The involvement of pineal gland and melatonin in immunity and aging. I. Thymus-mediated, immunoreconstituting and antiviral activity of thyrotropin-releasing hormone.

Circadian, continued treatment with melatonin during the dark cycle produces changes in the blood level of thyroid hormones in aging mice. Thyroid-stimulating hormone (TSH) and thyrotropin-releasing hormone (TRH) antagonize the involution of the thymus produced by prednisolone. This effect of TRH does not seem thyroid dependent. TRH restores antibody production in non-responder athymic nude mice but does not exert this effect in neonatally thymectomized mice. Moreover, this activity does not correlate with thyroxine levels. TRH exerts a powerful protective effect in mice challenged with encephalomyocarditis (EMC) virus. Presumably pineal melatonin exerts its varied regulatory functions via hypothalamic TRH.

The pineal gland and cancer. I. Pinealectomy corrects congenital hormonal dysfunctions and prolongs life of cancer-prone C3H/He mice.

Hormonal derangements almost invariably anticipate and signal the onset of tumors. Chronic, nocturnal melatonin administration delays aging in normal strains of mice. On the contrary it promotes and accelerates the onset of tumors in the cancer-prone strain of C3H/He mice. Grafting of a young pineal gland into aging mice prolongs their longevity and maintains juvenile circadian hormonal functions while pinealectomy (Px) does the opposite. We investigated if Px in C3H/He mice would modify their congenitally deranged pituitary function and affect their longevity. It was found that contrarily to Px in normal mice, Px in C3H/He mice remarkably maintains juvenile night levels of thyroid hormones and lipids, preserves a cell-mediated immune response and significantly prolongs their life. The pineal gland and its pathology may be the key for understanding, not only the causes of metabolic aging, but also the origin of those congenital or progressive aging-related hormonal alterations preceding onset of all tumors and thus allow preventive corrective interventions with pineal-derived agents.

Role of the pineal gland in immunity. Circadian synthesis and release of melatonin modulates the antibody response and antagonizes the immunosuppressive effect of corticosterone.

Inhibition of synthesis of the pineal neurohormone melatonin (MEL) in mice, by administration of propranolol (PRO) in the evening, and daily injections of p-chlorophenylalanine (PCPA), resulted in a significant depression of the primary antibody response to sheep red blood cells (SRBC). Spleen cells from these mice showed a reduced reactivity against antigens in the autologous mixed lymphocyte reaction (AMLR). In contrast, alloreactivity remained normal. Reconstitution of the night-time peak of plasma MEL by evening injections to the mice completely reversed the suppression of the humoral response and the AMLR. MEL administration was able to antagonize the depression of antibody production induced by corticosterone in vivo. These results suggest that the pineal gland has important immunomodulatory functions through its cyclic, circadian release of MEL.

The immuno-reconstituting effect of melatonin or pineal grafting and its relation to zinc pool in aging mice.

It has been demonstrated that melatonin, the main neuro-hormone of the pineal gland, affects thymic functions and the regulation of the immune system. In addition, experimental evidences indicate that melatonin can modulate zinc turnover. The knowledge that with advancing age both melatonin and zinc plasma levels decline, and that zinc supplementation in old mice is able to restore the reduced immunological functions, has prompted investigations on the effect of chronic melatonin treatment or pineal graft in old mice on the age-related decline of thymic endocrine activity, peripheral immune functions and zinc turnover. Both melatonin treatment in old mice and pineal graft into the thymus of old mice correct the reduced thymic endocrine activity and increase the weight of the thymus and its cellularity. A restoration of cortical thymic volume, as detected by the percentage of tissue in active proliferation, is also observed in old mice after both treatments. Thymocyte CD phenotype expression is also restored to young values. At peripheral level, recovery of peripheral blood lymphocyte number and of spleen cell subsets, with increased mitogen responsiveness also occurs. Melatonin treatment or pineal graft induce also a restoration of the altered zinc turnover in aged mice with an increment of the crude zinc balance from negative (-1.6 microgram/day/mouse) to positive value (+1.2 microgram/day/mouse), similar to that one of young mice (+1.4 microgram/day/mouse). The reduced zinc plasma level is restored to normal values. These findings support the idea that the effect of melatonin on thymic endocrine activity and peripheral immune functions may be mediated by the zinc pool.

Melatonin: a principal neuroimmunoregulatory and anti-stress hormone: its anti-aging effects.

Major environmental variables such as daily and seasonal changes of light and temperature regulate the daily circadian variations of synthesis and release of the pineal neurohormone N-acetyl-5-methoxytryptamine (melatonin). Melatonin has now been shown to be a potent immunoregulatory agent, and to be able to antagonize the immunosuppressive effects of acute anxiety stress in mice, as measured by antibody production, by thymus weight, and by the capacity of stressed- and evening-melatonin-treated mice to react against a lethal virus. Both psychogenic factors and infectious agents such as viruses can act as "stressors" and induce an immunosuppression. Their combination is a determinant for the course of infectious diseases and, perhaps, cancer. Circadian (evening) melatonin possesses thus the singular ability to up-regulate the immunosuppression of stressed mice. This effect of melatonin is not exerted directly on immunocompetent cells, but mediated via the endogenous opioid system upon antigen-activation of T cells. Melatonin being a short-lived hormone with negligible side-effects which is rapidly degraded and eliminated by the body, the use of melatonin offers a new approach to the physiological control of stress and stress-related infectious diseases. In addition, melatonin could be used for the potentiation of primary immunization (vaccination) against antigens of the most varied nature which do not evoke a robust or longlasting secondary (memory) response. The regulatory function of pineal melatonin is discussed also in relation to hematopoiesis, to its oncostatic effects, and to its possible dual role in reproduction physiology and generation of immunocompetence and tolerance during ontogeny.

Dominance and persistence of donor marrow in long-lived allogeneic radiation chimeras obtained with unmanipulated bone marrow.

Allogeneic, H-2-incompatible irradiation chimeras (H-2d leads to H-2b) constructed with normal, unmanipulated bone marrow and with marrow-derived factors live long and do not manifest a GvH disease. Their response to primary immunization is deficient but their alloreactivity is normal. This chimeric allotolerance cannot be passively transferred from chimeric donors to normal irradiated recipients. Passive transfer of both donor- or recipient-type immunocompetent T-cells into the chimeric mice does not lead to syngeneic reconstitution, rejection of the engrafted marrow or GvH disease and the mice maintain permanently their chimerism. This new model demonstrates that chimerism is not eradicable in long-lived chimeras reconstituted with unmanipulated bone marrow, and that the bone marrow itself plays a dominant role in maintenance of chimerism.

Transferrin treatment corrects aging-related immunologic and hormonal decay in old mice.

Experiments were conducted to study the effect of heterologous plasma transferrins separated and purified from human plasma pools on endocrine and immune functions of old, aging mice. Two similar experiments have shown that parenteral treatment with iron and zinc-free human transferrins produces a significant improvement of immunological and endocrine functions in the aging mice toward more juvenile values. Those changes occur in the thymus and its cell subsets, in peripheral blood lymphocytes, in the restoration of juvenile levels of thyroxine, in the increase of testis weight, and in the normalization of plasma zinc levels. These totally unsuspected effects of transferrin in aging mice suggest a most important role of endogenous transferrins in the maintenance of neuroendocrine and immune functions. The mechanism remains unexplained although the basic immunoenhancing and anti-apoptotic effect of transferrin-vehiculated zinc may be relevant.

Effects of melatonin in perimenopausal and menopausal women: a randomized and placebo controlled study.

In aging humans, night levels of melatonin (MEL) decline progressively. Also thyroid and gonadal functions decline during aging while gonadotropins (luteotropic hormone (LH) and follicle stimulating hormone (FSH)) steadily increase. A desynchronization of pineal circadian cyclicity as expressed by the progressive decrease of the MEL night peak may be permissively linked to the onset and progression of menopause. We studied the effects of exogenous, evening administration of MEL on the level of hormones which are known to be involved in the genesis and progression of menopause. Perimenopausal and menopausal women from 42 to 62years of age with no pathology or medication were selected. MEL was measured in saliva to divide them into low, medium and high-MEL patients. Half of them took 3mg MEL and half of them Placebo at bedtime (10-12p.m.) in a fully randomized and double-blind fashion. Three and six months later blood was taken for determination of pituitary (LH, FSH), ovarian, and thyroid hormones I(T3 and T4). All women taking MEL with low basal level of MEL and/or Placebo for three and six months showed a significant increase in levels of thyroid hormones. Before initiation of the study, a negative correlation was found in all women between LH, FSH and basal MEL levels. Within six months of treatment, MEL produced a significant diminution of LH in the younger women (43 to 49year-old), while no effect was seen in the older women (50-62years old). A decrement of FSH was observed in MEL-treated women with low basal MEL levels. In addition, most MEL-treated women reported a general improvement of mood and a significant mitigation of depression. MEL decline during aging may thus signal the derangement of pineal and pituitary-controlled ovarian cyclicity and the progressive quenching of fertility in women. These findings seem to show a recovery of pituitary and thyroid functions in MEL-treated women, towards a more juvenile pattern of regulation.

Circadian melatonin and young-to-old pineal grafting postpone aging and maintain juvenile conditions of reproductive functions in mice and rats.

Chronic, night administration of melatonin to aging mice and transplantation of a young pineal gland into the thymic rudiment of older mice and rats have been studied with the aim of evaluating their effects on aging of gonadal, sexual, and reproductive functions. Both melatonin administration and young-to-old pineal grafting positively affect size and function of testes and maintenance of juvenile hippocampal and testicular LHRH-receptors and beta-adrenergic receptors in the tests of old rats and mice. These results demonstrate that a pineal-directed circadian function and cyclicity is fundamental for the regulation of sexual, reproductive physiology, and that proper intervention with melatonin may potentially postpone aging of both neural and gonadal sexual function.

Allogeneic H-2d leads to H-2b irradiation bone marrow chimeras: a) failure to transfer chimerism adoptively and b) immune reactivity of immunocompetent lymphocytes adoptively transferred to chimeras.

Long-lived, GVHD-free, H-2 incompatible haemopoietic chimeras (P1 leads to P2) were constructed transfusing unmanipulated bone marrow cells together with recently identified marrow-regulating factors (MRF) in lethally irradiated recipients. The chimeric tolerance of P1 leads to P2 chimeras proved to be adoptively untransferable. Another peculiar property of established P1 leads to P2 allochimeras was their ability to "suppress" or reject passively transfused immunocompetent P1 or P2 lymphocytes. Even this "suppression" appeared to be untransferable and to operate in the chimera only in a fashion dependent upon the age of the established chimeras. This chimeric "unidentified suppressive principle" seems not to follow familiar immunologic lines. A relationship with the mechanism of chimeric tolerance is suggested.

Donor-derived plasma transferrin facilitates the engraftment of xenogeneic (rat) bone marrow in irradiated mice.

Endogenous factors originally found in the bone marrow (BM) and facilitating the engraftment of xenogeneic (rat) BM in lethally irradiated mice have been recently identified as transferrins (Tf). Tf have been separated and purified from plasma pools of inbred Rii/2 rats and injected in lethally irradiated BALB/c and C57BL/6 mice 1 h before the infusion of BM and for several days after BM transplantation. Other groups of irradiated mice have been similarly treated with human Tf, Tf from other strains of rats different from the BM donors and with human or rat serum albumin. A remarkable facilitation of BM engraftment and a durable graft-versus-host disease (GVHD)-free hemopoietic chimerism have been achieved in the irradiated mice when a combination of BM and Tf from the same donor rat (Rii/2) strain was used for transplantation. Durable survival and persistent chimerism were not observed in the control groups. It seems that donor Tf profoundly affects the outcome of BM transplantation when combined with donor BM. These results indicate that the mechanism by which Tf promotes engraftment of xenogeneic BM deserves investigation in order to improve this novel procedure and to extend it to other species and possibly to man.

Neuroimmunomodulation of aging. A program in the pineal gland.

We have investigated for 35 years the relationship between the neuroendocrine and the thymo-lymphatic, immune system. In the last decade we have shown that the pineal gland is a main adapter and fine synchronizer of environmental variables and endogenous messages into physiological modifications of basic functions. In particular the pineal gland itself seems to regulate, via circadian, night secretion of melatonin, all basic hormonal functions and also immunity. We have shown with several in vivo models that this fundamental role of the pineal gland decays during aging. Aging itself seems to be a strictly pineal-programmed event similar to growth and puberty. The continuation of our interventions with melatonin against the typical degenerative diseases of aging must be based on an accurate evaluation of its mechanisms of action. Melatonin being a ubiquitous molecule in nature, we suggest that it has acquired during evolution of the species numerous levels of activities. In fact, melatonin can be found in a large variety of cells and tissues, and bindings sites and "receptors" have been identified in many tissues and cells of the neuroendocrine and immune system. Therefore, the progressive understanding of the aging-programming role of the pineal gland also depends on studies of melatonin and its basic regulatory function. Our present studies will be described.

Pineal cross-transplantation (old-to-young and vice versa) as evidence for an endogenous "aging clock".

Circadian (night), chronic administration of melatonin and young-to-old pineal grafting into the thymus have provided evidence for the existence of an endogenous, primary and central "aging clock" in the pineal gland. The new model described here serves to definitely demonstrate that the replacement of the pineal gland of an old mouse with the pineal from a young, syngeneic donor mouse remarkably prolongs its life and, conversely, the "old" pineal transplanted into a younger mouse will considerably shorten its life span. Pineal cross-transplantation thus provides clear-cut evidence for the central role of the pineal gland in the initiation and progression of senescence. It offers a novel basis for interventions in the aging process.

Unresponsiveness to human leukocytes in immunosuppressed mice by combined donor-derived human transferrin and antigens.

Previous work on the facilitation of xenogeneic and allogeneic bone marrow engraftment in irradiated mice and dogs with transferrins allowed the development of a model for induction of an apparently durable state of immunological unresponsiveness or 'tolerance' in chemically immunosuppressed mice. The system is based on the simultaneous and combined administration of donor-derived cell antigens, namely human leukocytes, and specific donor-derived or plasma pool human transferrin into BALB/c or C57BL/6 mice previously treated with prednisolone and cyclophosphamide on day 0 and day 1 of the experiment. A properly timed presentation of both donor-specific or plasma pool transferrin and leukocyte antigens into the mice on day 3 and day 16 of the experiment, in the course of initial restoration of their lymphohaemopoietic tissues and cells after severe immunosuppression, results 1-3 months later, in their inability to 'recognize' human donor lymphocytes and to mount an immediate or a delayed-type immune response against human antigens. This durable state of unresponsiveness was evaluated by a complement-mediated cytotoxicity assay, with a mixed lymphocyte culture method and confirmed by the abrogation of the humoral (antibody response to human erythrocytes) and of the cell-mediated (popliteal lymph node test) immune responses in vivo. Our findings demonstrate the capacity of human plasma-derived transferrins to induce a state of durable unresponsiveness (xenogeneic tolerance?) in mice when administered with human antigens in the course of regeneration of stem cells in the bone marrow and lymphatic organs.

Facilitation of DLA-incompatible marrow grafts by donor-specific serum transferrin?

Studies in mice have shown that donor-specific plasma transferrin (TF) given to the recipient in the peritransplant period facilitates engraftment of marrow from histoincompatible donors. Dogs given 920 cGy of total body irradiation (TBI) and infused with marrow from an unrelated major histocompatibility complex (DLA) different donor generally fail to engraft; only approximately 20% of dogs achieve sustained engraftment. We have now investigated in this model whether the infusion of donor-specific plasma TF would facilitate engraftment. Ten dogs were given TBI, followed at 23 h by an intravenous dose of TF, at 24 h by marrow from the same donor, and another dose of TF at 48 h; six dogs also received postgrafting methotrexate (MTX). Seven dogs (three of four without MTX, four of six with MTX) had sustained engraftment, and three dogs failed to engraft. A single dog given third-party TF failed to engraft. Among five dogs not given TF two achieved sustained engraftment. This pilot study suggests that donor-specific TF facilitates engraftment of DLA-incompatible marrow. Further studies are warranted.

Overcoming the histocompatibility barrier: transferrins as carriers and modulators of immunogenic identity.

Proteic molecules were found in the bone marrow that were later identified as transferrins. When applied to transplantation of genetically incompatible bone marrow in supralethally irradiated recipient mice, the transferrins obtained from plasma of bone marrow donors promoted engraftment, permanent hemopoietic chimerism, and donor-type immune character. A combination of donor-matched transferrins and antigens was needed for induction of xenogeneic (interspecies) "tolerance" or unresponsiveness to donor antigens in chemically immunosuppressed mice treated with human transferrins and donor leucocytes. This novel and unique property of transferrins may explain the genesis and maintenance of immunogenic identity and allow a reshaping of the immune system.

[The effect of iron carrier proteins on the transplantation of H-2 locus-incompatible bone marrow in irradiated mice]. / Vliianie zhelezonesushchikh belkov na transplantatsiiu nesovmestimogo po H-2 lokusu kostnogo mozga obluchennym mysham.

Rabbit bone marrow supernatants were fractionated and purified by Ultrogel and Superose chromatography. A unique fraction promoted engraftment of allogenic bone marrow and enduring hemopoietic chimerism across the histocompatibility (H-2) barrier in lethally irradiated mice. This fraction analysed by reducing SDS-PAGE electrophoresis and transblotted on PVDF membrane or purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main pre-albumin band that was examined for primary structure by Edman degradation. It appeared to be rabbit transferrin. Iron saturated human transferrin, lactotransferrin and egg transferrin (conalbumin) were then tested in irradiated C57B1/6 mice transplanted with bone marrow from histoincompatible BALB/CJ donors. Most mice treated with iron-loaded transferrins survived and developed enduring allogeneic chimerism with no discernible signs of graft-versus-host disease at 10 months posttransplant. Observation of these animals is still carried on. Iron carrier proteins seem to provide a novel unexpected means for achieving a successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations and may open a new approach in the clinical area.