1 Institute for General and Experimental Pathology, and
2 Central Laboratory Animal Facilities, Medical School, University of Innsbruck, 6020 Innsbruck, Austria
3 Mucosal Immunity Group, German Research Centre for Biotechnology, 38124 Braunschweig, Germany
4 Laboratory of Immune Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA
Correspondence to: G. Wick, Institute for General and Experimental Pathology, Fritz-Pregl-Strasse 3/IV, 6020 Innsbruck, Austria
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Abstract |
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Keywords: B cell development, ectopic steroidogenesis, T cell development
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Introduction |
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However, GC do not necessarily always result in apoptosis; under certain circumstances they even seem to be necessary for specific steps in thymocyte maturation (11,12). In vitro data show that signaling via the GC receptor (GR) can produce either inhibitory or stimulatory effects, depending on GC concentration and incubation time (13,14). Experiments in transgenic mice in which antisense transcripts to the GR are expressed in immature thymocytes revealed that GC are essential for progression from the CD4CD8 (double-negative) to the CD4+CD8+ (double-positive) stage and maintenance of viability at the double-positive stage (12).
GC hormones are mainly synthesized in the adrenal glands from the precursor cholesterol through sequential modifications by members of the cytochrome P450 enzyme family and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). Corticosterone is the main GC formed in rodents and birds. Its synthesis and release from the adrenals is under the control of the hypothalamo-pituitaryadrenal axis, adrenocorticotropic hormone (ACTH) being the major mediator acting on the adrenals. In addition to this central endocrine signaling, locally produced steroids have been implicated in paracrine signaling. Thus, it has been suggested that steroids locally produced in the brain play a role, both in cognitive functions and survival of cells in the nervous system (1517). Furthermore, all enzymes required for the production of the major steroid hormones have been detected in the murine brain either on protein or at least the mRNA level (18,19).
The thymus has been shown to have endocrine features, e.g. a variety of hormones and corresponding receptors are known to be produced ectopically within the thymus (20). Previous studies have shown that murine thymic epithelial cells (TEC) possess enzymes necessary to produce GC (2124) and that addition of GC synthesis inhibitors to fetal thymic organ cultures enhanced TCR-mediated thymocyte deletion (21,22,25). Based on these results, it has been proposed that GC interfere with the antigen-driven development of thymocytes by preventing TCR-mediated deletion of cells bearing receptors with low-to-moderate avidity for self-antigenMHC (26). Another study by Xue et al. indicated that thymic apoptosis occurs independently of adrenal GC and that the GC antagonist RU486 inhibits apoptosis induced by a MHC class II-binding peptide, whereas the drug had no effect on deletion induced by a MHC class I-restricted peptide (27). However, a recent study put a question on these studies, as GC receptor knockout mice showed a normal embryonic development of the thymus and of intrathymic T cell development (28).
The aim of the present study was to investigate if intrathymic steroidogenic activity can also be found in a non-mammalian species like chickens and, more important, as immature B cells seem also to be especially GC-sensitive, if GC are also produced in the compartment responsible for B cell developmentin our case the bursa of Fabricius.
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Methods |
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Reagents
Trilostane, an inhibitor of 3ß-HSD, was kindly supplied by Dr B. D. Bond (Sanofi Winthrop, Newcastle-upon-Tyne, UK). Aminoglutethimide [an inhibitor of cholesterol side chain cleavage enzyme (P450scc)], 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone, an inhibitor of P450c11ß) as well as all steroid standards and precursors were purchased from Sigma (St Louis, MO).
[1,2-3H(N)]Corticosterone, [7-3H(N)]pregnenolone, [1,2,6,7-3H(N)]progesterone, [7-3H(N)]-17-hydroxypregnenolone, [1,2-3H(N)]-11-deoxycortisol and [1,2-3H(N)]-17
-hydroxy-progesterone were obtained from Du Pont (New England Nuclear, Boston, MA), and [1,2,6,7-3H(N)]cortisol from Amersham Life Science Les Ulis, France). Trasylol was purchased from Bayer (Leverkusen, Germany) and rabbit anti-corticosterone-21-thyroglobulin serum from Biomakor (Kiryat Weizmann, Rehovot, Israel).
Preparation of steroidogenic organelles
Thymus and bursa were used as primary lymphoid organs, and adrenal and heart as positive and negative controls respectively. In the murine system we analyzed splenic preparations as a control for a secondary lymphoid organ (24), detecting P450scc but no 3ß-HSD, P450c17, P450c11ß or P450c21 activity. Therefore, we wanted to investigate if this phenomenon can also be observed in chickens.
Organs were placed in ice-cold homogenization buffer [(HB) 50 mM PBS (pH 7.4), 1 mM EDTA, 250 mM sucrose and 0.04 U/ml Trasylol], trimmed of fat, minced finely with scissors and mixed with 5 volumes of HB. The mixture was homogenized with a glass homogenizer and centrifuged for 10 min at 800 g to pellet nuclei and unbroken cells; the resulting supernatant was centrifuged 20 min at 10,000 g, while the resulting pellet was recentrifuged for 20 min at 6000 g to sediment mitochondria. Both supernatants were pooled and centrifuged for 75 min at 105,000 g to pellet microsomes. Mitochondrial and microsomal pellets were resuspended in HB. All fractions were divided into aliquots and stored in borosilicate glass tubes at 20°C until use. The protein concentration was determined by precipitating the fraction with 1 volume 0.5 N TCA for 15 min at 4°C. After washing the precipitate 2 times with 2 ml 0.25 N TCA, samples were centrifuged for 10 min at 4°C at 1000 g. The pellet was resuspended in 100 µl 0.4 N NaOH. Final determination of protein concentration was performed with BCA protein assay reagent (Pierce, Rockford, IL) according to the manufacturer's instructions.
Determination of steroid enzyme activities (see Fig. 1)
P450scc.
Mitochondrial fractions were preincubated in HB with 10 mM glucose-6-phosphate, 1.5 U NAD(P)+-dependent glucose-6-phosphate dehydrogenase, 5 µM Trilostane and 5 µM 22-HO-cholesterol in a total volume of 0.9 ml for 20 min at 37°C. The enzymatic reaction was started by the addition of 0.6 mM NADPH (in 100 µl HB buffer), and stopped by addition of 4 ml ice-cold methylenechloride and vortexing.
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Further processing of the samples was performed as described below, with the difference that extracted and evaporated samples were dissolved in 250 µl PGB [0.05 M PBS (pH 7.4) and 0.1% (w/v) gelatine]. Since no suitable 3H-labeled 22-HO-cholesterol was available for the P450scc reaction, the product of this reaction, pregnenolone, was determined by radioimmunoassay as follows: 100 µl sample or standard (5-pregnen-3ß-ol-20-one) was mixed with 100 µl [7-3H(N)]pregnenolone (12,000 c.p.m./100 µl) and 100 µl of anti-pregnenolone-3-hemosuccinate serum (ICN, Costa Mesa, CA) diluted in PGB.
After an incubation for 30 min at 37°C and 60 min at 4°C, samples were incubated for 10 min at 4°C with 500 µl charcoal and centrifuged for 10 min at 4°C and 1000 g. Then 500 µl of supernatant was emulsified in 2.8 ml scintillation cocktail (Ultima Gold; Packard Instruments, Groningen, The Netherlands) and counted in a ß-counter (Tri-Carb 1900-TR; Canberra-Packard, Dreireich, Germany).
3ß-HSD.
Microsomal fractions were incubated in HB with 30 µM NAD+, 0.5 µl (~7.5 ng) [7-3H(N)]pregnenolone, in a total volume of 1 ml at 40°C. Trilostane was used as competitive inhibitor for 3ß-HSD. The reaction was stopped by the addition of 4 ml ice-cold methylene chloride and vortexing. Further processing of the samples was performed as described below.
P450c17.
Microsomal proteins were incubated in HB with 0.6 mM NADPH, 5.0 mM MgCl2, 4 mM glucose-6-phosphate, 1 U NAD(P)+-dependent glucose-6-phosphate dehydrogenase and [7-3H(N)]pregnenolone in a total volume of 1 ml at 37°C. Further sample processing was performed as described below.
P450c21.
Microsomal proteins were incubated in HB with 0.6 mM NADPH, 5.0 mM MgCl2, 10 mM glucose-6-phosphate, 2 U NAD(P)+-dependent glucose-6-phosphate dehydrogenase and [1,2,6,7-3H(N)]progesterone or [1,23H(N)]-17-hydroxyprogesterone in a total volume of 1 ml at 37°C. Further sample processing was performed as described below.
P450c11ß.
Mitochondrial fractions were incubated in HB with 0.3 mM NADPH, 5.0 mM MgCl2, 10 mM glucose-6-phosphate, 2 U NAD(P)+-dependent glucose-6-phosphate dehydrogenase and 1 µl (~0.4 ng) [1,2-3H(N)]11-deoxycortisol in a total volume of 1 ml at 37°C. Further processing of the samples was performed as described below. As inhibitor of this reaction we used metyrapone at final concentrations of 1050 µM.
Sample processing and thin-layer chromatography (TLC)
Samples were extracted 3 times with 4 volumes of methylene chloride. The extracts were combined, evaporated in a Turbo Vap LV evaporator (Zymark, Hopkinton, MA) and reconstituted in 10 µl ethanol. Steroids (substrates and products) were separated on silica gel TLC plates (Kieselgel 60 F254; Merck, Darmstadt, Germany) developed in different mixtures of organic solvents (see Results). Carrier unlabeled steroids were added to the samples before chromatography to aid steroid visualization on the TLC plates. The cold steroid spots were identified by UV absorption. 5 steroids (which cannot be visualized by UV absorption) were visualized by application of 3H-labeled standards to a separate marker lane. 3H products and markers were visualized after enhancement with EN3HANCE spray (New England Nuclear) and autoradiography on a Reflection film (New England Nuclear). Visualized radioactive spots were excised and radioactivity measured in a MR-300 ß-counter (Kontron, Neufahrn, Germany). Total radioactivity within a single lane was considered as 100% and equivalent to the total amount of steroids added to the reaction. The amount of steroid in each individual spot was calculated by its percentage of c.p.m. with respect to the total radioactivity and the amount of microsomal/mitochondrial protein used for the reaction. Therefore, data are given as ng/pg steroid formed/mg of microsomal/mitochondrial protein.
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Results |
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Steroidogenic enzyme activities
As mRNA expression does not necessarily correlate with protein levels, not to mention enzyme activities, we used a biochemical approach to monitor the instantaneous activities of the entire enzyme complexes, e.g. the functional activities and cooperation of ferridoxin reductase, ferridoxin and cytochrome P450scc. We isolated microsomes and mitochondria from different organs, and incubated these steroidogenic organelles with 3H-labeled substrates and an appropriate energy source. Typical autoradiographies are shown for the different tested enzymes.
P450scc (Fig. 2)
Thymus, spleen and bursa are able to convert 22-HO-cholesterol to pregnenolone, showing the presence of a functionally active P450scc system. The reaction could be inhibited by the P450scc-specific aminoglutethimide (50 µM) or by omission of NADPH (data not shown). Adrenal samples were positive and out of our measuring range (>1000 pg pregnenolone formed/mg of mitochondrial protein). Heart mitochondria showed no reactivity.
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P450c17/21 (Fig. 4
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P450c17/21 activity was measured by the ability to convert progesterone to 17
-HO-progesterone and subsequently to 11-deoxycortisol. Thymus proved to be clearly P450c17
+ and P450c21+, whereas bursa microsomes were clearly P450c17
+ and weakly P450c21+. It is noteworthy that progesterone is preferentially metabolized by P450c17
and not P450c21, indicating that the major GC production by the chicken thymus and bursa is cortisol. Bursa exhibits a ~3 times lower P450c17
and a ~30 times lower P450c21 activity/mg of microsomal protein respectively compared to thymic tissue. Adrenals were also positive for P450c21, and exhibited a ~17- and ~700-fold activity/mg of microsomal protein as calculated from the conversion rate when compared to thymus and bursa respectively. In contrast, no P450c17 activity could be measured with adrenal and spleen (data not shown).
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Discussion |
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The thymus has been identified as an active member of the immuno-neuroendocrine system. Different hormones and their corresponding receptors are produced ectopically within the thymus, including the hypothalamo-pituitaryadrenal axis hormones corticotropin releasing hormone and ACTH (20,3033). The latter two hormones were also found in the chicken bursa (32,33).
Recent results by our groups showed that the murine thymus possesses all steroidogenic activities necessary to produce GC, that this steroidogenic activity is located to TEC, and that these intrathymically produced GC are involved in positive and negative selection of thymocytes (21,24,26,34). Another study by the Ashwell group, finally, showed that thymocyte resistance to GC leads to antigen-specific unresponsiveness due to `holes' in the T cell repertoire (35).
However, the role of GC in thymopoiesis is still very controversial and seems to depend on the particular model used for the respective experiments. Recently, Purton et al. investigated this problem using GR-deficient mice. These animals were resistant to dexamethasone-mediated apoptosis, confirming the absence of GC responsiveness. Surprisingly, an absence of GR signaling had no impact on thymocyte development either in vivo or in vitro. T cell differentiation, including positive selection, was normal as assessed by normal development of double-positive, ßTCR+CD4+ and
ßTCR+CD8+ thymocytes. Negative selection, mediated by the superantigen staphylococcal enterotoxin B or anti-CD3/CD28, was also normal in the absence of GR signaling (28,36). Therefore, these data would demonstrate that GR signaling is not essential for intrathymic T cell development or selection. Further experiments will have to clarify these controversies.
The exact localization of intrathymic GC production is also still a subject of discussion. However, the localization of steroidogenic activity to murine TEC was described by different independent groups. In contrast to one single report by Jenkinson et al. (37), who described that the thymic epithelium has not the potential to synthesize GC. In our own experiments we clearly showed the presence of all enzyme activities and cofactors necessary for GC production in total thymus preparations. Additionally, we showed P450scc and 3ß-HSD activities in thymi of sublethally irradiated mice and a TEC cell line. Finally, immunofluorescence staining of thymus sections revealed that the P450scc protein is present in TEC and not thymocytes. In contrast to these data, Jenkinson et al. analyzed only one enzyme in the cascade at the mRNA level, namely P450scc.
Differences between strains of normal animals and spontaneous autoimmune models have been shown in the immuno-neuroendocrine communication along the hypothalamo-pituitaryadrenal axis (i.e. defective response of different endocrine parameters after stimulation with an antigen or cytokines) (3841). If these findings could be corroborated with respect to intrathymic GC production, this would provide a possible explanation for the survival of autoreactive T cell clones within the thymus. According to the `mutual antagonism' model, proposed by Ashwell et al., local concentrations of GC set the thresholds between positive and negative selection in thymocyte development (26). Thymocytes bearing TCRs with high avidity are negatively selected and those with intermediate avidity are positively selected. However, there is a large additional population of cells, with TCR that recognize self-antigenMHC with low, but biological significant avidity. GC inhibit the activation of all thymocytes in all TCR avidity groups. The fate of the activated cell is determined by the balance of the strength of the TCR signal(s) (dependent upon TCR affinity/avidity and the concentration of ligand) and the local concentrations of GC. Inhibition of intrathymic GC biosynthesis causes an increase in thymocyte apoptosis and a decrease in recovery that are directly proportional to the number of MHC-encoded molecules present and, therefore, the number of ligands available for TCR recognition (22). These results indicate that thymus-derived GC determine where the window of thymocyte selection occurs in the TCR avidity spectrum by dampening the biological consequences of TCR occupancy.
The aim of the present study was to investigate if the total synthetic pathway of GC observed in mice could also be found in the thymus of a non-mammalian species like chickens. Furthermore, the bursa of Fabricius, a paradigmatic lymphoid organ for B cell development and selection, was also analyzed for local GC production. A problem when working with chickens is the lack of avian nucleotide sequence information about genes of interest and suitable antibodies for immunohistological and functional assays. Beside this problem, mRNA levels measured by RT-PCR are not informative concerning the activity of a protein, especially if it is only part of an enzyme complex. Therefore, we used a method that had the advantage of monitoring instantaneous activities of the entire steroidogenic enzyme systems, i.e. in the case of cytochrome P450scc its functional activity and cooperation with ferridoxinreductase and ferridoxin. With these methods, we detected P450scc, 3ß-HSD, P450c17, P450c21 and P450c11ß activities in chicken thymi and bursae. Chicken spleen was also found to be able to metabolize 22-HO-cholesterol, i.e. it expressed P450scc reactivity, but no 3ß-HSD or P450c17 activity could be detected. Therefore, it can be concluded that a secondary lymphoid organ like the spleen is not able to synthesize GC hormones, a phenomenon common to distant species like mice and chickens.
Compared to murine intrathymic steroidogenic activity, two important differences could be observed. First, chicken 3ß-HSD activity seems to be mainly localized in the mitochondrial compartment, whereas murine 3ß-HSD activity is mainly restricted to microsomes (24). Second, chicken thymi and bursae also expressed P450c17 activity, and incubation of microsomes with progesterone resulted in the sequential synthesis of 17
-hydroxyprogesterone and 11-deoxycortisol (see Fig. 1
). Thus, chicken intrathymic GC synthesis seems to preferentially go in the direction of cortisol. This is surprising since the main adrenally derived serum GC in chickens and mice is corticosterone. In contrast, no P450c17
activity could be detected in murine thymus (24).
However, the most important result of the present work was the proof for the presence of all steroidogenic enzyme activities within the bursa. Again, the main bursal GC seems to be cortisol. Several studies showed that GC affect the bursa in a similar way as they affect the thymus (7,8,42). Treatment of different avian species, including chickens, with exogenous adrenal steroids or elevated endogenous GC results in a profound involution of the bursa, as a result of B cell apoptosis (7,8,43,44).
With the proof of the whole cascade of GC synthesizing enzymes one could speculate that endogenous bursal GC may have a similar importance for B cell development as they have for thymic T cell maturation. In this respect it is noteworthy that the chicken bursa contains a peptide, called bursal anti-steroidogenic peptide (BASP), with suppressive effects on B lymphocyte mitogenesis (45). BASP has been described to inhibit basal and ACTH-stimulated GC production from normal or tumor-derived adrenocortical cells in vitro (46).
In conclusion, we demonstrate that the entire biosynthetic pathway for GC hormone production, including all intermediary metabolites, is present in the chicken bursa and thymus. In contrast to the murine and chicken periphery and the situation in the murine thymus, where corticosterone represents the main GC, steroidogenic pathways in the chicken bursa and thymus lead toward cortisol, which has a higher affinity for the GR than corticosterone [inhibition constant (Ki) = 15 nM for cortisol compared to 20 nM for corticosterone].
Irrespective of the ongoing controversy about the role of GC in thymopoiesis, the evolutionary conserved or in parallel developed ability of the murine and chicken thymus to synthesize GC is a strong hint that these hormones must play an important role in this primary lymphoid organ. Further studies in different experimental systems will have to clarify the precise function of GC in the thymus.
The exact role of bursal GC production in B cell development will have to be elucidated by further experiments in the avian system. It will be interesting to find out if there exists a similar mutual antagonism between BCR and GC as observed in the thymus between TCR and GC. In parallel, experiments should be extended to the sites of mammalian B cell development in order to further clarify the implication of GC in B cell development.
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Acknowledgments |
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Abbreviations |
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3ß-HSD 3ß-hydroxysteroid dehydrogenase |
ACTH adrenocorticotropic hormone |
BASP bursal anti-steroidogenic peptide |
HB homogenization buffer |
GC glucocorticoid hormones |
GR glucocorticoid receptor |
TEC thymic epithelial cells |
TLC thin-layer chromatography |
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Notes |
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Transmitting editor: J. F. Bach
Received 13 October 2000, accepted 21 February 2001.
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References |
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