Cryptocrine signaling in the thymus network and T cell education to neuroendocrine self-antigens.

Both during phylogeny and ontogeny the thymus appears as a nodal point between the two major systems of cell-to-cell signaling, the neuroendocrine and immune systems. This review presents the experimental observations which support a dual role in T cell selection played by the thymic repertoire of neuroendocrine polypeptide precursors. Through the mode of cryptocrine intercellular signaling thymic neuroendocrine-related precursors synthesized in thymic epithelial cells have been shown to influence the early steps in T cell differentiation. In addition, thymic neuroendocrine-related polypeptides are a source of self-antigens which are presented by the major histocompatibility system of the thymic epithelium. Preliminary data also suggest that the intrathymic T cell education to neuroendocrine self-antigens is not strictly superimposible to the antigen presentation by dedicated presenting cells. Insulin-like growth factor-II (IGF-II) was identified as one dominant member of the insulin family expressed by thymic epithelial and nurse cells. The intrathymic presentation of IGF-II or IGF-II derived self-antigens is under current investigation. If further confirmed, the central tolerogenic properties of IGF-II could be considered in the elaboration of a strategy for an efficient and safe prevention of insulin-dependent diabetes.

In vitro stimulation of the prepubertal rat gonadotropin-releasing hormone pulse generator by leptin and neuropeptide Y through distinct mechanisms.

Leptin may act as a negative feedback signal to the brain in the control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine- and amphetamine-regulated transcript (CART), a new anorectic peptide. We aimed at studying whether leptin, NPY, and CART have related effects on the hypothalamic control of the pituitary-gonadal system and the developmental changes in NPY and CART effects. Using retrochiasmatic hypothalamic explants from prepubertal 15-day-old male rats, the GnRH interpulse interval (mean +/- SD: 62 +/- 5 min) was significantly reduced by 10(-7) M of leptin (46 +/- 3.3 min) as well as 10(-7) M of NPY (47 +/- 4.4 min) and 10(-6) M of CART (46 +/- 2.7 min), whereas the GnRH pulse amplitude was not affected. The stimulatory effects of different NPY receptor agonists [human PYY 3-36, porcine NPY 13-36, human (D-Trp 32) NPY, porcine (Leu 31 Pro 34) NPY, human pancreatic polypeptide (PP)], as well as the absent effects of rat PP were consistent with the involvement of the Y5-receptor subtype in mediation of NPY effects. Incubation with 10(-7) M of a Y5-receptor selective antagonist prevented the effect of NPY (61 +/- 4 vs. 46 +/- 2 min), whereas leptin and CART effects were not (47 +/- 3 vs. 46 +/- 3 min and 46 +/- 3 vs. 46 +/- 2 min, respectively), suggesting that NPY was not involved in leptin and CART effects. Using an anti-CART antiserum (1:1000), the reduction of GnRH interpulse interval caused by leptin was partially prevented (56.2 +/- 4 vs. 47.9 +/- 3.8 min), whereas the reduction of GnRH interval caused by NPY was not affected (45.9 +/-2.5 vs. 47.8 +/- 3.7). The GnRH interpulse interval was decreased by 10(-7) M of NPY at 5 days (72 +/- 3.8 vs. 91.9 +/- 3.5) as well as at 15 days, whereas such an effect was not observed anymore at 25 and 50 days. Similar effects were observed using 10(-6) M of CART-peptide. Using 10(-6) M of the Y5-receptor antagonist, the GnRH interpulse interval was significantly increased at 15 days (66.6 +/- 2.7 min), 25 days (56.5 +/- 39.9 min), and 50 days (52.5 vs. 38.2 min), whereas no change was observed at 5 days. Using the anti-CART antiserum, a significant increase of GnRH interpulse interval was observed at 25 days only. In conclusion, the stimulatory effects of leptin and NPY on the frequency of pulsatile GnRH secretion before puberty involve two distinct mechanisms. NPY causes acceleration of GnRH pulsatility via the Y5-receptor subtype, which is not involved in leptin effects while the CART is involved in leptin effects on GnRH secretion but not in NPY effects. The reduction of pulsatility by the Y5 antagonist provides evidence of endogenous NPY involvement in the control of GnRH secretion from the time of onset of puberty.

Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion: I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH.

GnRH[1-5], a subproduct resulting from degradation of GnRH by prolyl endopeptidase (PEP) and endopeptidase 24.15 (EP24.15) was known to account for an inhibitory autofeedback of GnRH secretion through an effect at the N-methyl-D-aspartate (NMDA) receptors. This study aimed at determining the possible role of such a mechanism in the early developmental changes in frequency of pulsatile GnRH secretion. Using retrochiasmatic explants from fetal male rats (day 20-21 of gestation), no GnRH pulses could be observed in vitro, whereas pulses occurred at a mean interval of 86 min from the day of birth onwards. This interval decreased steadily until day 25 (39 min), during the period preceding the onset of puberty. Based on GnRH[1-10] or GnRH[1-9] degradation and GnRH[1-5] generation after incubation with hypothalamic extracts, EP24.15 activity did not change with age, whereas PEP activity was maximal at days 5-10 and decreased subsequently until day 50. These changes were consistent with the ontogenetic variations in PEP messenger RNAs (mRNAs) quantitated using RT-PCR. Using fetal explants, the NMDA-evoked release of GnRH was potentiated in a dose-dependent manner by bacitracin, a competitive PEP inhibitor and the desensitization to the NMDA effect was prevented using 2 mM of bacitracin. At day 5, a higher bacitracin concentration of 20 mM was required for a similar effect. Pulsatile GnRH secretion from fetal explants was not caused to occur using bacitracin or Fmoc-Prolyl-Pyrrolidine-2-nitrile (Fmoc-Pro-PyrrCN), a noncompetitive PEP inhibitor. At postnatal days 5 and 15, a significant acceleration of pulsatility was obtained using 1 microM of Fmoc-Pro-PyrrCN or 2 mM of bacitracin. At 25 and 50 days, a lower bacitracin concentration of 20 microM was effective as well in increasing the frequency of GnRH pulsatility. We conclude that the GnRH inhibitory autofeedback resulting from degradation of the peptide is operational in the fetal hypothalamus but does not explain the absence of pulsatile GnRH secretion at that early age. After birth, PEP activity is high and may account for the low frequency of pulsatility. The potency of that effect decreases before the onset of puberty and may contribute to the acceleration of GnRH pulsatility.

Identification of neurotensin-related peptides in human thymic epithelial cell membranes and relationship with major histocompatibility complex class I molecules.

This study shows the expression at the cell surface of human thymic epithelial cells (TEC) of a neurotensin (NT)-like immunoreactivity. NT radio-immunoassay (RIA) revealed that cultured human TEC contain +/-5 ng immunoreactive (ir) NT/10(6) cells, of which 5% is associated with plasma cell membranes. HPLC analysis of NT-ir present in human TEC showed a major peak of NT-ir corresponding to NT1-13. NT-ir was not detected in the supernatant of human TEC cultures. Using an affinity column prepared with a anti-MHC class I monoclonal antibody, NT-ir-related peptides were retained on the column and eluted together with MHC class I-related proteins. According to the elution time on HPLC of these peptides, they correspond to intact NT1-13, as well as to smaller fragments of NT1-13.

Cocaine and amphetamine-regulated-transcript peptide mediation of leptin stimulatory effect on the rat gonadotropin-releasing hormone pulse generator in vitro.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using explants of the retrochiasmatic hypothalamus from prepubertal male and female rats. Leptin caused a dose-dependent reduction of the GnRH interpulse interval in both sexes. We studied the effects of cocaine- and amphetamine-regulated transcript (CART) since this peptide was shown recently to mediate the anorectic effects of leptin in the hypothalamus. CART caused a reduction of the GnRH interpulse interval. This effect was prevented using an anti-CART antiserum which could partially overcome leptin stimulatory effects as well. Using hypothalamic explants from Zucker rats homozygous for the leptin receptor mutation ( fa/fa), GnRH pulse frequency was not affected by leptin, while a significant acceleration was caused by the CART-peptide. In conclusion, leptin involves the hypothalamic CART-peptide to stimulate the prepubertal GnRH pulse generator in vitro.

Cellular and molecular aspects of thymic T-cell education in neuroendocrine self principles. Implications for autoimmunity.

Thymic epithelial and nurse cells from different species express a repertoire of neuroendocrine polypeptide precursors. This repertoire exerts a dual role in T-lymphocyte selection according to their status either as cryptocrine signals or as neuroendocrine self-antigens of the peptide sequences that are processed from those precursors then presented to pre-T cells. Thymic neuroendocrine self-antigens correspond to peptide sequences highly conserved throughout evolution of their family. Though thymic MHC class I molecules are involved in the processing of thymic neuroendocrine self-antigens, preliminary data show that their presentation to pre-T cells is not allelically restricted. Thymic T-cell education in neuroendocrine families also implies that the structure of a given family may be presented to pre-T cells. Our studies have evidenced the homology between thymic neuroendocrine-related self-antigens and dominant T-cell epitopes of peripheral neuroendocrine signals (neuroendocrine autoantigens). The biochemical difference between neuroendocrine autoantigens and homologous thymic self-antigens might explain the opposite immune responses evoked by those two types of antigens (activation and memory induction vs. tolerogenic effect). Altogether, these studies support the therapeutic use of thymic neuroendocrine self-antigens in reprogramming the immunological self-tolerance that is broken in autoimmune endocrine diseases like insulin-dependent diabetes type I. As recently stated by P. M. Allen in an important review, the fate of developing T lymphocytes in the thymus is influenced by the numerous types of peptidic interactions within the thymic cellular environment. To define the precise nature of thymic cells and naturally occurring biochemical peptide signals involved in positive and negative selection of immature T cells has become a prominent objective for the future research efforts in thymic physiology. This paper will try to show how thymic neuroendocrine-related peptides synthesized and processed within the thymic microenvironment indeed can play a role both in the development of the peripheral T-cell repertoire and in the death of randomly rearranged, self-reactive T cells.

Leptin effects on pulsatile gonadotropin releasing hormone secretion from the adult rat hypothalamus and interaction with cocaine and amphetamine regulated transcript peptide and neuropeptide Y.

Leptin may act as a negative feedback signal to the hypothalamic control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine and amphetamine regulated transcript (CART). We aimed at studying the effects of leptin, CART and NPY on the hypothalamic control of the pituitary-gonadal system. Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using retrochiasmatic hypothalamic explants from adult rats. In the female, GnRH pulse amplitude was significantly increased by leptin (10(-7) M) and CART (10(-6) M) irrespective of the estrus cycle phase while no such effects were seen in the male. The GnRH interpulse interval was not affected in both sexes. Passive immunoneutralization against CART caused a reduction in GnRH pulse amplitude in the female. A slight but significant increase in GnRH pulse amplitude was caused by NPY (10(-7) M) in the female. However, GnRH pulse amplitude was not affected by a Y5-receptor antagonist (10(-6) M) while the interpulse interval was significantly increased as shown previously in the male. The increase in GnRH pulse amplitude caused by leptin was totally prevented by coincubation with an anti-CART antiserum whereas it was not affected by coincubation with the NPY Y5-receptor antagonist (10(-7) M). In conclusion, leptin and NPY show separate permissive effects on GnRH secretion in the adult rat hypothalamus. In both sexes, NPY is prominently involved in the control of the frequency of pulsatile GnRH secretion through the Y5 receptor subtype. Leptin causes a female-specific facilitatory effect on GnRH pulse amplitude which is mediated by CART and which occurs irrespective of the estrus cycle phase.

Further data to the aetiology, pathogenesis and therapy of infectious bovine keratoconjunctivitis.

Out of a total of 224 bovine eye secretions, 126 Moraxella bovis and 64 Neisseria ovis strains were isolated. The pathogenesis and histological lesions caused by Neisseria ovis have been studied on the eyes of three calves naturally affected with IBK, using electron microscopy. Neisseria ovis caused in 1-12 weeks old calves acute, transient and mostly benign serous conjunctivitis with only slight affection of the cornea. More rarely erosions and even ulceration of the cornea have been observed. Moraxella bovis and Neisseria ovis strains proved nearly unanimously sensitive in vitro to chloramphenicol, neomycin, oxytetracyclin, nitrofurantoin, erythromycin and cefoperazone. Other antibiotics and chemotherapeutics inhibited the growth of these agents only partly or were ineffective. Experimental therapy has been carried out using a single i.m. injection of Terramycin/LA inj. (Pfizer) in a dose of 20 mg/kg body mass, repeated if necessary after 72-96 h. This formulation proved more effective and practical than treatments used earlier.

[Central function of the thymus in the recognition of neuroendocrine functions by T lymphocytes during their development]. / La fonction centrale du thymus dans la reconnaissance des fonctions neuroendocrines par les lymphocytes T au cours de leur développement.

The dual physiological role of the thymus in T cell positive and negative selection appears prominent in the establishment of appropriate host immune defenses. However, the cellular and molecular mechanisms underlying those thymic functions begin only to be understood. On the basis of our previous investigations about the thymic expression of different neuroendocrine-related signals, we have advanced a model which transposes at the peptide level the intervention of this primary lymphoid organ in both T cell positive and negative selective processes. There is now ample evidence that the thymic subcapsular and medullary epithelium is the site for synthesis of neurohypophysial (NHP)-related peptides (reviewed in Geenen et al., 1992a). We have also demonstrated that the epithelial component of thymic "nurse" cells (TNC) synthesizes NHP-related peptides and expresses a neuroendocrine-like phenotype (Geenen et al., 1988a). This observation was a remarkable example of the intimate neuroendocrine-immune interactions that take place during T cell ontogeny. Further immunocytochemical analyses have confirmed that one dominant NHP-related epitope belongs to the oxytocin (OT) lineage of the NHP peptide superfamily (Robert et al., 1991, 1992). The intrathymic coexpression of this OT-like epitope with a neurophysin protein domain is a strong argument for a local synthetic process similar to the hypothalamo-NHP one. However, the absence of ir-OT in secretory granules of thymic epithelial cells (TEC), as well as of NHP-related peptides in the supernatant of TEC cultures questioned the application to the thymus of the classical neurosecretory model established for hypothalamic magnocellular neurons (Scharrer & Scharrer, 1944).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that insulin-like growth factor 2 (IGF2) is the dominant thymic peptide of the insulin superfamily.

The central T-cell tolerance of neuroendocrine functions has been proposed to be primarily induced by the thymic repertoire of neuroendocrine self antigens. The present study aimed at characterizing the human thymic insulin-related autoantigen able to represent the pancreatic B-cell function in face of the developing T-cells. Immunofluorescence studies were performed on human and rat thymic sections, as well as on the rat IT-45R1 thymic epithelial cell line using several antibodies to epitopes of the insulin peptide superfamily. These studies identify beyond any doubt that insulin-like growth factor 2 (IGF2) is the dominant thymic peptide of the insulin family. The sequence of an insulin-derived autoantigen is proposed. This autoantigen is a nonamer and has a hydrophobic residue leucine (L) at position 9. In the human species, this autoantigen would primarily be tolerogenic for the pancreatic B-cell endocrine function during fetal development.

Membrane translocation and relationship with MHC class I of a human thymic neurophysin-like protein.

Thymic epithelial and nurse cells (TEC/TNC) synthesize an oxytocin (OT)-like peptide in association with a neurophysin (NP)-related protein in a way similar to in the hypothalamo-neurohypophysial (NHP) system. The central T-cell tolerance of the NHP neuroendocrine functions have been proposed to be mediated through these thymic NHP-related peptides due to their close homology with the NHP neurohormones OT and vasopressin (VP). In order to investigate their putative presentation by proteins of the major histocompatibility complex (MHC), human thymic membranes were purified and passed through an immunoaffinity column using mAb B9.12 directed to the monomorphic determinant of human MHC class I proteins. This methodology provided the following observations: (1) a NP-like protein is translocated in human thymic membranes and is retained by B9.12 on the column; (2) the MW of this NP-like material (50-55 kD) is quite different from the MW of hypothalamic NP proteins (10 kD), and (3) this thymic NP-like protein could be identified on Western blots with mAb B9.12. The precise extent of this relationship between the thymic NP-like protein and the Ig/MHC superfamily is actually investigated through the characterization of the genetic mechanisms responsible for the thymic expression of NHP-related peptides. Given the physiological importance of OT and of its binding to NP for transport along the axonal processes of the NHP tract, we postulate that, somewhat analogously, the thymic NP-/MHC class I-related protein could be involved in the presentation of the OT-like peptide to immature T-cells.