (IL-6) in serum and urine of renal transplant recipients. Clin Exp Immunol 71:314 Houssiau FA, Bukasa K, Sindic CJM, Van Damme J, Van Snick J 1988 Elevated levels of the 26K human hybridoma growth factor (interleukin-6) in cerebrospinal fluid of patients with acute infection of the central nervous system. Clin Exp Immunol 71:320 Van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubira MR, Simpson RJ 1986 Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA 83:9679 Sironi M, Breviario F, Proserpio P, Biondi A, Vecchi A, Van Damme J, Dejana E, Mantovani A 1989 IL-I stimulates IL-6 production in endothelial cells. J Immunol 142:549 Mosmann T 1983 Rapid colorimetric assay for cellular growth and survival:, application to proliferation and cytotoxicity assays. J Immunol Methods 65:55 Helle M, Brakenhoff JPJ, De Groot ER, Aarden LA 1988 Interleukin 6 is involved in interleukin 1-induced activities. Eur J Immunol 18:957 Chiu C-P, Moulds C, Coffman RL, Rennick D, Lee F 1988 Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells. Proc Natl Acad Sci USA 85:7099 Elias JA, Trinchieri G, Beck JM, Simon PL, Sehgal PB, May LT, Kern JA 1989 A synergistic interaction of IL-6 and IL-I mediates the thymocyte-stimulating activity produced by recombinant IL-Istimulated fibroblasts. J Immunol 142:509 Vankelecom H, Carmeliet P, Van Damme J, Billiau A, Denef C 1989 Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 49:102 Baes M, Allaerts W, Denef C 1987 Evidence for functional communicaiion between folliculo-stellate cells and hormone-secreting cells in perifused anterior pituitary cell aggregates. Endocrinology 120:685 Helfgott DC, May ILT, Sthoeger Z, Tamm I, Sehgal PB 1987 Bacterial lipopolysaccharide (endotoxin) enhanced expression and secretion of /32 interferon by human fibroblasts. J Exp Med J 66:1300 Revhaug A, Michie HR, Manson IM, Watters JM, Dinarello CA, Wolff AM, Wilmore DW 1988 Inhibition of cyclooxygenase attenuates the metabolic response to endotoxin in humans. Arch Surg 123:162 Fong Y, Moldawer ILL, Marano M, Wei II, Tatter SB, Clarick RH, Santhanam U, Sherris D, May ILT, Sehgal PB, Lowry 1989 Endotoxemia elicits increased circulating /32-IFN/IL-6 in man. J Immunol 142:2321 Jones TH, Brown BIL, Dobson PRM 1988 Evidence that angiotensin II is a paracrine agent mediating gonadotrophin-releasing hormone-stimulated inositol phosphate production and prolactin secretion in the rat. J Endocrinol 116:367 Nagy G, Mulchahey JJ, Neill ID I 988 Autocrine control of prolactin seretion by vasoactive intestinal peptide. Endocrinology 122:364 Denef C 1986 Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab 15:1 Denef C, Andries M 1983 Evidence for paracrine interaction between gonadotrophs and lactotrophs in pituitary cell aggregates. Endocrinol J 12:813
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