Thymus-pituitary interactions during ageing.
In recent years a growing body of evidence has accumulated suggesting that the immune system is functionally linked to the nervous and endocrine systems, thus constituting an integrated homoeostatic network |1, 2~. During early life the thymus gland appears to be essential for this integration as suggested by the extensive derangements of the hormonal and immune balance caused by neonatal thymectomy or congenital absence of the thymus in various animal species |3, 4~. Interestingly, it has been reported that perinatal administration of certain thymic preparations can prevent the immune and endocrine consequences of thymus removal |5, 6~.
It is now well established that the endocrine thymus produces a number of immunoregulatory substances, some of which are also active on nervous and endocrine circuits |7~. Recent evidence suggests that in old rodents there is, in addition to a reduced activity of the endocrine thymus, a significant desensitization of the neuroendocrine system to thymic signals. Thus, we have reported that thymosin fraction five (TF5) and homoeostatic thymus hormone (HTH), two partially purified thymic preparations of bovine origin, possess thyrotropin (TSH)-inhibiting activity in young but not in old rats |8-10~. When administered intravenously to young rats, HTH markedly reduced growth hormone (GH) secretion, an effect that was much weaker in old animals |10~. Intravenous HTH was also shown to activate the adrenal axis in young and, to a lesser extent, old rats |11~. The age-dependency of this adreno-stimulating activity of HTH can be appreciated in Figure 1 which shows the effect of a single i.v. dose of HTH on the plasma levels of corticosterone in young and old rats.
Considering that thymus removal induces generalized endocrine alterations, the multi-hormone changes elicited by HTH in vivo suggest that this preparation may contain some of the chemical messengers linking the thymus with the neuroendocrine system. The HTH preparation used in the above in vivo studies was a chromatographically homogeneous extract from calf thymus, also known as the Bernardi-Comsa preparation |12~. In 1985, Reichhart et al. |13, 14~ further purified this preparation and established that HTH consisted of two polypeptide chains, HT|H.sub.|Alpha~~ and HT|H.sub.|Beta~~, whose primary structures were found to be identical to those of histones H2A and H2B, respectively. These findings prompted us to explore the activity of histones on pituitary hormone secretion as well as the impact of ageing on these effects. Since the adrenal axis had been shown to be responsive to HTH in an age-dependent manner |11~, it was of interest to assess the corticotropin (ACTH)-releasing activity of histones and other related peptides on pituitary cells from young and mature animals. We report here our findings.
Materials and Methods
Protein preparations tested: The following histones, prepared from calf thymus, were purchased from Sigma Chemical Co., St. Louis, MO, USA: H1, H2A, H2B, H3, Sigma type IIA, and Sigma type IIS. Nucleohistone from calf thymus, histone-free protamine from salmon, salmon testis nucleoprotamine and ubiquitin from bovine red cells were also obtained from Sigma. Peptide MB35 was a generous gift from Dr Allan Goldstein, Department of Biochemistry, The George Washington University Medical Center, Washington DC, USA. Human/rat corticotropin-releasing factor (CRH) was purchased from Peninsula Laboratories, Palo Alto, CA, USA.
Cell perifusion system: Eight to ten rat pituitaries were cut into 8-10 pieces each with a razor blade and placed together in a Petri dish where they were washed twice with Earle's balanced salt solution containing 1 g/l NaC|O.sub.3~H, 0.5% bovine serum albumin, 30 |Micron~g/ml ascorbic acid, 50 IU/ml aprotinin and antibiotics (perifusion medium). The pieces were transferred to a plastic tube containing 10 ml perifusion medium with 30 mg collagenase and 1 mg DNase. After 1 h incubation under constant shaking the cell suspension was repeatedly flushed with a Pasteur pipette to complete the dispersion process. The suspension was filtered through a 100 |Micrometer~ mesh gauze to remove tissue fragments and large cell aggregates, and then centrifuged at 150 g for 20 min at 4 |degrees~ C. The cell pellet was gently resuspended in 4 ml perifusion medium. An aliquot was mixed with an equal volume of 0.4% Trypan Blue in saline and the mix used for cell viability assessment. Cell viability ranged from 88 to 96% and was similar for young and mature animals.
Dispersed cells obtained as described above were mixed with preswollen Bio Gel P-2 and packed in a 2.5-ml disposable plastic syringe (column). The rubber plunger of the syringe was pierced with a needle to which a plastic cannula was attached. A 10-|Micrometer~ nylon gauze was placed over the plunger in order to prevent leakage of cells and gel particles through the needle. The syringe was kept with the nozzle upwards and the perifusion medium was pumped through the column by means of a peristaltic pump which kept a constant perifusion flux of 0.5 ml/min. The medium reservoir and the column were immersed in a water bath at 37 |degrees~ C. The cell column was perifused for 1 h with perifusion medium alone. Then, each of the substances to be tested (stimuli), dissolved in perifusion medium, were pumped through the column for 3 min leaving a 20-min interval (incubation medium alone pumped) between successive stimuli. A fraction collector at the end of the perifusion system collected 1-ml fractions of the column perifusate. These fractions were frozen at -20 |degrees~ C until ready for hormone assays.
Cell incubation system: Cells were dispersed as described above, their concentration adjusted to about 2 x |10.sup.5~ cells/ml and preincubated 60 min at 37 |degrees~ C under continuous shaking in a 95% air: 5% C|O.sub.2~ atmosphere. After incubation, cells were centrifuged, the medium discarded and the same volume of fresh medium added to the cell pellet which was then gently resuspended. This suspension was rapidly distributed in plastic tubes (1 ml/tube) which contained the appropriate stimuli in 100 |Micron~l medium. The tubes were incubated for 3 h under the above conditions. At the end of incubation, tubes were centrifuged at 300 g for 15 min, the cell supernatants collected and frozen at -20 |degrees~ C until ready for hormone assays.
CRH-Immunoradiometric assay (IRMA): The human/rat (h/r) CRH-IRMA has been described in detail elsewhere |15~. Briefly, triplicate 200-|Micron~l aliquots of samples or of standard (1-10 000 pg/ml) diluted in 0.05 M sodium phosphate buffer (pH 7.4), 0.25% (w/v) BSA, were incubated overnight at room temperature with 200 |Micron~l of the CRH-IRMA reagent mixture. This consisted of 125I-labelled rabbit anti-CRH (36-41)-N|H.sub.2~ IgG (100 000 c.p.m./tube) and 1/5000 guinea pig anti-CRH-(1-20) serum (referred to as 'CRH linker') in 0.05 M sodium phosphate buffer (pH 7.4) containing 0.5% (w/v) human serum albumin, 1% (v/v) normal rabbit serum and 0.01% (w/v) sodium azide. Separation of CRH-bound from unbound labelled IgG was performed with sheep anti-guinea pig Fc region IgG coupled to a dynosphere solid phase. The radioactivity of the bound IgG was measured in a |Gamma~-counter.
ACTH-IRMA: This assay has been described in detail elsewhere |16~. Briefly, triplicate 200-|Micron~l aliquots of samples or standards (1-10 000 pg/ml range) diluted in 0.05 M sodium phosphate buffer (pH 7.4), 0.25% (w/v) BSA, were incubated overnight at room temperature with 200 |Micron~l of the ACTH-IRMA reagent mixture. This consisted of 125I-labelled sheep anti-ACTH-(1-24) IgG (100 000 c.p.m./tube) and 1/5000 rabbit anti-ACTH-(25-39) antiserum (referred to as 'ACTH linker') in 0.05 M sodium phosphate buffer (pH 7.4) containing 0.5% (w/v) human serum albumin, 1% (v/v) normal rabbit serum, 2% (v/v) normal sheep serum, 2% (w/v) polyethylene glycol (PEG) and 0.01% (w/v) sodium azide. Separation of ACTH-bound from unbound labelled antibody was achieved by precipitating the bound complex with 200 |Micron~l sheep anti-rabbit IgG serum diluted 1:10 in 0.05 M sodium phosphate buffer (pH 7.4). The mixture was centrifuged at 4000 g for 30 min at 4 |degrees~ C after the addition of 2 ml 2.0% (w/v) PEG solution in 0.05 M sodium phosphate buffer (pH 7.4). The supernatant was aspirated and the radioactivity in the pellet measured in a |Gamma~-counter.
Statistical analysis: For each stimulus, differences in hormone release between young and mature groups were assessed by one-way ANOVA.
Interference of histones with IRMAs
Initial experiments revealed that histones and protamine generated a dose-dependent signal in the ACTH-IRMA. The shape and magnitude of the corresponding dose-response curves were not significantly affected when the ACTH linker antiserum was eliminated from the reagent mixture, a manipulation that completely flattened the ACTH standard curve (data not shown). A similar although weaker positive interference was observed in the CRH-IRMA. The average |ACTH signal/CRH a signal~ ratio for histones H1, H2A, H2B, H3 and protamine (at 1 mg protein/ml) was: 4.1; 2.6; 3.3; 4.4 and 23.7, respectively. Nucleohistone, nucleoprotamine, peptide MB35 and ubiquitin did not produce any detectable interference at the dose levels tested in this study.
ACTH and CRH signal profiles in pituitary cell perifusates
In the experiments with perifused pituitary cells reported below the CRH-IRMA was used TABULAR DATA OMITTED to assess the levels of IRMA-interfering activity in the cell perifusates, thus attempting to discriminate true ACTH release from nonspecific interference. Figure 2 shows the ACTH and CRH signals detected in the perifusates from pituitary cells of young (3-4 months) male rats perifused with different doses of median eminence (ME) extracts, CRH, histones H2A and H2B, and ubiquitin. The |ACTH signal/CRH signal~ ratios in the H2A and H2B perifusates were markedly higher than those of the original histone solutions (stimuli).
Figure 3 shows a similar ACTH and CRH signal profile in perifusates of pituitary cells from young rats perifused with a number of histones and related preparations. Histones and protamine generated a significant signal in both IRMAs. On the other hand, nucleohistone, nucleoprotamine and peptide MB35 only generated an ACTH signal.
Studies with incubates of pituitary cells from young and mature rats
Table I shows the levels of ACTH release from dispersed pituitary cells of young (2-4 months) and mature (16-18 months) rats, after incubation with hypothalamic secretagogues, nucleoproteins or peptide MB35. There were no age-related differences in the ACTH response to ME or CRH. On the other hand, nucleoproteins and MB35 induced a lower ACTH response in cells from mature animals.
There is increasing evidence that in addition to their structural role in chromosomal architecture, histones may possess hormone-like properties when present in extracellular fluids. Thus, histones H1, H2A, H2B and H3 have been reported to inhibit adenylate cyclase in canine renal cortical membrane preparations |17~. Also, a gonadotropin-releasing hormone-binding inhibitor from bovine ovaries was purified and identified as histone H2A |18~. Peptide MB35, which is an active component of the thymic preparation TF5 and is identical to the fragment 86-120 of histone H2A, has been recently reported to stimulate prolactin and GH release from cultured pituitary cells |19~. Our previous studies (see above) reporting the effects of HTH in vivo on pituitary hormone secretion lend further support to this line of evidence.
Our initial experiments revealed that histones and protamines generate a significant interaction with the IRMA for CRH and ACTH. That this is a nonspecific phenomenon is suggested by the observation that the dose-dependent signal generated by histones in the IRMA standard curves was not affected by removal of the linker antiserum from the reagent mixture (data not shown). Since the linker antiserum is essential for the immunoprecipitation of the hormone-|125I~IgG complex, any radioactivity precipitated in the absence of linker must be nonspecific. The fact that histones and protamines bound to DNA (nucleohistone and nucleoprotamine complexes, respectively) are no longer able to interfere with the ACTH-IRMA suggests that free histones and protamines may possess a significant electrostatic affinity for the IgG molecules, with which they could form protein aggregates of low solubility. On the other hand, the peptide ubiquitin, an adenylate cyclase activator |20~ which is usually present in histone preparations, did not show any interfering activity in our IRMAs, thus serving as a negative control. Histones and protamine also interfered with the ACTH-RIA (unpublished observations) thus making it difficult to assess their true ACTH-releasing activity. Nevertheless, the marked increase of the ratios |ACTH signal/CRH signal~ in the elution profiles (as compared with those of the original stimuli) after perifusion of pituitary cells with histones (particularly H2A and H2B) and protamines suggests, although does not prove, that these preparations stimulated ACTH release to some extent. This hypothesis is also supported by the fact that a histone fragment devoid of interfering activity, namely peptide MB35, caused a small but significant increase of the ACTH signal in the cell perifusates. More clear-cut are the results with histone--DNA and protamine-DNA complexes which induced a definite ACTH signal increase in the cell perifusates despite their lack of interference with the ACTH- and CRH-IRMA. There is in fact previous evidence that nucleoprotein complexes released into the extracellular milieu can elicit definite cellular responses. Thus, it has been reported that the mono- and oligo-nucleosomes released by spleen and thymic T cells undergoing programmed death (apoptosis) in short-term tissue culture have mitogenic and polyclonal effects on normal B lymphocytes |21~. This results in a generalized enhancement of Ig synthesis and anti-DNA antibody responses in vitro |22~. It is therefore possible that during physiological or pathological processes involving massive programmed cell death (like postpubertal involution of the thymus or autoimmune diseases), the nucleoproteins released by the dying cells may convey important signals to the neuroendocrine and immune systems. Within this context, our data indicating a diminished responsiveness of the pituitary cells from mature rats to nucleoprotein complexes may represent just one facet of a generalized age-related desensitization of the neuroendocrine system to feedback signals from peripheral structures.
Although further work is necessary to clarify the physiological significance of our findings, the present results nevertheless open challenging conceptual avenues. Thus, if such highly conserved proteins as histones do play a role as extracellular messengers, this is bound to be a fundamental one, comparable perhaps in importance to the role of histones as structural components of chromatin. Certainly, an age-dependent desensitization of the neuroendocrine system and other integrative centres of the body to these putative signals, could act as an ancestral pacemaker of homoeostatic decline.
One of the authors (MGC) wishes to thank the Dundee Institute of Technology Research Committee, the Industrial Development Fund and the Department of Molecular and Life Sciences for their invaluable support. During the present study RGG was recipient of a Senior Fellowship from the European Economic Community as well as a grant from the Sandoz Foundation for Gerontological Research.
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|Author:||Goya, Rodolfo G.; Castro, Maria G.; Saphier, Peter W.; Sosa, Yolanda E.; Lowry, Philip J.|
|Publication:||Age and Ageing|
|Date:||Jan 1, 1993|
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